Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling

Mechanisms of drug-induced apoptosis in human leukemia

RIJKSUNIVERSITEIT GRONINGEN

Mechanisms of drug-induced apoptosis in human leukemia

Proefschrift

ter verkrijging van de graad van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts,

in het openbaar te verdedigen op woensdag 11 oktober 2006

om 16.15 uur

door

Alexandre D. Martins Cavagis

geboren op 22 april 1974 te Piracicaba, Brazilië

Promotor Prof. dr. Maikel P. Peppelenbosch co-promotor Prof. dr. Carmen Veríssima Ferreira Beoordelingscommissie Prof. dr. H. Hollema Prof. dr. H.H. Kampinga Prof. dr. E. Vellenga

To Alessandra

The work described in this thesis was carried out at the departament of Biochemics (laboratory of Signal Transduction), Institute of Biology, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil; the laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands and at the section Immunology of the department of Cell Biology, University Medical Center Groningen, Groningen, the Nethe rlands. The printing of this thesis was financially supported by the University of Groningen and the University Medical Center Groningen, the Netherlands.

ISBN 9036727812 (Also available as hard copy: printed by Wöhrman Print Service, Zutphen, the Netherlands; ISBN nr. 9036727804)

Contents 1 General overview: apoptosis and mitogen activated protein kinases (MAPKs) 7 2 Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling 27 3 Kinome profiling for uncovering the molecular mode of pharmacon action 51 4 Programmed cell death of HL60 leukaemia cells by the photosensitizer riboflavin is mediated by FasL-Fas induction and ceramide 67 5 Summary 91 6 Dutch summary 93 7 Curriculum Vitae 95 8 Word of thanks 97 9 Publications 99 10 Appendix 1 – Riboflavin: a multifunctional vitamin (translation - article published in Portuguese) 101 11 Appendix 2 – Overview sequences 119

Chapter 1

General Overview: Apoptosis and Mitogen-

activated protein kinases (MAPKs)

Chapter 1

8

1. Programmed Cell Death

At present, cell death is a field that has been attracting much attention

leading to several new and important progresses in cell biology. Nevertheless,

the recruitment of many new researchers to the field has been leading to some

confusion in terms and in the precise classification of the different categories of

cell death.

More precisely, cell death has been subdivided into three categories:

apoptosis (Type I), autophagic cell death (Type II) and necrosis (Type III).

Although the boundaries among these different categories are not always

distinct, the different types of cell death may be summarized in the following

scheme:

Programmed cell death (PCD) is a natural death of cells during

development and/or homeostasis, and it is important for sculpting tissues and

destroying harmful cells such as autoreactive immune cells and tumor cells.

Excessive PCD may contribute to various degenerative pathologies, whereas

lack of PCD can lead to the development of proliferative disorders such as

cancer.

Apoptosis is a type of PCD mediated by caspase activation and

particular morphological changes. In all works shown in the experimental

chapters of this thesis, we will focus on the caspase-dependent apoptosis and

use the term apoptosis referring to the classical approach of the caspase-

dependent apoptotic way of cell death.

Cell Death

Physiological

Apoptosis- caspase independent or- caspase-dependent

- receptor-linked (caspase 8)- mitochondria-linked (caspase 9)

Autophagy (caspase-independent)

Necrosis

General Overview: Apoptosis and MAPKS

9

2. Apoptosis

According to the classical approach, apoptosis is a morphologically and

biochemically distinct form of eukaryotic cell death that occurs under a variety of

physiological and pathological conditions. Apoptotic cells can be recognized by

characteristic morphological features: the cell shrinks, shows deformation and

looses contact to its neighbouring cells. The chromatin condenses and

marginates at the nuclear membrane, the plasma membrane is blebbing or

budding and finally the cell is fragmented into compact membrane-enclosed

structures which are called "apoptotic bodies" containing cytosol, the

condensed chromatin and organelles. The apoptotic bodies are engulfed by

macrophages and subsequently removed from the tissue without leading to an

inflammatory response. Those morphological characteristics are consequences

of biochemical events taking place in the apoptotic cell, such as the activation of

proteolytic enzymes that eventually mediate the cleavage of DNA into

oligonucleosomal fragments as well as the cleavage of a multitude of specific

protein substrates which usually determine the integrity and shape of the

cytoplasm and its organelles. Apoptosis is in contrast to the necrotic mode of

cell death in which situation the cells suffer a major insult, resulting in a loss of

membrane integrity, swelling and disrupture of the cells. During necrosis, the

cellular contents are released uncontrolled into the cell's environment which

results in damage of surrounding cells and a strong inflammatory response in

the corresponding tissue. As consequence, necrotic cell death causes an

inflammatory response with cytokine release by the surrounding macrophages.

The cell death with morphological features related to apoptosis may

occur in response to various stimuli, such as intracelular stress and cell

signalling mediated by specific receptors whereas, any failure on this system of

cell death has been associated to a wide range of human diseases such as

cancer and the resistance to chemoterapy.

During development many cells are produced in excess which

eventually undergo programmed cell death and thereby contribute to sculpturing

various tissues and organs and allow the organism to get rid of harmful or

potentially malignant cells. Apoptosis represents a process of great biological

Chapter 1

10

importance, being involved also in differentiation, development, proliferation,

homoeostasis, regulation and function of the immune system. Thus, failures in

apoptosis may result in autoimmune diseases, neoplasia and spreading of viral

infections. Conversely, excessive apoptosis has been associated with other

sorts of diseases, such as AIDS, neurodegenerative disorders and ischaemic

diseases.

3. Caspases

The cell death by apoptosis may be executed by external or internal

stimuli. The two main pathways of apoptosis induction are the receptor (or

extrinsic) pathway and the mitochondrial (or intrinsic) pathway. Both apoptotic

signaling pathways converge at the level of the specific proteases: the

caspases. The caspases are synthesized in the cell as inactive zymogens

called procaspases which at their N-terminus have a prodomain followed by a

large and a small subunit. Upon maturation, the procaspases are proteolytically

processed, resulting in a small and a large subunit, generating a heterotetramer

consisting of each two small and two large subunits, forming the active

caspase. There are 14 mammalian caspases identified which usually undergo

proteolysis and activation by other caspases in a cascade. Peptide caspase

inhibitors can inhibit downstream caspase activation and subsequently

apoptosis. The caspases may be grouped into subclasses in various ways and

the three different classes of caspases are the following:

- initiator caspases that are characterized by long prodomains (more

than 90 amino acids) containing either death effector domains (DEDs) or a

caspase recruitment domain (CARD);

- effector or executioner caspases containing short prodomains

(caspase-3, caspase-6 and caspase-7);

- the remaining caspases which the principal function is related to

cytokine maturation rather than apoptosis. Once activated, the prodomains are

removed and the large and small subunits are separated by caspase action (all

cleavages occur after Asp residues). The active site is formed by the interface


11

of the two subunits by 1 Arg, 1 His, 1 Cys of the large subunit and 1 Arg of the

small subunit. Then, the so activated caspases cleave cellular substrates

leading to all fashions of the apoptotic morphology. When activated, the effector

caspase-3 cleaves important cellular substrates leading to those morphological

features related to apoptosis discussed earlier. Several cellular and viral

proteins act as caspase inhibitors. For example, cells contain inhibitor of

apoptosis proteins (IAPs) that can inhibit activated caspases. Neuronal cells

typically contain such proteins (neuronal apoptosis inhibitory protein, NAIP) to

protect them from premature apoptosis. Many viruses also contain viral IAPs,

viral anti-apoptotic Bcl-2 proteins or other inhibitors of apoptosis in order to

prevent infected cells from dying.

4. The intrinsic (mitochondrial) pathway

Intrinsic apoptosis pathways involve procaspase-9 which is activated

downstream of mitochondrial proapoptotic events at the so called apoptosome,

a cytosolic death signalling protein complex that is formed upon release of

cytochrome c from the mitochondria. In oncology, the intrinsic pathway is

activated by DNA damage resulting from cytotoxic chemotherapy as well as

irradiation, and also mediates apoptosis from other stimuli including hypoxia,

defective cell cycle events, and deprivation of growth factors. Following an

apoptotic stimulus, Bax (the prototypic proapoptotic protein), undergoes

homodimerization and oligodimerization through interaction of BH3-regions and

associates with the mitochondrial membrane with insertion in the mitochondrial

membrane, permeabilization resulting in loss of membrane potential, and

consequently release of apoptogenic factors including cytochrome c, ATP, and

SMAC/DIABLO (second mitochondria-derived activator of caspase/direct IAP

binding protein with low pI). ATP binds to the nucleotide-binding domain of

apoptotic protease-activating factor 1 (APAF1) resulting in the formation of large

oligomers (heptamers). Cytochrome c binds to APAF1 and through a caspase-

associated recruitment domain (CARD) in APAF1, binds to a complementary

CARD on procaspase 9 leading to activation of caspase 9 and of the

downstream “executioner” caspases 3, 6, and 7. These activated caspases

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12

auto-induce activation of themselves as well as downstream caspases and the

proteolytic cascade, that ultimately cleaves substrates essential for cell viability,

results in the characteristic biochemical and morphological changes of

apoptosis.

5. The extrinsic (receptor) pathway

The extrinsic apoptosis pathway is mediated by the activation of death

receptors which are at the cell surface and transmit apoptotic signals after

binding to specific ligands. The extrinsic apoptosis pathway involves

procaspase-8 which is recruited by its DEDs to the death inducing signalling

complex (DISC), a membrane receptor complex formed following to the ligation

of a member of the tumor necrosis factor receptor (TNFR) family. Once bound

to the DISC, several procaspase-8 molecules are joined in close proximity and

then are capable to activate each other by autoproteolysis. Death receptors

belong to the tumor necrosis factor receptor (TNFR) gene superfamily, including

TNFR-1, Fas/CD95 and the TRAIL receptors DR-4 and DR-5. Thereafter, the

signaling is transmitted through the cytoplasmic part of the death receptor which

contains a conserved sequence termed the death domain (DD). Adapter

molecules like FADD or TRADD have their own DDs by which they are recruited

to the DDs of the activated death receptor, thereby forming the DISC and the

local concentration of several procaspase-8 molecules at the DISC leads to

their autocatalytic activation and release of active caspase-8. Active caspase-8

then processes downstream effector caspases which subsequently cleave

specific substrates resulting in apoptosis. Cells that possesses the capacity to

induce this direct and mainly caspase-dependent apoptosis pathways are

classified as type I cells. On the other hand, in type II cells, the signal coming

from the activated receptor does not mediate a caspase signaling cascade

strong enough for execution of cell death on its own. In this case, the signal

needs to be amplified via mitochondria-dependent apoptotic pathways. The link

between the caspase signalling cascade and the mitochondria is provided by

the Bcl-2 family member Bid. Bid is cleaved by caspase-8 into the truncated

form (t-BID) which translocates to the mitochondria where it acts in concert with


13

the proapoptotic Bcl-2 family members Bax and Bak to induce the release of

cytochrome c and other mitochondrial proapoptotic factors into the cytosol.

Cytosolic cytochrome c is binding to monomeric Apaf-1 which then oligomerizes

to assemble the apoptosome, the already mentioned complex of wheel-like

structure with 7-fold symmetry that triggers the activation of the initiator

procaspase-9. Then, the activated caspase-9 starts a caspase cascade

involving downstream effector caspases such as caspase-3, caspase-7, and

caspase-6, eventually leading to apoptosis.

The initial attempts to engage the extrinsic pathway therapeutically

used systemic and local administration of TNF-a and FAS ligand. However, the

occurrence of a constellation of adverse clinical events that were consistent with

a septic shock-like syndrome precluded further development of these agents as

systemic therapy. Recent drug discovery efforts have focused on the TRAIL

family of receptors as a target for therapeutic intervention with the goal of

restoration of normal cellular apoptosis and enhancement of the effectiveness

of chemotherapeutic agents.

6. Apoptosis regulation

The activation of procaspases is regulated by the Bcl-2 family of

intracellular proteins. Some members of this family, like Bcl-2 itself or Bcl-XL,

inhibit apoptosis by preventing the release of cytochrome c from mitochondria.

Other members of the Bcl-2 family, coversely, are not death inhibitors but

instead lead to procaspase activation and apoptosis. As an example, BAD

associates to death-inhibiting members of the family inactivating them, whereas

others, like Bax and Bak, stimulate the release of cytochrome c from

mitochondria. If the genes encoding Bax and Bak are both inactivated, cells

become strongly resistant to most apoptosis-inducing stimuli, indicating the

pivotal importance of these proteins in apoptosis induction. Bax and Bak are

themselves activated by other apoptosis-promoting members of the Bcl-2 family

such as Bid.

In a viable cell, the proapoptotic Bcl-2 family members Bax, Bak, and

BH3-only proteins are antagonized by antiapoptotic members such as Bcl-2. In

Chapter 1

14

response to an apoptotic stimulus, BH3-only members are activated by

transcriptional upregulation (Bax, Noxa, Puma), subcellular relocalization (Bim,

Bmf), dephosphorylation (Bad) or proteolysis (Bid). Activated BH3-only proteins

prevent antiapoptotic Bcl-2 members from inhibiting proapoptotic members.

Moreover, they might directly induce a conformational change of Bax and Bak

which subsequently oligomerize and insert into the mitochondrial membrane

where they form pores either by themselves or by associating with the

permeability transition pore complex. In consequence, proapoptotic factors are

released from the inner mitochondrial membrane into the cytosol, such as

cytochrome c which contributes to the formation of the apoptosome and the

subsequent activation of the caspase cascade.

Another important family of intracellular apoptosis regulators is the IAP

(inhibitor of apoptosis) family. These proteins are thought to inhibit apoptosis in

two ways: they bind to some procaspases to prevent their activation, and they

bind to caspases to inhibit their activity. IAP proteins were originally discovered

as proteins produced by certain insect viruses, which use them to prevent the

infected cell from killing itself before the virus has had time to replicate. When

mitochondria release cytochrome c to activate Apaf-1, they also release a

protein that blocks IAPs, thereby greatly increasing the efficiency of the death

activation process.The intracellular cell death program is also regulated by

extracellular signals, which can either activate apoptosis or inhibit it. These

signal molecules mainly act by regulating the levels or activity of members of

the Bcl-2 and IAP families. We see in the next section how these signal

molecules help multicellular organisms regulate their cell numbers. Beyond that,

the signalling involving the Mitogen-activated Protein Kinases (MAPKs) has an

important role in apoptosis regulation as we will discuss in section 8.


15

7. Programmed cell death type II and III : autophagy and necrosis

Cell death has been subdivided into the categories apoptosis (Type I),

autophagic cell death (Type II), and necrosis (Type III). The boundary between

Type I and II has never been completely clear and perhaps does not exist due

to intrinsic factors among different cell types and the crosstalk among

organelles within each type. Apoptosis can begin with autophagy, autophagy

can end with apoptosis, and blockage of caspase activity can cause a cell to

default to Type II cell death from Type I. Furthermore, autophagy is a normal

physiological process active in both homeostasis (organelle turnover) and

atrophy. "Autophagic cell death" may be interpreted as the process of

autophagy that, unlike other situations, does not terminate before the cell

collapses. Since switching among the alternative pathways to death is relatively

common, interpretations based on knockouts or inhibitors, and therapies

directed at controlling apoptosis must include these considerations.

The autophagic type of death, which is typically seen in large,

cytoplasm-rich post-mitotic or only slowly mitotic cells, is characterized by

autophagic capture of organelles and particles, substantial expansion of the

lysosomal compartment including primary lysosomes, autophagic vacuoles, and

secondary lysosomes, and belated collapse of the nucleus. Often organelles

appear to be eliminated in waves, for instance one wave in which mitochondria

were seen in autophagic vacuoles and afterwards nearly eliminated, and

another in which ribosomes or glycogen particles were the primary occupants of

autophagic vacuoles. In some instances the lysosomes reside in attacking

phagocytes. In others, cell organelles such as mitochondria are sequestered,

with any apoptotic morphology delayed until the cytoplasm was nearly

completely destroyed. Thus this cells death appears distinct from apoptosis.

"Necrosis" is today the catch-all term for any deaths that do not fit in

the other categories described here. Typically, cells entering necrosis lose

control of their ionic balance, imbibe water, and lyse. Intracellular proteins in

new ionic milieus, often in the presence of high ionic calcium and acid or other

abnormal pH, often precipitate. The lysis releases many intracellular

Chapter 1

16

constituents, attracting Mast cells and provoking an inflammatory response.

Consequently, the morphology of necrosis is variable and poorly defined.

8. Mitogen-activated protein kinases

Protein kinases and other messenger systems form highly interactive

networks to achieve the integrated function of cells in an organism.

To understand the signaling mechanism for any agent, its repertoire of

signal transducers and their interactions within this network must be defined in

the cellular context. This includes the production of second messengers,

activation of protein kinases and the subcellular distribution of these

transducers to bring them into contact with appropriate targets. In this context,

mitogen-activated protein kinases (MAPKs) exert an essential and

multifunctional action.

MAPKs respond to extracellular stimuli and regulate various cellular

activities, such as gene expression, mitosis, differentiation, and cell

survival/apoptosis. Extracellular stimuli lead to activation of a MAPK via a

signaling cascade composed of MAPK, MAPK kinase (MAPKK), and MAPKK

kinase (MAPKKK). A MAPKKK that is activated by extracellular stimuli

phosphorylates a MAPKK on its serine and threonine residues, and then this

MAPKK activates a MAPK through phosphorylation on its serine and tyrosine

residues. This MAPK signaling cascade has been evolutionarily well-conserved

from yeast to mammals. This cascade not only conveys information to the target

effectors but also coordinate incoming information from parallel signaling

pathways. Such mechanism allow for signal amplification and the generation of

a threshold and are subject to multiple activation cascades.

To date, three distinct groups of MAPKs have been characterized in

mammals: the extracellular regulated kinase (ERK or p42/p44 MAPK), c-Jun N-

terminal kinase (JNK or SAPK1), and p38 MAPK.

In general, ERK1 and ERK2 are key transducers of prolifera-

tion/differentiation signals and are often activated by growth factors and phorbol

ester (a tumor promoter). In contrast, SAPKs/JNKs and p38 are poorly activated

by mitogens but strongly activated by cellular stress inducers such as cytokines,


17

ultraviolet irradiation, heat shock, and osmotic shock, and are involved in cell

differentiation and apoptosis.

The interactions between MAPK and its immediate upstream kinase

(MAPKK) are highly specific: for instance, p42/p44 MAPKs are selectively

activated by MEK1 and MEK2, p38 MAPK is selectively activated by MKK3 and

MKK6, and JNK is specifically activated by MKK7 and MKK4 under

physiological conditions; however, when overexpressed, MKK4 can also

activate p38 MAPK.

9. MAPK regulation

Phosphorylation/dephosphorylation - MAPKs are regulated by

phosphorylation cascades. In all currently known MAPK cascades, the kinase

immediately upstream of MAPK is dual specificity enzyme that can

phosphorylate hydroxyl side chains of serine/threonine and tyrosine residues in

their MAPK substrate. The duration and amplitude of MAPK activation

represents the balance between the activating signal and inactivation

mechanisms. The removal of one or both of these phosphates by tyrosine,

serine/threonine, or dual-specificity phosphatases dramatically decreases

MAPK activity.

Protein complex - scaffolding proteins can bind and organize multiple

signaling proteins in a complex by non-catalytic protein–protein interactions; the

non-catalytic docking interactions involve protein recognition modules for

organization of MAPKs in signaling complexes.

10. MAPKs and cancer therapy

Antitumor agents, despite having diverse primary mechanisms of

action, mediate their effects by inducing apoptosis in tumor cells. Cellular

commitment to apoptosis, or the ability to evade apoptosis in response to

damage, involves the integration of a complex network of survival and death

pathways.

Among the best-characterized pathways regulating cell survival and cell

death are those mediated by the MAPK family. Not surprisingly, MAPK signaling

Chapter 1

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pathways have been implicated in the response of tumor cells to

chemotherapeutic drugs. While the activities of the major MAPK subgroups are

subject to modulation upon exposure of different types of cancer cell lines to

diverse classes of antitumor agents, the response tend to be context-

dependent, and can differ depending on the system and conditions. Despite

these complexities, some important trends have surfaced, and molecular

connections between MAPK signaling pathways and the apoptotic regulatory

machinery are beginning to emerge. With increased evidence supporting a role

for MAPK signaling in antitumor drug action, MAPK modulators may have

potential as chemotherapeutic drugs themselves or as chemosensitizing

agents. The ability of MAPK/ERK kinase (MEK) inhibitors to block survival

signaling in specific contexts and promote drug cytotoxicity represents an

example, and recent knowledge of the pro-apoptotic functions of JNK and p38

suggests possible new approaches to targeted therapy.

11. Leukemia

Adult myelopoiesis is a very well regulated cell system with remarkable

cellular turnover that constantly regenerates from very few hematopoietic stem

cells with self-renewal capacity through a process of cell division and

differentiation. While the process is driven by early and lineage-specific growth

factors and their receptors, the decisions of differentiation are governed by a set

of early acting and lineage-specific transcription factors that regulate the

expression of lineage-specific genes. This cell system sets the stage for the

pathogenesis of acute myeloid leukemia (AML).

AML is characterized by the clonal growth of immature progenitor cells.

Increased proliferation and apoptosis resistance, as well as the inhibition of

differentiation are in the center of this pathogenetic event, and it seemed very

likely a priori that constitutive and/or aberrant activation of growth factor

receptor signaling pathways should contribute. Indeed, both aberrant and

constitutive activation of signal transduction molecules have been found in

about 50% of primary AML bone marrow samples. The most common of these

activating events were observed in the RTK Flt3, N-Ras and K-Ras, and


19

sporadically in other RTKs. The nature is an inexhaustible source of natural

compounds with therapeutic properties for different diseases including cancer.

These compounds have shown several interesting effects in animal models and

in vitro systems.

Studies to date have demonstrated that natural compounds can have

complementary and overlapping mechanisms of action, including antioxidant

activity and scavenging free radicals, regulation of gene expression in cell

proliferation, cell differentiation, oncogenes and tumor suppressor genes,

induction of cell cycle arrest and apoptosis.

In this context, violacein and tetrahydroxiquinone present a promissory

potential as a coadjuvant of leukemia treatment. The purple-coloured pigment

violacein [3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ilydene)-

1,3-dihydro-2H-indol-2-one], produced mainly by bacteria of the genus

Chromobacterium, has attracted increased interest due to its important

biological activities and pharmacological potential. The biosynthesis of this

indole derivative has been extensively studied and interesting reviews

illustrating its production and industrial perspectives, as well as the biological

interest in violacein from Chromobacterium violaceum have been published

12. Aim of this thesis

Societal significance in a broader context

Among the most important relative competitive advantages for a country

like Brazil is the availability of the enormous Amazon rain forest with its vast and

largely untapped resources. At present the Amazon is mainly used for poorly-

sustainable low-technology economic uses, whereas exploiting the jungle for

possible medicinal uses is often done by multi national companies offering little

benefit for the country itself. I feel that it may be important to exploit the

Amazon for the natural products with possible medicinal or other use and build

a local knowledge-based industry for such development. If successful, such an

industry may also become an important factor in Brazil in the efforts to protect

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the forest from lodging. In this thesis I shall embark investigating a few

compounds from the Amazonia, focusing on unravelling the molecular mode of

action, for their action in leukaemia.

Scope limitation

As may have become clear from this chapter, it is my feeling that

successful treatment of leukemia may come from induction of differentiation and

apoptosis of the malignant cells, and targeting MAP kinases and survival

pathways may be the way forward. To prove this point, I shall address the

molecular mode of action for three important compounds, tetrahydroxyquinone,

violacein and riboflavin. In chapter 2, the effect of tetrahydroxyquinone (THQ)

was investigated in HL60 cells. THQ caused substantial cytotoxicity that

coincided with HL60 cell apoptosis through the mitochondrial pathway and was

followed by reduced activity of various anti-apoptotic survival molecules,

including the protein kinase B pathway. Importantly, transfection of protein

kinase B into HL60 cells leading to an artificial increase in protein kinase B

activity inhibited ROS-dependent cytotoxicity. More generally, this chapter

shows that specific interference of signalling pathways for clinically potentially

beneficial effects is possible. Nevertheless, from this work also the limitations of

the candidate approach became evident, prompting the development of

approaches in which no a priori assumptions as to the biochemical mechanisms

mediating pharmacon action are made. To this end in chapter 3, a truly novel

approach was taken. Peptide arrays were used to generate comprehensive

descriptions of cellular metabolism. The results unambiguously identified the

MAP kinase pathway as a major target for violacein, the anti-leukemic purple-

colored pigment produced by Chromobacterium violaceum from the Amazon

River that stimulates HL60 cells to differentiate into monocytes and

granulocytes. That natural compounds may also employ other mechanisms

becomes clear in chapter 4, where it is shown that irradiated riboflavin-

dependent cytotoxicity is the result of a Fas and FasL-dependent activation of

caspase 8. Remarkably, like seen in chapter 2 for THQ, the activation of this


21

cascade led to an inhibition of survival mediators (PKB and IAP1), as well as

downregulation of cell cycle progression regulators. Taken together, these

results provide a molecular approach characterizing the riboflavin-mediated

apoptosis after photodynamic therapy.

Conclusion

Together, this work shows that natural compounds as derived from the

Amazonia forest have potential in anti-leukaemia therapy and that it is possible

to deduce their mechanism of action. It is likely that such compounds are also

useful for other diseases. Thus, the Amazonia jungle may prove a big asset in

to Brazil in this respect.

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Chapter 2

Tetrahydroxyquinone induces apoptosis of

leukemia cells through diminished survival

signaling

Alexandre D. Martins Cavagis1,2, Carmen Veríssima Ferreira1,2,

Henri H. Versteeg2, Cristiane Fernandes Assis1, Carina L. Bos2, Sylvia A.

Bleuming2, Sander H. Diks3, Hiroshi Aoyama1,

Maikel P. Peppelenbosch3,4

1 Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de

Campinas (UNICAMP), Campinas, São Paulo, Brazil. 2 Department of Experimental Internal Medicine, Academic Medical Center (AMC)

University of Amsterdam, The Netherlands. 3Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1

NL-9713 AV Groningen, The Netherlands

4corresponding author (M.P.Peppelenbosch)

Tel: 31-50-363 2522 Fax: 31-50-363 2512

e-mail address: [email protected]

Experimental Hemato logy (2006) 34 (2) 188-196.

Chapter 2

28

ABSTRACT Objective Tetrahydroxyquinone is a molecule best known as a primitive anti-cataract drug, but is

also a highly redox active molecule which can take part in a redox cycle with

semiquinone radicals, leading to the formation of reactive oxygen species (ROS). Its

potential as an anticancer drug has not been investigated.

Methods The effects of tetrahydroxyquinone on HL60 leukaemia cells are investigated using

FACS-dependent detection of phosphatidylserine exposure combined with 7-amino-

actinomycin D (7-AAD) exclusion, via Western blotting using phosphospecific

antibodies, and by transfection of constitutively active protein kinase B (PKB).

Results We observe that in HL60 leukaemia cells tetrahydroxyquinone causes ROS production

followed by apoptosis through the mitochondrial pathway, whereas cellular physiology

of normal human blood leukocytes was not affected by tetrahydroxyquinone. The anti-

leukaemic effect of tetrahydroxyquinone is accompanied by reduced activity of various

anti-apoptotic survival molecules including the protein kinase B pathway. Importantly,

transfection of protein kinase B into HL60 cells and thus artificially increasing protein

kinase B activity inhibits tetrahydroxyquinone-dependent cytotoxicity.

Conclusion

Tetrahydroxyquinone provokes cytotoxic effects on leukaemia cells by reduced protein

kinase B-dependent survival signalling followed by apoptosis through the

mitochondrial pathway. Thus, tetrahydroxyquinone may be representative of a novel

class of chemotherapeutic drugs, inducing apoptosis in cancer cells through diminished

survival signalling possibly as a consequence of ROS-generation.

Keywords: HL60 cells; tetrahydroxyquinone; protein phosphatases; MAPKs; oxidative stress.

THQ induces apoptosis of leukemia cells through diminished survival signaling

29

INTRODUCTION

Chemotherapy for the treatment of some types of neoplastic disease has been

one of the success stories of medicine. However, the chemotherapeutic treatment

outcome of most adult acute myeloid leukemia (AML) remains unacceptable [1].

Among AMLs, acute promyelocytic leukemia can be successfully treated with all-trans-

retinoic acid (ATRA). The development, however, of resistance to a wide spectrum of

cytotoxic drugs frequently impedes the successful treatment of AML either in the

beginning of disease or following primary or subsequent relapses. Moreover, ATRA

resistance in acute promyelocytic leukemia is rare but markedly increases in frequency

after relapses from chemotherapy-induced clinical remission [2, 3]. Hence, novel

avenues for the treatment of AML are required.

It is now generally recognised that the reactive oxygen species (ROS) play an

important role as regulatory mediators in signalling processes [4]. Accordingly, it has

now been shown that a multitude of physiological processes are under the direct control

of ROS, the most important being the regulation of vascular tone, the sensing of oxygen

tension, the enhancement of leukocyte signal transduction and the induction of

apoptosis, the latter as an essential component of the tumour necrosis factor α-

dependent signal transduction [5-9]. Therefore, ROS generation is an important element

in the control of cellular biochemical processes. In addition, ROS generation is

important for inducing cytotoxicity in cancer cells, but the molecular mechanism

underlying these cytotoxic effects remains unclear, hampering the development of more

effective drugs.

ROS formation depends on the univalent reduction of triplet-state molecular

oxygen [10]. This process is mediated by enzymes such as NAD(P)H oxidases and

xanthine oxidase or nonenzymatic by redox-reactive compounds such as

tetrahydroxyquinone [11-13]. Superoxide dismutases convert superoxide enzymatically

into hydrogen peroxide or non-enzymatically into non-radical species, hydrogen

peroxide and singlet oxygen. Hydrogen peroxide may be converted into water by the

enzymes catalase or glutathione peroxidase. This latter enzyme oxidizes glutathione to

glutathione disulfide, which can be converted back to glutathione by glutathione

reductase in an NADPH-consuming process [14, 15]. Thus, the biochemistry of ROS

generation is relatively well understood.

Chapter 2

30

The biological effects of ROS production, however, are less clear. At high

concentrations ROS are dangerous for living organisms, damaging virtually all cellular

constituents [10]. Nevertheless, at moderate concentrations, ROS play important roles in

the control of various cellular functions and cellular generation of ROS is actively

induced under various conditions [4, 10]. The archetypal example is NADPH oxidase

activation upon immune stimulation of the phagocytic cells of the myeloid lineage,

resulting ROS production which, apart from its bactericidal function, is also

instrumental for the induction of pro-inflammatory gene transcription. Hence,

phagocyte ROS production is pivotal for proper function of the innate immune system

[16-18].

Various studies have shown that ROS producing drugs can exert important

cytotoxic effects in leukaemia cells, although the molecular details by which such drugs

mediate cancer cell death remain obscure [15]. This consideration prompted us to test

the effect of as a cytotoxic agent for HL60 leukaemia cells. Tetrahydroxyquinone is a

compound best known as a primitive anti-cataract drug, but expected to act as a redox

active benzoquinone [13]. In the present study we observe that tetrahydroxyquinone

indeed efficiently induces ROS generation in turn responsible for HL60 cell apoptosis

through the mitochondrial pathway. This apoptosis is accompanied by reduced activity

of the anti-apoptotic PKB [14] and nuclear factor (NF)-κB pathways [15] while

concomitantly specific activation of Jun-N-terminal kinase and protein phosphatases

(PPs) is observed [16]. Importantly, forced expression of PKB counteracts the effect of

tetrahydroxyquinone on HL60 cell apoptosis. Thus the diminished survival signalling is

essential for tetrahydroxyquinone-dependent cytotoxity and tetrahydroxyquinone may

be representative of a novel class of chemotherapeutic drugs, inducing apoptosis in

cancer cells through diminished survival signalling. The tetrahydroxyquinone pathways

mediating this effect involve at least in part ROS-generation.


31

MATERIALS AND METHODS

Cell line and reagents

HL60 cells were purchased from the American Type Culture Collection

(ATCC, Rock-ville, MD). Polyclonal antibodies anti-caspase 3, anti-BAD, anti-

phospho-BAD, anti-phospho-p38 MAPK, anti-phospho-IκB, anti-IκB, anti-phospho-

PKC delta, anti-phospho-PKB, anti-phospho-Raf, anti-phospho-p42/44, anti-phospho-

JNK, anti-phospho-CREB, anti-rabbit and anti-mouse peroxidase-conjugated anti-

bodies were purchased from Cell Signaling Tech-nology (Beverly, MA). The antibodies

against phos-pho-PP2A, phosphotyrosine, phospho-threonine and NF-κB (p50 and p65)

were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Tetrahydroxy -

quinone was purchased from Sigma Chemical Com-pany.

Leukocyte Culture

Human blood was collected from healthy donors and human peripheral blood

mononuclear cells were isolated by Ficoll/Hypaque gradient centrifugation. Leukocytes

were cultured at the same conditions described for HL60 cells, the only difference was

the addition of 5 µg/ml phytohemaglutinin in each well. Cells were plated at density of 1

x 106 plating/ml in 24-well plate. The medium was removed 48h after cell seeding and

replaced with medium containing tetrahydroxyquinone.

Cell Culture and viability assays

HL60 cells were routinely grown in suspension in RPMI 1640 medium

supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and

10% heat-inactivated fetal bovine serum, at 37oC in a 5% CO2 humidified atmosphere.

In all experiments, 3 x 105 cells/ml were seeded and, after 72 h, treated with different

tetra-hydroxyquinone concentrations for the specified periods of time.

Cell viability was assessed based on trypan blue dye exclusion and three

additional parameters: MTT reduction, protein phosphatase activity and determination

of the total protein amount.

Chapter 2

32

MTT reduction assay

The medium containing tetrahydroxyqui-none was removed and 1.0 ml of

MTT solution (0.5 mg MTT/ml of culture medium) was added to each well. After

incubation for 4 h at 37oC, the medium was removed and the formazan released by

solubilisation in 1.0 ml of ethanol. The plate was shaken for 5 min on a plate shaker and

the absorbance was measured at 570 nm (15, 16).

Protein phosphatase assay

The phosphatase extract was obtained by lysing the cells with acetate buffer

0.1 mM (pH 5.5). Then, the enzyme activity was measured in a reaction medium (final

volume, 1.0 ml) containing 100 mM acetate buffer (pH 5.5), 5.0 mM pNPP and cell

extract enzyme. After a 30 min incubation at 37°C, the reaction was stopped by adding

1.0 ml of 1.0 M NaOH. The amount of pNP released was measured at 405 nm (17).

Protein quantification

Protein concentrations were determined by a modification of the Lowry

method as described by Hartree (18).

Reduced glutathione determination

The concentration of reduced glutathione in HL60 cells was determined after

treatment of the cells for 24 h. The cells (3×105/ml) were washed with physiological

solution and lysed with water; 3 ml of precipitant solution (1.67 g glacial

metaphosphoric acid, 0.2 g ethylenediaminetetraacetic acid (EDTA) and 30 g NaCl in

100 ml MilliQ water) was added to the lysate (2 ml). After 5 min, this mixture was

centrifuged and 0.4 ml of the supernatant was added to 1.6 ml of reaction medium

(0.2 M Na2HPO4 buffer, pH 8.0; 0.5 mM DTNB dissolved in 1% sodium citrate).

Subsequently, the absorbance of the product (NTB) was measured at 412 nm and

reduced glutathione concentration calculated using the extinction coefficient E =

13.6 mol-1 cm-1 (19).


33

Spectrofluorometric determination of ROS

HL60 cells were treated with 100 µM tetra-hydroxyquinone or the

combinations tetrahydroxy -quinone/10 mM glutathione and tetrahydroxyqui-none/15

mM N-acetyl-L-cysteine for 24 h in RPMI 1640 (without phenol red) containing 5%

serum. Afterwards, the cells (2×106/10 ml) were washed with PBS and resuspended in

RPMI 1640 (without phenol red, with 5% FBS) containing 20 µM DCFH-DA

(dichlorofluorescein diacetate). After 30 min of incubation at 37°C, the cells were

washed three times and resuspended in RPMI 1640 (without phenol red, with 5% FBS).

The fluorescence of the suspension was measured using a RF-5300 PC Shimadzu

spectrofluorometer with excitation at 485 nm and emission at 530 nm.

DNA fragmentation analysis

The cell pellets (5x106) were lysed in 0.5 ml of lysis buffer containing 5 mM

Tris -HCl, 20 mM ethilenediaminetetraacetic acid (EDTA) and 0.5% Triton X 100. After

centrifugation at 1,500 x g for 10 min, the pellets were resuspended in 250 µL of lysis

buffer and, to the supernatants (S), 20 µL of 6 M perchloric acid was added. Then, 500

µL of 10% trichloroacetic acid (TCA) were added to the pellets (P), the samples were

centrifuged for 10 min at 5,000 rpm and the pellets were resuspended in 250 µL of 5%

TCA followed by incubation at 100oC for 15 min. Subsequently, to each sample, 500

µL of solution (15 mg/ml DPA in glacial acetic acid), 15 µL/ml of sulfuric acid and 15

µg/ml acetaldehyde were added and incubated at 37 oC for 18 h (20). The proportion of

fragmented DNA was calculated from the absorbance at 594 nm using the following

formula:

Fragmented DNA (%) = 100 x (amount of the fragmented DNA in the

supernatant) / (amount of the fragmented DNA in the supernatant + amount DNA in the

pellets).

Western blotting

Cells (3 x 107) were lysed in 200 µL of lysis buffer [50 mM Tris –HCl (pH

7.4), 1% Tween 20, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM

Na3 VO4, 1 mM NaF and protease inhibitors (1 µg/ml aprotinin and 1 µg/ml 4-(2-

aminoethyl) benzenesulfonyl fluoride)] for 2 h on ice. Protein extracts were cleared by

Chapter 2

34

centrifugation and protein concentrations were determined using a Lowry protein assay.

An equal volume of 2X sodium dodecyl sulfate (SDS) gel loading buffer [100 mM

Tris–HCl (pH 6.8), 200 mM DTT, 4% SDS, 0.1% bromophenol blue and 20% glycerol]

was added to the samples, which were subsequently boiled for 10 min. Afterwards, cell

extracts were resolved by SDS-polyacry lamide gel (12%) electrophoresis (PAGE) and

transferred to PVDF membranes. Membranes were blocked in 1% fat-free dried milk or

bovine serum albumin (2%) in Tris -buffered saline (TBS)-Tween 20 (0.05%) and

incubated overnight at 4°C with appropriate primary antibody at 1:1000 dilution. After

washing in TBS-Tween 20 (0.05%), mem-branes were incubated with anti-rabbit or

anti-mouse horseradish peroxidase conjugated secondary anti-bodies, at 1:2000

dilutions, in blocking buffer for 1h. Detection was performed using enhanced

chemiluminescence (ECL).

Mitochondrial extract preparation (cytochrome c release)

For cytochrome c analysis, the cells were washed with ice cold PBS and

resuspended in lysis buffer containing 220 mM mannitol, 68 mM sucrose, 50 mM

Hepes–NaOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and

protease inhibitors (1 µg/ml aprotinin, 10 µg/ml leupeptin and 1mM 4-(2-amino-ethyl)-

benzolsulfonyl-fluoride-hy-drochloride). After incubation on ice for 1 h, the lysate was

centrifuged at 14,000 x g for 15 min. Then, the supernatant was resolved by SDS-

PAGE.

Annexin V and 7-amino-actinomycin D assays

Control and tetrahydroxyquinone-treated cells were collected and resuspended

in 1X binding buffer (0.01 M Hepes/NaOH, pH 7.4, 0.14 mM NaCl and 2.5 mM CaCl2)

at a concentration of 1 x 106 cells/ml. Subsequently, 100 µl of cell suspension were

transfered to a 5 ml tube and Annexin V FITC (5 µl) and 7-amino-actinomycin D (7-

AAD) - (10 µl) were added. The cells were incubated at room temperature for 15 min,

after which 400 µl of 1X binding buffer was added and apoptosis then analyzed by flow

cytometry.


35

Transient transfection of HL60 cells

HL60 cells (4 x 106) were transfected with 0.4 µg of a plasmid expressing

GAG-PKB, a constitutive form of PKB. Cells were cotransfected with 0.1 µg of a pUT-

galactosidase to normalize for transfection efficiency. After transfection, the cells were

cultured for 24h, harvested, lysed in commer-cially available reporter lysis buffer

(Promega) and β-galactosidase activity was determined using chloro-phenol red-β-D-

galactopyranoside (Roche) as subs-trate.

Transfected cells were treated with tetrahydroxyquinone for 24 h and the cell

viability was assessed by MTT reduction. The expression of PKB was evaluated by

western blotting.

Statistical evaluation

All experiments were performed in triplicate and the results shown in the

graphs represent the mean and standard deviation. Cell viability data were expressed as

the means ± standard errors of three independent experiments carried out in triplicates.

Data from each assay were analyzed statistically by ANOVA followed by a Dunnett´s

test. Multiple comparisons among group mean differences were checked with Tukey

post hoc test.

Differences were considered significant when the p value was less than 0.05.

Western blottings represent three independent experiments.

RESULTS

Tetrahydroxyquinone is cytotoxic for HL60 leukaemia cells

As evidence has been presented that the redox state-altering agents are potent

cytotoxic agents in leukaemia cells, although acting through as yet unknown molecular

mechanisms [23, 24], we decided to test the possible cytotoxic effects of

tetrahydroxyquinone on HL60 cells. To this end, HL60 were treated with various

concentrations of tetrahydroxyquinone for 24 hours and cell viability was determined

using total protein content, cellular phosphatase activity, or mitochondrial function

(MTT reduction, Fig. 1A) as a measure. It appeared that using either measure,

tetrahydroxyquinone was highly cytotoxic to the leukaemia cells, the apparent IC50 of

Chapter 2

36

0 100 200 300 400 5000

20

40

60

80

100

120

140

160

Protein MTT phosphatase

Cel

l via

bilit

y (%

)0 100 200 300 400 500

0

20

40

60

80

100

120

140

160

Protein MTT phosphatase

Cel

l via

bilit

y (%

)

Human leukaemia cells Human peripheral bloodmononuclear cells

C

WithROSscavenger

WithoutROSscavenger

tetrahydroxyquinone-induced cytotoxicity being similar whether assessed by total

protein content (IC50 20 µM), phosphatase activity (IC50 40 µM), or by MTT assay

(IC50 45 µM).

Figure 1. Effect of tetrahydroxyquinone on HL60 cell viability versus effects on

untransformed cells. HL60 cell viability was evaluated using three different parameters (protein

content, MTT reduction and phosphatase activity) after treatment with tetrahydroxyquinone for

24 h, in the absence (A) or presence of the ROS scavenger gluthathion (10 mM; B). The lack of

effect of tetrahydroxyquinone on normal peripheral blood mononuclear cell leukocytes is also

depicted (C). Each point represents the mean ± standard deviation of three independent

experiments.


37

Importantly, when healthy human leukocytes were exposed to tetrahydroxy -

quinone no apparent toxicity was apparent, even at concentrations 10 times as high as

the IC50 for leukaemia cells, when assayed by protein content, phosphatase activity, or

MTT (fig 1C). Thus tetrahydroxyquinone is specifically cytotoxic for HL60 leukaemia

cells but not for the corresponding untransformed counterparts.

Tetrahydroxyquinone-dependent ROS generation mediates cytotoxity

Tetrahydroxyquinone is a highly redox active molecule, expected to induce the

formation of ROS by taking part in a redox cycle with semiquinone radicals. We

decided to investigate whether the tetrahydroxyquinone-mediated cytotoxic effects are

mediated by ROS generation. In agreement with a role of ROS generation in

tetrahydroxyquinone cytotoxicity, we observed that the compound substantially

increases the cellular levels of ROS, as determined by dichlorofluorescein diacetate-

dependent spectrophotometry (Fig. 2). Importantly, treatment with reduced glutathione

or N-acetyl-L-cysteine abolished the capacity of HL60 cells to react to

tetrahydroxyquinone with ROS production (Fig. 2). This allowed us test the importance

of tetrahydroxyquinone-induced ROS formation for its cytotoxic effects, and a

significant rightward shift of the dose-response curve with respect to

tetrahydroxyquinone-induced cytotoxicity was observed in the presence of ROS

generation inhibitors whether assessed by total protein content (IC50 from 20 µM to 45

µM ), phosphatase activity (IC50 from 40 µM to 140 µM), or by MTT assay (IC50 from

45 µM to 140 µM) (Fig. 1A,B). Thus tetrahydroxyquinone is an efficient inducer of

ROS production in HL60 leukaemia cells and ROS generation is essential for the

cytotoxic effect of this compound.

Chapter 2

38

RO

S p

rod

uct

ion

Figure 2. Tetrahydroxyquinone causes production of reactive oxygen species. The

production of reactive oxygen metabolites was determined using dichlorofluorescein diacetate-

loaded cells and a spectrofluorometer (excitation at 485 nm and emission at 530 nm). 1 - Non-

treated HL60 cells (control); 2 – tetrahydroxyquinone (100 µM) - treated cells; 3 - HL60 cells

pre- treated with N-acetyl-L-cysteine (10 mM) and 4 - HL60 cells pre-treated with reduced

glutathione (10 mM) for 30 min. For conditions 3 and 4, the cultures were further incubated with

100 µM tetrahydroxyquinone for 24h. Afterwards, a 30 min incubation with DCFH-D was carried

out and the fluorescence intensity was recorded by spectrofluorometry. Results were expressed as

the relative fluorescence intensity with respect to untreated cells. 5 - As a positive control - H2O2-

treated cells were used to monitor the level of ROS. Bars represent mean ± standard deviation.

Tetrahydroxyquinone induces cell death by apoptosis

Generally speaking cell death is brought about either via necrosis or via

apoptosis. The former process is associated with relatively large damage to the

surrounding tissue. The latter is associated with controlled elimination of cancer cells.

Thus for the possible treatment of leukaemia cytotoxic compounds should preferentially

act via apoptosis. To address the question whether tetrahydroxyquinone induces cell

death via apoptosis we measured three cellular processes associated with apoptosis

rather as necrosis: caspase 3 activation, DNA fragmentation, and phosphatidylserine

exposure.


39

Figure 3. Apoptosis induction by tetrahydroxyquinone. (A) Cells were treated with

tetrahydroxyquinone as indicated and lysates were resolved by immunoblotting as described in

methods to assess the level of active caspase 3. (B) The samples were prepared as described by

Zhu and collaborators (23) and the proportion of fragmented DNA was calculated from

absorbance at 594 nm using the approach described in the method section. The bars represent the

mean ± standard deviation of three experiments. (C) Cell samples were prepared as described in

Methods and Annexin V-positive, 7-AAD-positive and Annexin V/7-AAD-positive populations

were analyzed by flow cytometry.

It appeared that tetrahydroxyquinone efficiently activates caspase 3 (Fig. 3A)

in concentration in excess of 25 µM, stimulates DNA fragmentation at the same

concentration (Fig. 3B) and provoke phosphatidylserine exposure (Fig. 3C) when

applied in a concentration of 25 µM or more. Importantly, induction of apoptosis as

measured by DNA fragmentation was not detected when the formation of ROS was

blocked (Fig 3B). Thus apoptosis is the main route to cell death in tetrahydroxyquinone-

treated cells.

Chapter 2

40

JNK and protein phosphatases activation in HL60 cells

To investigate the molecular mechanism underlying tetrahydroxyquinone-

dependent apoptosis induction we studied the activation status of the MAP kinase

family, since this family of kinases is well known to be involved in the control of a

variety of cell survival-controlling pathways [25]. In myeloid leukemia cell lines the

p42/p44 MAP kinase cascade positively regulates differentiation into the monocyte

lineage and is instrumental for phorbolester-dependent differentiation of this cell type,

while inhibition of the JNK has been implicated in 1,25-dihydroxyvitamin D3-

dependent HL60 cell differentiation. Conversely, in HL60 cells JNK activation is linked

to apoptosis [23,24]. We observed that tetrahydroxyquinone treatment strongly

activated JNK. Unexpectedly, only a modest increase in the phosphorylation of p38

MAP kinase with little effect on the activation state of p42/44 MAP kinase was

observed, even at concentrations as high as 100 µM (Fig. 4A). Thus,

tetrahydroxyquinone-induced changes in the activation of MAP kinase family members

are discordant with an effect on differentiation induction, and is consistent with an

effect on apoptosis in this cell type [23, 24].

Despite the induction of oxidative stress by tetrahydroxyquinone, unusual

activations of both protein tyrosine phosphatases and protein serine/threonine

phosphatases coinciding with the activation of PP2A were observed (Fig. 4B).


41

Figure 4. Effect of tetrahydroxyquinone on MAP kinase and protein phosphatase activities.

The phosphorylation and total protein level of MAPK family members (A), phosphoprotein

profiles (employing anti-phospho-amino acid antibodies and phospho-PP2A (B) were evaluated

by immunoblotting. The panel shows tetrahydroxyquine-induced chances in phosphorylation state

(see arrows).

Chapter 2

42

Tetrahydroxyquinone-induced apoptosis coincides with activation of the mitochondrial pathway through diminished protein kinase B activation

Apoptosis can be excecuted through two basic signalling pathways: the

extrinsic pathway and the mitochondrial intrinsic pathway. We observed that

application of tetrahydroxyquinone induced the release of cytochrome c from the

mitochondria at concentration as low as 25 µM (Fig. 5), demonstrating that

tetrahydroxyquinone activates the mitochondrial pathway. Importantly, the

phosphorylation of Bad Ser112 (which leads to cell survival by inhibiting the

mitochondrial pathway) was concomitantly decreased. In agreement,

tetrahydroxyquinone treatment also caused increase of phosphorylation of Ser473 in

protein kinase B (the Bad kinase for Ser112), demonstrating that tetrahydroxyquinone

signalling reduces activity of this anti-apoptotic kinase and, consequently, leads to

apoptosis.

Figure 5. Participation of mitochondrial pathway of apoptosis in tetrahydroxyquinone-

dependent cytotoxicity. Cells were treated with tetrahydroxyquinone as indicated and lysates

were prepared as appropriate (see methods) and analysed by immunoblotting for PKB, phospho-

Bad and total Bad proteinlevels.


43

The importance of mitochondria for apoptosis -induction by

tetrahydroxyquinone was also confirmed by a dramatic increase in total Bad protein

levels (Bad phosphorylation is followed by its ubiquitination and proteolysis and

mediates the inhibitory effect of protein kinase B on apoptosis through the

mitochondrial pathway). In addition, inhibition of two other kinases involved in the

survival signalling: Raf and PKC delta, reinforced our hypothesis that

tetrahydroxyquinone inhibits cell survival signalling pathways (Fig. 5). Furthermore,

additional evidence for survival pathway inhibition by tetrahydroxyquinone was

provided through down regulation of NF-κBp65 and a decrease of phosphorylated IκB

(Fig. 6).

Figure 6. Diminished levels of nuclear factor kappa B by tetrahydroxyquinone. HL60 cells

were treated with tetrahydroxyquinone as indicated and the level of both subunits of NF-κB (p65

and p50) and its phosphorylated inhibitory protein (IκB) were analysed by western blotting

Independent support for this notion was obtained in experiments in which the

protein kinase B activity was artificially increased by transfection with GAG-protein

kinase B, a constitutive active form of protein kinase B, expression construct. As it has

been shown in the Figure 7, HL60 cells transfected with protein kinase B became

resistant to tetrahydroxyquinone, even when treated with 500 µM of the compound.

Chapter 2

44

Figure 7. Viability analysis of HL60 cells transfected with PKB. HL60 cell viability was

evaluated using MTT assay after treatment with tetrahydroxyquinone for 24 h in cell transfected

with a PKB expression construct (filled circles) or empty vector (open circles).

DISCUSSION It is now generally recognised that novel compounds are called for treating

leukaemic disease. This consideration prompted us to investigate the consequences of

tetrahydroxyquinone on HL60 leukaemia cells, a compound most readily known as a

primitive anti-cataract drug, but also expected to act as a redox active molecule. In

agreement with the latter notion, we observed that tetrahydroxyquinone led to ROS

production that coincided with apoptosis through the mitochondrial pathway, and

corresponded with reduced activity of various anti-apoptotic survival molecules

including the protein kinase B pathway in HL60 leukaemia cells but not their

untransformed counterparts. Importantly, transfection of protein kinase B into HL60

cells and thus artificially increasing protein kinase B activity inhibited ROS-dependent

cytotoxicity. We concluded that the remarkable cytotoxic effects tetrahydroxyquinone

on HL60 leukaemia cells are dependent on reduced protein kinase B-dependent survival


45

PKBPKB

BadBad BadP

BadBadP

BadP

BadBadP

Bad

destructionin the proteosome

mitochondrialpathway

apoptosis

NF-κBJNKJNK

Tetrahydroxyquinone

PKCδ

signalling followed by apoptosis through the mitochondrial pathway, probably as a

consequence of tetrahydroxyquinone-dependent ROS generation. Thus tetrahydroxyqui-

none may be representative of a novel class of chemotherapeutic drugs, inducing

apoptosis in cancer cells through diminished survival signalling as a consequence of

ROS production. Proof of this notion awaits in vivo experiments in which the direct

potential of tetrahydroxyquinone as an anticancer drug is directly demonstrated.

Figure 8. Schematic representation of tetrahydroxyquinone effects on HL60 leukaemia cells.

Tetrahydroxyquinone inhibits the anti-apoptotic PKB/Bad, NF-κB, and PKCδ signalling cassettes

and stimulates the pro-apoptotic JNK cassette. Of these effects transfection studies reveal

diminished activation of the anti-apoptotic PKB/Bad signalling cassette to be most important

effect.

Increased ROS production by NADPH oxidase upon macrophage activation is

well established. Earlier studies have shown that such ROS production upon immune

stimulation of the phagocytic cells, apart from its bactericidal function, is also

instrumental for the induction of pro-inflammatory gene transcription [16-18]. Among

the cellular responses to macrophage activation is also cell death, a response probably

required to rid the body from cells damaged by the immune response. HL60 cells share

important characteristics with monocytes and macrophages and the data obtained in the

Chapter 2

46

present study may indicate that this apoptosis is a direct consequence of ROS

production followed by reduced survival signalling and activation of mitochondrial

pathway of cell death, is also important in the induction of apoptosis following

macrophage activation. Final proof of this notion, however, awaits experiments in

which activation-induced cell death is investigated in the presence and absence of

glutathione and N-acetyl-L-cysteine.

The molecular mechanisms by which ROS participate in inflammatory gene

expression are obscure at best. In the present study we observed that ROS reduced the

apparent activity of the NF-κB pathway, as deduced from the decrease in expression of

both the ratio between p65 expression and I-κB as well as the reduced phosphorylation

of I-κB. As the NF-κB pathway is a potent survival signal, this down regulation

corresponds well with the reduced survival signalling induced by ROS formation

detected in this study. It is, however, more difficult to reconcile with the role of ROS

generation in mediating inflammatory gene expression, as NF-kB is in general a strong

pro-inflammatory transcription factor. Importantly, however, a recent study by

Blanchette et al. [29] indicates that the contribution of NF-κB to macrophage activation-

associated gene expression is minimal, hence our results are not necessarily at bay with

the established role of ROS in the induction of inflammatory gene expression.

Interestingly, increased JNK activity has recently come up as an important mediator of

inflammatory gene expression in inflammatory bowel disease in vivo [30]. Hence, the

ROS-induced activation of JNK may function in the induction of inflammatory gene

expression.

Despite the intricacies of ROS action in cellular physiology, the present study

has shown a remarkable action of tetrahydroxyquinone on HL60 leukaemia cells, acting

through a novel survival signalling-inhibiting mechanism. Recent data indicate that

impaired survival signalling through PKB/Akt has substantial clinical promise for the

treatment of chemotherapy-resistant leukaemia [31]. Together, these observations make

tetrahydroxyquinone a candidate drug for the treatment of leukaemia and further studies

are under progress addressing its potential usefulness for combating disease.


47

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1. Larson RA, Daley GQ, Schiffer CA, et al. Treatment by design in leukemia, a meeting

report, Philadelphia, Pennsylvania, December 2002. Leukemia 2003;17: 2358-2382.

2. Hisatake J, O´Kelly J, Uskokovic MR, Tomoyasu S, Koeffler P. Novel vitamin D3 analog,

21-(3-methyl-3-hydroxy -butyl)-19-nor D3, that modulates cell growth, differentiation,

apoptosis, cell cycle, and induction of PTEN in leukemic cells. Blood 2001;97:2427-2433.

3. Tsuzuki S, Kitajima K, Nakano T, Glasow A, Zelent A, Enver T. Cross talk between retinoic

acid signaling and transcription factor GATA-2. Mol Cell Biol. 2004; 24:6824-6836.

4. Allen RG, Tresini M. Oxidative stress and gene regulation.

Free Radic Biol Med. 2000;28(3):463-499.

5. MacDonald GM, Steenhuis JJ, and Barry BA. A difference Fourier transform infrared

spectroscopic study of chlorophyll oxidation in hydroxylamine-treated photosystem II. J Biol

Chem. 1995;270:8420-8428.

6. Daou GB, Srivastava AK. Reactive oxygen species mediate Endothelin-1-induced activation

of ERK1/2, PKB and Pyk2 signalling, as well as protein synthesis, in vascular smooth

muscle cells. Free Radic Biol Med. 2004;37:208-215.

7. Shen HM, Lin Y, Choksi S, et al. Essential Roles of Receptor-Interacting Protein and

TRAF2 in Oxidative Stress-Induced Cell Death. Mol Cell Biol. 2004;24(13):5914-5922.

8. Jang BC, Paik JH, Jeong HY, et al. Leptomycin B-induced apoptosis is mediated through

caspase activation and down-regulation of Mcl-1 and XIAP expression, but not through the

generation of ROS in U937 leukemia cells. Biochem Pharmacol. 2004;68(2):263-274.

9. Murray J, Walmsley SR, Mecklenburgh KI, et al. Hypoxic regulation of neutrophil apoptosis

role: of reactive oxygen intermediates in constitutive and tumor necrosis factor alpha-

induced cell death. Ann NY Acad Sci. 2003;1010:417-425.

10. Ryter SW, Tyrrell RM. Singlet molecular oxygen ((1)O2): a possible effector of eukaryotic

gene expression. Free Radic Biol Med. 1998:24(9):1520-1534.

11. Wiemels J, Wiencke JK, Varykoni A, Smith MT. Modulation of the toxicity and

macromolecular binding of benzene metabolites by NAD(P)H:Quinone oxidoreductase in

transfected HL-60 cells. Chem Res Toxicol. 1999:12(6):467-475.

12. Chen Q, Vázquez EJ, Moghaddas S, Hoppel CL, Lesnefsky EJ. Production of reactive

oxygen species by mitochondria: central role of complex III. J Biol Chem.

2003;278(38):36027-36031.

13. Tudor G, Gutierrez P, Aguilera-Gutierrez A, Sausville EA. Cytotoxicity and apoptosis of

benzoquinones: redox cycling, cytochrome c release, and BAD protein expression. Biochem

Pharmacol. 2003;65:1061-1075.

Chapter 2

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14. Anderson ME. Glutathione: an overview of biosynthesis and modulation. Chem Biol

Interact. 1998;111-112:1-14.

15. Matés JM, Sánchez-Jiménez FM. Role of reactive oxygen species in apoptosis: implications

for cancer therapy. Int J Biochem Cell Biol. 2000;32(2):157-170.

16. Schoonbroodt S, Piette J. Oxidative stress interference with the nuclear factor-kappa B

activation pathways. Biochem Pharmacol. 2000;60(8):1075-1083.

17. Victor VM, De la Fuente M. N-acetylcysteine improves in vitro the function of

macrophages from mice with endotoxin-induced oxidative stress. Free Radic Res.

2002;36(1):33-45.

18. Ryan KA, Smith MF Jr, Sanders MK, Ernst PB. Reactive oxygen and nitrogen species

differentially regulate Toll-like receptor 4-mediated activation of NF-kappa B and

interleukin-8 expression. Infect Immun. 2004;72(4):2123-2130.

19. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to

proliferation and cytotoxicity assay. J Immunol Methods 1983;65:55-63.

20. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. modifications to

the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol

Methods 1986;89:271-277.

21. Aoyama H, Melo PS, Granjeiro PA, Haun M and Ferreira CV. Cytotoxicity of okadaic acid

and kinetic characterization of protein tyrosine phosphatase activity in V79 fibroblasts.

Pharm Pharmacol Comm. 2000;6:331-334.

22. Hartree EF. Determination of proteins: a modification of the Lowry method that gives a

linear photometric response. Anal Biochem. 1972;48:422-427.

23. Zhu W-H., Majluf-Cruz A, Omburo GA. Cyclic AMP-specific phosphodiesterase inhibitor

rolipram and RO-20-1724 promoted apoptosis in HL-60 promyelocytic leukemic cells via

cyclic AMP-independent mechanism. Life Sci. 1998;63(4):265-274.

24. Freire AG, Melo PS, Haun M, Dúran N, Aoyama H, Ferreira CV. Cytotoxic effect of the

diterpene lactone dehydrocrotonin from Croton cajucara on human promyelocytic leukemia

cells. Planta Medica 2003;69:67-69.

25. Versteeg HH, Evertzen MW, van Deventer SJ, Peppelenbosch MP. The role of

phosphatidylinositide-3-kinase in basal mitogen-activated protein kinase activity and cell

survival. FEBS Lett. 2000 465:69-73

26. Wang XN, Studzinski GP. Inhibition of p38MAP kinase potentiates the JNK/SAPK pathway

and AP-1 activity in monocytic but not in macrophage or granulocytic differentiation of

HL60 cells. J Cell Biochem. 2001;82:68-77.


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27. Sthadeim TA, Kucera GL. c-Jun N-terminal kinase/stress-activated protein kinase

(JNK/SAPK) is required for mitoxantrone and anisomycin-induced apoptosis in HL-60 cells.

Leuk Res. 2002;26:55-65.

28. Chénais B, Andriollo M, Guiraud P, Belhoussine R, Jeannesson P. Oxidative stress

involvement in chemically induced differentiation of K562 cells. Free Radical Biol Med.

2000;28:18-27.

29. Blanchette J, Jaramillo M, Olivier M. Signalling events involved in interferon-gamma-

inducible macrophage nitric oxide generation. Immunol. 2003; 108:513-22.

30. Hommes D, van den Blink B, Plasse T, Bartelsman J, Xu C, Macpherson B, Tytgat G,

Peppelenbosch M, Van Deventer S. Inhibition of stress-activated MAP kinases induces

clinical improvement in moderate to severe Crohn's disease. Gastroenterology. 2002 122:7-

14

31. Martelli AM, Tazzari PL, Tabellini G, et al. A new selective AKT pharmacological inhibitor

reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing

radiation of human leukemia cells. Leukemia 2003;17:1794-1805.

Chapter 2

50

Chapter 3

Kinome profiling for uncovering the molecular

mode of pharmacon action

Carmen Veríssima Ferreira1,7, Alexandre D. Martins Cavagis1, Sander H.

Diks2,7, Francesca Milano3 ,

Carina L. Bos3, Giselle Z. Justo1, Nelson Durán4,5 and Maikel P.

Peppelenbosch2,6

1Departamento de Bioquímica, Universidade Estadual de Campinas São Paulo, Brazil 2Department of Cell Biology, Faculty of Medicine

University of Groningen, A. Deusinglaan 1 NL-9713 AV Groningen, The Netherlands,

Tel: 31-50-363 2522, Fax: 31-50-363 2512

E mail : [email protected] 3Experimental Internal Medicine, Academic Medical Center

Amsterdam, The Netherlands 4Núcleo de Ciências Ambientais, Universidade de Mogi das Cruzes.

São Paulo, Brazil 5Biological Chemistry Laboratory, Instituto de Química,

Universidade Estadual de Campinas

São Paulo, Brazil

6To whom correspondence should be addressed 7Contributed equally to this work

Chapter 3

52

ABSTRACT

Defining the molecular effects of compounds with clinically useful properties

remains exceedingly challenging if no a priori assumptions can be made as to the

biochemical details of the biological effect observed. We set out to identify the

molecular target of violacein, a purple-coloured pigment produced by the Amazon

River Chromobacterium violaceum, which is used by indigenous Indians in the

Amazon forest to treat a variety of inflammatory conditions and attracts

substantial interest as a consequence of its anti-leukaemic properties. To this end,

we compared lysates of violacein-treated cells and vehicle-treated cells for in vitro

phosphorylation of peptide arrays containing 1152 different kinase consensus

substrates. The results provide a wealth of data on violacein-dependent

biochemical effects. From this kinome profiling effort, the p42/p44 MAP kinase

pathway emerged as a major target for violacein. Subsequent studies revealed that

activation of the p42/p44 MAP kinase pathway is essential for violacein-dependent

effects, triggering differentiation of the leukaemia cells.

Kinome profiling for uncovering the molecular mode of pharmacon action

53

INTRODUCTION

Establishing the molecular mode of action compounds with clinically useful properties

is difficult if there is no information as to the biochemical details of the biological effect

observed. Over the last 5 years array and mass spectrometry technologies have enabled

the determination of the transcriptome and proteome, and such information will likely

be of significant value to our elucidation of the molecular mechanisms that govern the

effects of pharmacological compounds. However, defining those proteins that

participate in signalling pathways affected by such compounds may provide more direct

insight into the mechanism underlying the clinical effects of such compounds. Enzymes

that phosphorylate tyrosine, serine and threonine residues on other proteins play a major

role in signalling cascades that determine cell cycle entry, survival and differentiation

fate in the tissues of the mammalian body and traditional genetic and biochemical

approaches can certainly provide answers as to the molecular details that underlie

pharmacon action, but for technical and practical reasons there are typically pursued one

gene or pathway at a time. Thus, a more comprehensive approach is needed in order to

reveal all signalling pathways influenced by a pharmacological compound in a single

experiment. Adaptation of array technology for measuring enzymatic activity in a

parallel fashion seems a obvious solution and recently progress in this direction has

been made with the preparation of protein chips for the assessment of protein substrate

interactions 1–3 and the generation of peptide chips for the appraisal of ligand-receptor

interactions and enzymatic activities.4–7 Houseman and Mrksich8 showed that peptide

chips, prepared by the Diels -Alder-mediated immobilization of one kinase substrate for

the non-receptor tyrosine kinase c-Src) on a monolayer of alkanethiolates on gold,

allows quantitative evaluation of kinase activity. We recently showed that 33P-?γ-ATP

phosphorylation of arrays consisting of 192 peptides (substrates for kinases) spotted on

glass by cell lysates from human peripheral blood mononuclear cells allowed the

simultaneous description of the temporal kinetics of a multitude of kinase activities

following stimulation with lipopolysaccharide.9 It appeared, however, that the amount

of substrates on this array was insufficient to allow comprehensive descriptions of the

effects of pharmacological intervention on cellular signal transduction.

Chapter 3

54

This consideration prompted us to study the effectiveness of an array with substantially

increased numbers of substrates for obtaining complete descriptions of the effect of

pharmacological intervention on the kinome. To this end we selected a set substrates

covering almost the entire mammalian kinome from the Phosphobase resource

(http://phospho.elm.eu.org)10,11 (a full list of the peptides and the proteins from which

they are derived is listed in the supplementary data) and arrays were constructed arrays

by chemically synthesizing soluble peptides, which were covalently coupled to glass

substrates as described earlier. Arrays consisted of 1152 different nonapeptides,

providing kinase substrate consensus sequences across the entire mammalian kinome.

On each separate carrier, the array was spotted two times, to allow assessment of

possible variability in substrate phosphorylation. The final physical dimensions of the

array were 25 x 75 mm, each peptide spot having a diameter of approximately 250 µm,

and peptide spots being 620 µm apart. Employing this design, we have recently been

able to identify Fyn and Lck as kinase inhibited during glucocorticoid treatment of

human peripheral blood lymphocytes.12

We decided to explore the effects of violacein (Figure 1a), a purple -colored pigment

cum antibiotic produced by Amazon River Chromobacterium violaceum, which is used

by indigenous Indians in the Amazon forest to treat a variety of inflammatory

conditions and attracts substantial interest as a consequence of its anti-leukemic

properties. Recently we showed that violacein is a member of a novel class of cytotoxic

drugs mediating apoptosis of HL60 cells, probably via the induction of TNF receptor

signal transduction.13 When lysates of violacein-treated HL60 cells were investigated on

Western blot, using anti-phospho-amino acid antibodies, numerous changes in global

phosphorylation patterns are detected (Figure 1b) and thus it is reasonable to assume

that at least some kinase activities are affected by the violacein treatment. Thus,

employing the peptide array, the effect of violacein on the HL60 cell kinome we set out

to generate a comprehensive description of violacein effect on leukaemia cell kinases.


55

STUDY DESIGN

Cell Culture

HL60 cells were grown in suspension in RPMI 1640 medium according to routine

procedures.13 Violacein dissolved in dimethyl sulfoxide (DMSO) was added to the

culture medium which had the final DMSO concentration adjusted to 0.1% (v/v).

Peptide arraying

The production of the array and the protocol of the kinome array have been described in

detail earlier.9,12 In short, cells were washed in PBS and lysed in a non-denaturing

complete lysis buffer. The peptide arrays (Pepscan, Lelystad, the Netherlands),

containing up to 1152 different kinase substrates in duplo, were incubated with cell

lysates for 2 hours in a humidified stove at 37C°. Subsequently, the arrays were washed

in 2M NaCl, 1% triton-x-100, PBS, 0.1% tween and H2O, where after slides were

exposed to a phospho-imaging screen for 24-72 hours and scanned on a phospho-imager

(Fuji, Stamford, USA).

Western Blotting

Cells (3 ×107) were lysed in 200 µl of denaturing lysis buffer ans subjected to SDS-

PAGE and Western blotting as described in detail previously.12,13 Detection was

performed using enhanced chemiluminescence (ECL).

Cell differentiation Assay

Control and violacein-treated cells were collected and resuspended in 1X binding buffer

(0.01 M Hepes/NaOH, pH 7.4, 0.14 mM NaCl and 2.5 mM CaCl2) at a concentration of

1 x 106 cells/ml. Subsequently, 100 µl of cell suspension was transferred to a 5-ml tube

Chapter 3

56

and CD11 FITC (5 µl) and CD66 - (10 µl) were added. The cells were incubated at

room temperature for 15 minutes, after which 400 µl of 1X binding buffer was added,

and differentiation analysed by flow cytometry.

Statistical evaluation

The western blots represent 3 independent experiments. Cell viability was expressed as

the means ± standard errors of 3 independent experiments carried out in triplicate. Data

for each assay were analysed statistically by ANOVA


57

RESULTS AND DISCUSSION

To investigate the effects of violacein on the kinome, 2.5 x 105 HL60 cells were

incubated for 24 hrs with vehicle (DMSO) or violacein and subsequently analyzed using

the peptide array. An example is shown in figure 2a. Subsequently, we analyzed the

radio-activity incorporated in substrates phosphorylated by vehicle treated cells against

the radio-activity incorporated in substrates phosphorylated by violacein-treated cells.

The relationship between the two data-sets is roughly linear, suggesting that most

cellular kinases are not directly influenced by the violacein treatment, in apparent

agreement with its mild effect on primary non-transformed human leukocytes.13

Nevertheless, interesting differences are observed, phosphorylation of 49 peptides being

significantly different when violacein-treated and vehicle-treated HL60 cells were

compared (p<0.05; see supplementary information for a full description of results

obtained). These peptides include 2 substrates (#6 and #868 in the supplementary data)

annotated as targets for c-fms proto-oncogene tyrosine kinase activity, which is an

important regulator of myeloid cellular physiology. In addition, phosphorylation of

peptides acting as substrates for the Src-like kinases c-Hck (#395), c-Lck (#361), and c-

Src (#700 and #755) itself were substantially down regulated. Also increased

phosphorylation of a peptide of a Jun-N-terminal kinase (JNK)-derived peptide that

serves as a substrate of MKK 4/7 was observed, whereas also increased phosphorylation

of p53. In addition, peptides that are substrates for p34cdc2 (#172, #461, #462, #643,

#744, #904, and #1121) were less phosphorylated by cell lysates of violacein-treated

cells. Thus violacein influences various cellular signalling systems that could be

important for mediating its anti-leukaemic effect.

Strikingly, however, the most up-regulated substrate d substrate phosphorylation events

occurred in peptides specific for p42/p44 MAP kinase (#181, #460, #625),

Phosphorylation of a peptide specific for the upstream activator of MAPK, c-Raf (#863)

was also highly up-regulated by the violacein treatment, suggesting activation of the

MAPK signalling cassette in leukaemia cells by violacein and this notion was

confirmed in Western blot experiments (figure 2c) employing phosphospecific

antibodies. These experiments demonstrated enhanced immunoreactivity for pS338 c-

Chapter 3

58

Raf, pS221MEK and pT202/pY204 p42/p44 MAP kinase, thus violacein stimulates the

three major components of the MAP kinase signalling cassette. Concomitantly,

immunoreactivity towards pT180/pY182 was unchanged and thus the effects of violacein

on the MAP kinase cascade are not the result of aspecific kinase activation although at

the same time immunoreactivity for pT183/pY185 JNK did increase, but this was confirm

expectation from the peptide array results. Thus activation of the p42/p44 MAP kinase

cascade is a prominent effect of violacein on cellular physiology.

Activation of the MAK kinase cascade by a anti-leukaemic compound was surprising,

but this signalling pathway has been associated with leukaemic cell differentiation and

cell cycle arrest.14 Indeed, the down regulation of cdc2 enzymatic activity observed in

the peptide array experiments suggests cell cycle arrest in response violacein and such

cell cycle arrest was confirmed on Western blot employing an antiserum against the cell

cycle inhibitor p21 (Figure 2a) and down regulation of c-Myc immunoreactivity an

event closely associated with differentiation and inhibition of cycling in leukaemia

cells.15 Furthermore, we observed up-regulation of expression of both the p50 and p60

isoforms of NF-κB, an event closely associated with differentiation of HL60 cells.16

Direct assessment of CD14 (a cell surface marker for both granulocyte and monocyte

differentiation) and CD66b (a marker for granulocyte differentiation only) demonstrated

that a 72 hr treatment with violacein induces terminal commitment of the leukemia cells

into both lineages at level comparable to DMSO treatment, generally considered a

powerful differentiation agent in this model system (Figure 2b). Hence, it is possible

that activation of the MAP kinase cassette by violacein produces cell differentiation.

To directly test this hypothesis, we employed the specific p42/p44 MAP kinase inhibitor

UO126. In the presence of this inhibitor violacein was no longer capable of inducing

differentiation, providing strong support for the notion that activation of the MAP

kinase cascade by violacein is required for the induction of differentiation (Figure 2c).

Taken together, we feel that increased cell differentiation via MAP kinase activation

may contribute to the anti-leukaemic effects of violacein (in addition to the induction of

cytotoxicity13,18), although final confirmation of this hypothesis will require experiments

in patients in which the effect of violacein on leukaemia cell MAP kinase activity and


59

differentiation are monitored on a single basis. The mechanism by which violacein

produces MAP kinase activation remains only partly resolved, the present study shows

that it is mediated via activation of the Raf/MEK/MAPK cascade but the signals

operating upstream of this cassette are unclear but may well be secondary to the

induction of TNF receptor signalling19. In that case, the role of TNF receptor signalling

in the anti-leukaemic effect of violacein would dichotomic, inducing both direct

cytotoxicity via caspase 8 activation and differentiation via activation of MAPK.

Disregarding, however, the exact importance for MAP kinase-dependent leukaemia cell

differentiation for the anti-leukaemic actin of violacein we feel that the present study

shows that kinome profiling using metabolic arrays is a highly promising tool for the

rapid evaluation of the effect of pharmacological intervention on signalling pathways.

Chapter 3

60

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8. Houseman BT, Mrksich M. Towards quantitative assays with peptide chips: a surface

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9. Diks SH, Kok K, O'toole T, Hommes DW, van Dijken P, Joore J, Peppelenbosch

MP. Kinome profiling for studying lipopolysaccharide signal transduction in human

peripheral blood mononuclear cells. J. Biol. Chem. 2004;279:49206-49213.

10. Blom N, Kreegipuu A, Brunak S, Conway T, Schoolnik GK. PhosphoBase: a

database of phosphorylation sites. Nucleic Acids Res. 1998;26:382–386.

11. Kreegipuu A, Blom N, Brunak S. PhosphoBase, a database of phosphorylation sites:

release 2.0. Nucleic Acids Res. 1999;27:237–239.

12. Lowenberg M, Tuynman J, Bilderbeek J, Gaber T, Buttgereit F, van Deventer S, et

al. Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn.

Blood. 2005;106:1703-1710.


61

13. Ferreira CV, Bos CL, Versteeg HH, Justo GZ, Duran N, Peppelenbosch MP.

Molecular mechanism of violacein-mediated human leukemia cell death. Blood.

2004;104:1459-1464.

14. Das D, Pintucci G, Stern A. MAPK-dependent expression of p21(WAF) and

p27(kip1) in PMA-induced differentiation of HL60 cells. FEBS Lett. 2000;472:50-52.

15. Xu D, Popov N, Hou M, Wang Q, Bjorkholm M, Gruber A, et al. Switch from

Myc/Max to Mad1/Max binding and decrease in histone acetylation at the telomerase

reverse transcriptase promoter during differentiation of HL60 cells. Proc. Natl. Acad.

Sci. USA. 2001;98:3826-3831.

16. Kido S, Inoue D, Hiura K, Javier W, Ito Y, Matsumoto T. Expression of RANK is

dependent upon differentiation into the macrophage/osteoclast lineage: induction by

1alpha,25-dihydroxyvitamin D3 and TPA in a human myelomonocytic cell line, HL60.

Bone. 2003;2:621-629.

17. Rettori D, Durán N. Production, Extraction and Purification of Violacein: An

antibiotic Pigment Produced by Chromobacterium violaceum. World J. Microbiol.

Biotechnol. 1998;14:685-688.

18. Kodach LL, Bos CL, Duran N, Peppelenbosch MP, Ferreira CV, Hardwick JC.

Violacein synergistically increases 5-fluorouracil cytotoxicity, induces apoptosis and

inhibits Akt-mediated signal transduction in human colorectal cancer cells.

Carcinogenesis. 2005 Dec 12; [Epub ahead of print]

19. Boone E, Vandevoorde V, De Wilde G, Haegeman G. Activation of p42/p44

mitogen-activated protein kinases (MAPK) and p38 MAPK by tumor necrosis factor

(TNF) is mediated through the death domain of the 55-kDa TNF receptor. FEBS Lett.

1998; 441:275-80

Chapter 3

62

Figure 1. Effects of violacein. A. Chemical structure of violacein. B. HL60 cells were

incubated with different concentrations of violacein and subsequently investigated for

global phosphorylation patterns employing an anti-pY antibody and Western blotting.

HO

H

HO

N

N

N

HO

A B

D

MS

O

100

nM

1000

nM

D

MS

O

100

nM

1000

nM


63

Figure 2. Peptide arrays reveal activation RAF/MEK/MAPK cascade by violacein..A. Example of a peptide array substrate phosphorylation by lysates of HL60 cells treated with 1000 nM violacein. B. The relationship between radio-activity incorporated in substrates phosphorylated by vehicle treated cells against the radio-activity incorporated in substrates phosphorylated by violacein-treated cells. Note the strong violacein-induced effect on the phosphorylation of kipeptides of RAF/MEK/MAPK cascade. C. Analysis of activation of MAPK-associated signalling elements by Western blotting and phosphospecific antibodies.

Chapter 3

64

Figure 3. Violacein induces differentiation of HL60 leukaemia cells. a. HL60 cells were incubated with violacein for 24 hrs and the levels of differentiation associated proteins were investigated using Western blotting. b. Direct assessment of HL60 differentiation using FACS and CD14 (a differentiation marker for both granulocytes and monocytes) and CD66b (a marker for granulocyte differentiation only) after 72 hrs of violacein treatment. Treatment with violacein induces terminal commitment of the leukemia cells into both lineages at level comparable to DMSO treatment c. p42/p44 MAP kinase enzymatic activity is essential for violacein-dependent leukaemia differentiation.


65

Figure 4. p42/p44 MAP kinase enzymatic activity is essential for violacein-dependent leukaemia differentiation. HL60 cells were incubated with violacein in the presence and absence of 1 ?M of the p42/p44 MAP kinase inhibitor U0126 and investigated for differentiation.

0

10

20

30

40

50

60

70

80

90

100

0 100 1000

Violacein (nM)

% d

iffe

rent

iate

d ce

lls

Vehicle

U0126

Chapter 3

66

Chapter 4

Programmed cell death of HL60 leukaemia cells by

the photosensitizer riboflavin is mediated by

FasL-Fas induction and ceramide

Alexandre D. Martins Cavagis1, Ana Carolina Santos de Souza1,

Liudmila Kodach2, Carina L. Bos2, , Hiroshi Aoyama1,

Maikel P. Peppelenbosch3, Carmen Veríssima Ferreira1

1Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas

(UNICAMP), Campinas, São Paulo, Brazil. 2 Department of Experimental Internal Medicine, Academic Medical Center (AMC)

University of Amsterdam, The Netherlands. 3Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1

NL-9713 AV Groningen, The Netherlands

Correspondence: Dr. Carmen Veríssima Ferreira, Department of Biochemistry,

State University of Campinas; email: [email protected]

Accepted for publication in Apoptosis

Chapter 4

68

ABSTRACT Riboflavin is a photosensitizer which causes cell death in mammalian cancer after

photodynamic therapy. The molecular mechanism mediating its cytotoxicity, however,

remains unclear. In this work, it is shown that irradiated riboflavin-dependent

cytotoxicity in HL60 cells occured concomitantly with activation of caspase 8 caused

by overexpression of Fas and FasL and also stimulation of ceramide. The activation of

this cascade led to an increase of JNK activity and inhibition of survival mediators

(PKB and IAP1), as well as downregulation of cell cycle progression regulators. Taken

together, these results provide a molecular approach characterizing the riboflavin-

mediated apoptosis after photodynamic therapy.

KEYWORDS

Riboflavin; photosensitizer; apoptosis; myeloid leukemic cells; signal transduction;

MAPKs; protein phosphatases.

ABBREVIATIONS

ERK, extracellular signal-regulated kinase; Fas (CD95), Fas receptor; FasL (CD95L),

Fas ligand; HL60, human myeloid leukemia cell line; IAP1, inhibitory apoptosis protein

type 1; JNK, c-jun-NH2-terminal protein kinase; MAPK, mitogen-activated protein

kinase; MEK, MAPK/ERK kinase; PCNA, proliferating cell nuclear antigen; PKB,

protein kinase B; PP2A, phosphoprotein phosphatase 2A; RF, riboflavin; STAT, signal

transducer and activator of transcription; TNF, tumo r necrosis factor; TNFR, tumor

necrosis factor receptor; TRAF, receptor-associated factor.

INTRODUCTION

Photodynamic therapy (PDT) is a treatment that makes association of a

photosensitizer with light to generate oxygen-dependent photochemical destruction of

diseased tissue. This modality has been approved worldwidely since 1993 for the

treatment of several oncological and nononcological disorders (Peng and Nesland,

2004). PDT is based on the activation of a photosensitizer by illumination with visible

or UV light, leading to photochemical tissue destruction. Nevertheless, the molecular

PCD of leukaemia cells by irradiated riboflavin

69

details by which the photostimulation of photosensitizers induces cell death remain only

poorly understood, but are of obvious importance for devising therapy having enhanced

specificity for deviant tissue as to non-pathological cells.

Riboflavin (Figure 1), 7,8-dimethyl-10-ribityl-isoalloxazine, one of the

components of B2 vitamin complex, is an efficient photosensitizer and has been target of

several studies with photodynamic therapy (Edwards et al.,1999b). Riboflavin is

particularly sensitive to UV and visible light and acts via the generation of singlet

oxygen (type II mechanism) or via radical species (type I mechanism), including

reactive oxygen species such as the superoxide ion, hydroxyl radical and hydrogen

peroxide (Souza et al, 2005). Although the reduction potential of riboflavin is -0.3V at

pH 7.0, the redox potential of the excited triplet flavin can be shifted to 1.7V (Meisel

and Neta, 1975) which is higher than the redox potential of important biomolecules

such as amino acids, proteins, lipids and nucleic acids which can undergo

photodegradation in the presence of riboflavin.

Recently, important advances in the insight as to the effects of radiation on

riboflavin chemistry have been achieved (Ahmad et al., 2004a; Ahmad et al., 2004b;

Holzer, et al., 2005), demonstrating that this flavin is degraded through a variety of

reactions including photolysis (intramolecular photoreduction) and photoaddition

(intramolecular photoaddtion) as major reactions. The photodegradation of riboflavin in

aqueous solutions results in a number of products such as 7, 8-dimethyl-10-

(formylmethyl) isoalloxazine, lumichrome, lumiflavin and cyclodehydroriboflavin.

Additionally, near UV light and daylight fluorescent light irradiated riboflavin produces

lethal effects on mammalian cells in culture and this cytotoxicity has been attributed to

products of the riboflavin-sensitized photooxidation of Trp and/or Tyr such as a

complex mixture of indolic, flavinic and indolic-flavin type aggregates (photoadducts) –

(Silva et al., 1994; Edwards and Silva, 2001). Thus, the chemical effects of riboflavin

photostimulation are fairly well characterised.

The biological effects following these reactions are, however, much less clear.

It has been suggested that riboflavin irradiation-induced formation of reactive oxygen

species and other stable photoproducts leads to cell death, although clear cut evidence is

lacking (Edwards et al., 1999a; Edwards et al., 1999b; Edwards and Silva, 2001;

Ahmad et al., 2004a). These considerations prompted us to investigate the effects of

Chapter 4

70

H3C

H3C N

N

N

N

CH3

O

O

H

pre-irradiated riboflavin on HL60 cells. In the present work we observed Fas and FasL

overexpression followed by activation of caspase 8 , mo dulation of JNK, protein

phosphatase and ceramide synthase activities and apoptosis. Thus, our results can be

taken together to outline a molecular approach in order to characterise biochemically the

riboflavin-mediated apoptosis after photodynamic therapy.

Lumichrome Riboflavin Lumiflavin 7,8-dimethyl-10-formylmethyl-isoalloxazine

Figure 1 - Chemical structure of riboflavin and its photoproducts

N

N

NH

N

O

O

O

OH OH

OH

H

1

2

3456

7

89 10

1' 2'3' 4'

5'

N

N

N

NH3C

H3C

H

O

O

H

N

N

NH

N

CHO

O

O


71

MATERIALS AND METHODS

Cell line and Reagents

HL60 cells were purchased from the American Type Culture Collection

(ATCC, Rockville, MD). Polyclonal antibodies against, antiphospho-p38 mitogen-

ativated protein kinase (MAPK), antiphospho-p42/44 (ERK1/2), antiphospho-c-jun-

NH2-terminal protein kinase (JNK), antiphospho-MAPK/ERK Kinase 1/2 (MEK1/2),

antiphospho-PKB, cleaved caspase 8, antirabbit and antimouse peroxidase-conjugated

antibodies were purchased from Cell Signaling Technology (Beverly, MA). Antibodies

against phospho-PP2A, phosphotyrosine, phosphothreonine, p21, proliferating cell

nuclear antigen (PCNA), signal transducer and activator of transcription 1 (STAT1) and

2 (STAT2), receptor-associated factor 1 (TRAF1) and 2 (TRAF2) and inhibitory

apoptosis protein type 1 (IAP1) were purchased from Santa Cruz Biotechnology (Santa

Cruz, CA). anti-Fas receptor (Fas), anti-Fas ligand (FasL) - (BD Biosciences, San

Diego, CA); Fas neutralizing was from Upstate Biotechnology, Inc.(Lake Placid, NY).

Riboflavin and Fumonisin B1 were from Sigma. Caspase 3 and Caspase 9

Colorimetric Assay Kits were obtained from R&D Systems (Minneapolis).

Cell Culture

HL60 cells were routinely grown in suspension in RPMI 1640 medium

supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and

10 % heat-inactivated fetal bovine serum, at 37oC in a 5 % CO2 humidified atmosphere.

In all experiments, 7 x 105 cells/ml were treated for 24h with different concentrations of

irradiated riboflavin.

Irradiation of riboflavin and treatment of HL60 cells

Solutions of riboflavin (25, 50, 75, 100, 150, 200 and 250 µM) were diluted in

RPMI 1640 culture medium and irradiated with UV light. Afterwards, a suspension of 7

x 105 cells/ml was added to the samples so that the following final concentrations of

riboflavin: 5, 10, 15, 20, 30, 40 and 50 µM were reached. The cells were also pre-

treated with 25 µM fumonisin B1 or anti-Fas 10µg/mL for 1h and, afterwards, treated

Chapter 4

72

with riboflavin. Cell viability was assessed by trypan blue dye exclusion and MTT

reduction assays.

MTT reduction assay

The medium containing irradiated riboflavin was removed and 1.0 ml of MTT

solution (0.5 mg MTT/ml of culture medium) was added to each well. After incubation

for 4 h at 37oC, the medium was removed and the formazan released by solubilisation in

1.0 ml of ethanol. The plate was shaken for 5 min on a plate shaker and the absorbance

measured at 570 nm (Mosmann, 1983; Denizot et al., 1986).

Western blotting analysis Cells (3 x 107) were lysed in 200 µL of lysis buffer [50 mM Tris –HCl (pH

7.4), 1% Tween 20, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM

Na3 VO4, 1 mM NaF and protease inhibitors (1 µg/ml aprotinin, 10 µg/ml leupeptin, and

1 mM 4-(2-aminoethyl)benzenesulfonyl-fluorid -hydrochloride)] for 2 h in ice. Protein

extracts were cleared by centrifugation and protein concentrations were determined

using Lowry method (Hartree, 1972). An equal volume of 2X sodium dodecyl sulphide

(SDS) gel loading buffer [100 mM Tris –HCl (pH 6.8), 200 mM DTT, 4 % SDS, 0.1 %

bromophenol blue and 20 % glycerol] was added to samples which were subsequently

boiled for 10 min. Cell extracts, corresponding to 3 x 105 cells, were resolved by SDS-

polyacrylamide gel (12%) electrophoresis (PAGE) and transferred to PVDF

membranes. Membranes were blocked in 1% fat-free dried milk or bovine serum

albumin (2%) in Tris -buffered saline (TBS)-Tween 20 (0.05 %) and incubated overnight

at 4 °C with appropriate primary antibody at 1:1000 dilution. After washing in TBS-

Tween 20 (0.05 %), membranes were incubated with antirabbit or antimouse

horseradish peroxidase-conjugated secondary antibodies, at 1:2000 dilutions (in all

Western blotting assays), in blocking buffer for 1 h. The detection was made using

enhanced chemiluminescence (ECL).

Caspases 3 and 9 activity assays Caspase activities were determined by the measurement at 405 nm of p-

nitroanaline released from the cleavage of caspase 3 (Ac-DEVD-pNA) and caspase 9


73

(LEHD-pNA) substrates. The enzyme activities were expressed in pmol/min and the

extinction coefficient of pNA was 10,000 M -1 cm-1.

Annexin V and 7-amino-actinomycin D Assays Control and riboflavin-treated cells were collected and resuspended in 1X

binding buffer (0.01 M Hepes/NaOH, pH 7.4, 0.14 mM NaCl and 2.5 mM CaCl2) at the

concentration of 1 x 106 cells/ml. Subsequently, 100 µl of cell suspension were

transferred to a 5-ml tube and Annexin V FITC (5 µl) and 7-amino-actinomycin D (7-

AAD) - (10 µl) were added. The cells were incubated at room temp erature for 15

minutes, after which 400 µl of 1X binding buffer were added and apoptosis assessed by

flow cytometry.

Statistical evaluation All experiments were performed in triplicate and the results shown in the

graphs represent the means and standard errors. Cell viability data were expressed as the

means ± standard errors of 3 independent experiments carried out in triplicates. Data

from each assay were analyzed statistically by ANOVA. Multiple comparisons among

group mean differences were checked with Tukey post hoc test. Differences were

considered significant when the p value was less than 0.05. Western blottings represent

3 independent experiments.

RESULTS

Irradiated riboflavin but not non-radiated riboflavin affects HL60 cells

viability A model system for investigating the effects of a photosensitizer on cancer cell

signal transduction should fulfil two important requirements: 1) when cells are treated

with the non-radiated form of the compound, cellular physiology should not be affected,

and 2) pre-irradiation of the compound should result in metabolites which are cytotoxic

when added to the model system, to prevent irradiation of the cell itself with its

associated confounding effects. In order to find out whether HL60 cells fit in such a

model, the cells were treated with riboflavin in concentrations up to 50 µM, whereas a

Chapter 4

74

0 10 20 30 40 500

20

40

60

80

100

120

MTT

Red

uctio

n (%

)

[Riboflavin] / µM

Irradiated Riboflavin Non-irradiated Riboflavin

Control RF 10 µM RF 50 µM0

20

40

60

80

100

MT

T R

educ

tion

(%) 0 min

60 min 24 h

negligible cell death was observed at lower concentrations. Conversely, the pre-

irradiated riboflavin demonstrated a strong cytotoxic effect on HL60 cells, with an IC50

value of 10µM (Figure 2A). Therefore, in accordance with the observations of Edwards

and Silva (1999b), HL60 cells consists of a suitable model for studying the effects of a

photosensitizer.

A

B

Figure 2 – Irradiated riboflavin presents toxic effect on HL60 cells. The cells were treated

with different concentrations of irradiated and non-irradiated riboflavin in RPMI medium (A).

The effect of photoproducts was also evaluated after 1h, 24h and 7 days after irradiation (B). In

the absence of compounds, the MTT (3-(4, 5-dimethylthiazol-2, 5-diphenyl-tetrazolium bromide)

reduction was considered as 100%. The experiment was performed in a 24 wells plate. Results

represent t he means ± standard error of 3 experiments run in triplicate (P < 0.05).


75

0 10 20 30 40 500

5

10

15

20

25

30

35

Cel

l Dea

th (%

)

[Riboflavin] µM

Annexin V FITC Annexin V FITC + 7-AAD 7-AAD

Control RF 5 µM RF 10 µM RF 50 µM0

50

100

150

200

250

Cas

pase

act

ivity

(pm

ol/m

in)

caspase-3 caspase-9

Additionally, the effect of irradiated riboflavin was evaluated after 1h, 24h and

7 days after irradiation. Interestingly, the results show that the cytotoxic profile

remained the same, indicating that the anti-proliferative activity of pre-irradiated is

related to the formation of stable photoproducts (Figure 2B).

Irradiated riboflavin-dependent cytotoxicity is mediated by apoptosis Cytotoxicity can proceed either via necrosis, which may produce substantial

collateral damage, or apoptosis when cells dispose of themselves in an orderly fashion.

The morphological observation of Edwards and Silva is more consistent with an

apoptotic rather than a necrotic response to pre-irradiated riboflavin. In agreement,

when analyzed by flow cytometry, the exposure of phosphatidyl serine concomitantly

with the 7-AAD exclusion of the HL60 cells population demonstrated that apoptosis is

the principal mode of cell death induced by pre-irradiated riboflavin. However, when

the cells were treated with higher concentrations, an increase of later apoptosis and

necrosis was observed (Figure 3A). Moreover, the activation of direct markers of

apoptosis: caspase 9 and 3 (Figure 3B) was also observed as well as the downregulation

of inhibitory apoptosis protein type 1 (IAP1), an indirect marker of apoptosis – (Figure

3C).

A B

Figure 3 - Apoptosis analysis of HL60 cells treatment with riboflavin. (A) Cell samples were

prepared as described in Methods and Annexin V-positive, 7-AAD-positive and Annexin V/7-

AAD-positive populations were analyzed by flow cytometry. (B) Colorimetric assay was

Chapter 4

76

TRAF 1

TRAF 2

TRAF 1

TRAF 2

5 µM

Con

trol

10 µ

M

50 µ

m

performed to determine caspases 9 and 3 activities. (C) Western blot analysis was performed to

assess IAP1, caspase 8, Fas, FasL, Traf1 and 2 levels.

C

Irradiated riboflavin-induced apoptosis is mediated via the extrinsic

pathway and ceramide Apoptosis can occur as a response to external clues, mediated via death

receptors or be a consequence of intracellular events, such as the p53-mediated

apoptosis in response to DNA damage. The strong activation of caspase 8 detected in

response to irradiated riboflavin (Figure 3C) allows us to infer that the extrinsic

pathway mediates, at least, part of the pro-apoptotic effects of irradiated riboflavin. A

strong support for this notion can be taken from the observation that the expression

levels of both the death receptor Fas and FasL were considerably increased in response


77

Control

Riboflavin 5 µM

Riboflavin 10 µM

Riboflavin 50 µM

0 20 40 60 80 100

MTT Reduction (%)

Without anti-Fas With anti-Fas

Control

Riboflavin 5 µM

Riboflavin 10 µM

Riboflavin 50 µM

0 20 40 60 80 100

MTT Reduction (%)

Without Fumonisin B1 With Fumonisin B1

to stimulation with irradiated riboflavin (Figure 3C). Confidence in the specificity of

this effect was bolstered by the observation that expression of TRAF2, which

participates in the signalling downstream of the distinct death receptor TNFR, was

down regulated in response to pre-irradiated riboflavin (Figure 3C). Thus, our results

demonstrate a specific activation of FasL/Fas signalling in HL60 cells in response to

pre-irradiated riboflavin. Importantly, a pre-treatment of the cells with Fas neutralising

prevented the apoptosis induction by riboflavin (at 5 and 10 µM) - (Figure 4A).

A

B

Figure 4 - Effects of Fas neutralising and fumonisin B1 on riboflavin action. Cells at the

density of 7 x 106 were incubated with antibody Fas neutralising 10µg/ml (A) or 25 µM

Fumonisin B1 (B) for 1h and after treated with irradiated riboflavin for 24 h and the number of

viable cells was determined by MTT reduction assay. Results represent the means ± standard

error of 3 experiments run in triplicate (P < 0.05).

Chapter 4

78

Co

ntr

ol

5 µM

10 µ

M

50 µ

M

Phospho-MEK1/2

Phospho-ERK1/2

Phospho-JNK

Phospho-p38

On the other hand, fumonisin B1 (ceramide synthase inhibitor) also prevented

HL60 cells death when riboflavin was used at the concentrations of 5 and 10 µM

(Figure 4B).

Mitogen activated protein kinases regulation by riboflavin photoproducts Since the previous results predict an increased activity of kinases in response to

irradiated riboflavin, we examined the status of some members of the MAP kinases

family. In Figure 5, a strong increase in phospho-JNK level is observed, while ERK and

p38 MAP kinase levels remained unchanged after the treatment. These results are

consistent with the participation of the FasL/Fas system in mediating the cytotoxic

effects of irradiated riboflavin on HL60 cells viability.

Figure 5 - Effects of flavins on quinases phosphorylation and expression in HL60. Cells were

treated with flavins (5, 10 and 50 µM) and the phosphorylation of quinases were evaluated by

immunoblotting. Soluble lysates were matched for protein content and analyzed on Western blot.


79

STAT1

STAT2

Co

ntr

ol

5 µM

10 µ

M

50 µ

M

PCNA

Irradiated riboflavin causes cell cycle arrest The effects of irradiated riboflavin on the expression of a selected set of

proteins involved in the regulation of the cell cycle progression were investigated by

Western Blotting. An expressive up-regulation of p21 was observed, concomitantly

with down-regulation of PCNA and STATs 1 and 2 (Figure 6).

Figure 6 - HL60 cells cycle arrest by riboflavin. The expression of p21, PCNA, STAT1 and 2

was evaluated by immunoblotting. Soluble lysates were matched for protein content and analyzed

on Western blot.

Effect of irradiated riboflavin in protein phosphatases and PKB activities The analysis of tyrosine phosphorylation status in cytoplasmic proteins shows a

strong increase of tyrosine phosphorylation after treatment of cells with 5 µM of

irradiated riboflavin. Interestingly, the treatment with higher concentrations of irradiated

riboflavin showed a dose-dependent decrease in tyrosine phosphorylation associated

Chapter 4

80

with the activation of proteins tyrosine phosphatases (Figure 7A). Moreover, an increase

in the threonine phosphorylation (Figure 7B) was also observed. In agreement, Figures

7C and D show the inhibition of the serine/threonine phosphatase PP2A and the anti-

apoptotic kinase PKB.

Figure 7 - Protein phosphatase and protein kinase B activities are affected by flavins.

The profile status of protein phosphorylation in tyrosine (A) and threonine (B) residues and

PKB (D) as well as inhibitory phosphorylation of PP2A (C) was evaluated by

immunoblotting.

A B

5 µM

Con

trol

10 µ

M

50 µ

M

5 µM

Co

ntr

ol

10 µ

M

50 µ

M

5 µ

M

Co

ntr

ol

10 µ

M

50 µ

M

PP2A

PKBD

C


81

DISCUSSION Chemotherapy for the treatment of some types of neoplastic disease has been

one of the most intrigued challenge for the scientists. In relation to leukemia, the

chemotherapeutic treatment outcome for most adults with acute my eloid leukemia

remains unacceptable. In this way, new agents which act against cancerous cells at low

concentrations and presenting high specificity are desirable. For this purpose, HL60

cells have been employed as a suitable model system for studying cell differentiation

and apoptosis in vitro (Ferreira et al., 2004).

Some authors have been demonstrating that pre-irradiated riboflavin leads

HL60 cells to die by apoptosis. These previous results suggested the irradiation of

riboflavin induces to the formation of reactive oxygen species and other stable

photoproducts (Edwards et al., 1999a; Edwards et al., 1999b; Edwards and Silva, 2001;

Ahmad et al., 2004a). However, we demonstrate here that the effect of irradiated

riboflavin on HL60 cells treated for 24h is not due to oxidative stress induction since,

even after 24h of irradiation, the effect observed stays the same. This result agrees with

that showed by the Edwards’ group (Edwards et al., 1994; Edwards et al., 1999b) which

demonstrates that oxygen reactive species generated during irradiation cannot affect the

cells after the irradiation is interrupted, due either to their short lifetimes in aqueous

solution or to the low concentration, as the case of H2O2.

Additionally, the possible mechanisms by which irradiated riboflavin provokes

HL60 cells death were investigated. The results obtained confirm data from literature

(Edwards et al., 1994; Massey, 2000) which stablish relationship between the action of

irradiated riboflavin and alterations in signal transduction responsible for cell survival.

In the present results inhibition of PKB was observed, demonstrating that pre-irradiated

riboflavin reduces activity of this anti-apoptotic kinase and, consequently, leads cells to

apoptosis. Apoptosis, in turn, was detected by phosphatidylserine exposure, activation

of caspases 8, 9 and 3 and downregulation of IAP1. These results are in accordance with

other works which demonstrate that pre-irradiated riboflavin induces morphological and

biochemical changes similar to those of apoptotic cells (Edwards et al., 1994; Edwards

et al., 1999b; Edwards and Silva, 2001).

The capacity of irradiated riboflavin to induce HL60 to apoptotic cell death

suggests its potential use as photosensitizer agent, whereas photosensitising becomes

Chapter 4

82

increasingly important for the treatment of cancer especially for the treatment of

tumours not easily irradiated due to their location in the body, such as in leukemia. Here

we delineate the molecular details concerning the cell death induced by

photosensitization through the investigation of molecular mechanisms related to the

induction and execution of apoptosis by irradiated riboflavin.

Our results show strong activation of caspases 3 and 8 when compared with

caspase 9 indicating the intrinsic pathway is not the major signalling responsible for the

induction of the apoptotic process. In fact, we observed that the treatment of HL60 cells

with riboflavin photoproducts induced the up-regulation of both Fas and FasL

expression whereas the inhibition of death receptor with anti-Fas neutralizing prevented

the apoptosis induced by irradiated riboflavin, suggesting that death receptors play a

pivotal role in the induction of apoptosis by activating the extrinsic pathway.

Furthermore, our results show that the inhibition of ceramide synthase by Fumonisin B1

was able to avoid cell death provoked by riboflavin at concentrations of 5 and 10 µM,

suggesting that riboflavin-induced HL60 cell death is associated to activation of Fas

receptor induced via a ceramide- dependent pathway. These results are in accordance

with previous data from the literature showing the importance of ceramide as a second

lipid messenger involved in the reorganization of lipid rafts into ceramide-enriched

platforms responsible for increase of Fas clustering and its activation (Cremesti et al.,

2002; Huang et al., 2004; Miyaji et al., 2005; Rotolo et al., 2005) and also as a regulator

of JNK (Verheij et al., 1996).

Thus, cell death induced by this photosensitizer is not the consequence of a

specific adduct-induced apoptosis but follows an orderly apoptotic program that leads to

an orderly reduction in tumour mass and better clinical results since massive necrosis is

avoided.

Additionally, we observed that the irradiated riboflavin leads to cell cycle

arrest in HL60 as shown by the activation of p21 and down-regulation of PCNA. p21

binds to and inactivates a variety of cyclin/CDK complexes and thereby regulates the

cell cycle. PCNA is a protein synthesized in early G1 and S phases of the cell cycle and

functions in the cell cycle progression, DNA replication and DNA repair (Woods et al.,

1991; Xia et al., 2005). As HL60 cells are p53 negative of homozygous deletions

(Steinman et al., 1998) and p53 is one of the activators of p21, we investigated if the


83

expression of p21 could be regulated through STATs which mechanism is p53-

independent and can occur in response to TNFR1 signaling (Wang, et al., 2000). Unlike

to this observation, our results show that the activation of p21 in HL60 cells treated with

irradiated riboflavin is independent of STATs and TNFR1 signaling. Interestingly,

activation of p21 in myelocytic cell lines such as HL60 are also related to terminal

differentiation but the mechanism that regulates p21 expression in these p53-

independent circumstances is not clear (Das et al., 2000; Asada et al., 2004).

The reversible phosphorylation of proteins, regulated by protein kinases and

protein phosphatases, influences virtually all cellular functions and it is an essential

mechanism in the control of proliferation, differentiation and transformation (Zhang,

2003; Aoyama et al., 2003). Riboflavin photoproducts cause an increase in the

phosphorylation of serine/threonine residues and, on the contrary, a decrease in

phosphotyrosine residues. Interestingly, the decrease in phosphotyrosine residues

indicates an activation of protein tyrosine phosphatases and reinforce that the cytotoxic

effects of irradiated riboflavin are mediated overcoat by the production of stable

photoproducts, since it is known that tyrosine phosphatases are inhibited by oxidative

stress.

The inhibition of the serine/treonine phosphatase PP2A observed after the

treatment and is in agreement with other works that show induction of apoptosis by

PP2A inhibitors (McCluskey et al., 2003). Moreover, the inhibition of PP2A is

correlated to the differentiation of HL60 cells (Uzunoglu et al., 1999; Bhoola and

Hammond, 2000) and indicates that the induction of death may be a result of a terminal

differentiation induced by irradiated riboflavin. The activation of protein tyrosine

phosphatases also indicates a differentiation process induced by irradiated riboflavin

since the increase in phosphotyrosine residues has been shown in the terminal stages of

differentiation of HL60 cells (Treigyté et al., 2000).

The family of serine/threonine protein quinases MAPKs regulates diverse

cellular activities running the gamut from gene expression, mitosis and metabolism to

motility, survival and apoptosis (Brantley-Finley, 2003). In order to evaluate the role of

MAPKs in the induction of HL60 death by riboflavin the activation status of ERK, JNK

and p38 MAPK was analyzed and our results show that the cytotoxic effects of

Chapter 4

84

riboflavin photoproducts are related to the activation of JNK and down-regulation of

ERK1/2, whereas no changes in p38 MAPK status was observed.

ERK1/2 signaling has been implicated as a key regulator of cell proliferation

and, for this reason, inhibitors of the ERK pathway are entering clinical trials as

potential anticancer agents (Lee and McCubrey, 2002; Roux, and Blenis, 2004). In this

paper we show that riboflavin photoproducts caused a slight reduction of ERK activity,

which may also be related to the entry in the terminal stage of differentiation in HL60

cells (Chen et al., 2004).

The activation of JNK is in accordance with the increase in Fas and FasL

expression. In fact, JNK activity has been reported to mediate drug-induced caspase

activation and to be requested for up-regulation of FasL expression in HL60 cells

(Treigyté et al., 2000, O’Gorman et al., 2001, Barthelemy et al., 2004, Peng et al.,

2005, Su et al., 2005). Furthermore, ERK1/2 pathway has been related to resistance of

cell death triggered by death receptors and the inhibition of this cascade shows to

sensitize cells to Fas signaling (Holmstrom et al., 2000, Tran et al., 2001). HL60 cells

line are relatively resistant to Fas -induced death, hence, the down-regulation of ERK1/2

by irradiated riboflavin is an important mechanism to sensitization of these cells by

overexpression of Fas and FasL induced by JNK activity. Moreover, our results show

inhibition of the PKB pathway, which is also important to influence in the sensitization

of cell death by Fas (Treigyté et al., 2000).

The present work demonstrates that irradiated riboflavin is a potent inducer of

apoptotic cell death in HL60 by activating the extrinsic pathway in a mechanism

dependent of up-regulation of Fas and FasL expressions induced by activation of JNK.

Furthermore, our study indicates that the apoptotic process is preceded by cell cycle

arrest and terminal differentiation but additional studies are necessary to give more

detailed evidences about the riboflavin action (Figure 8). Nevertheless, the results here

presented reinforce the potentiality of riboflavin as adjuvant therapeutic for myeloid

leukemia and contribute to the better knowledge towards the rational use of the

photosensitization for cancer treatment.


85

Figure 8 - Schematic representation of the mechanism of flavins-induced HL60 cell death. Data

presented in this report revealed that riboflavin induces apoptosis in HL60 cells through activation of the

Fas signaling cascade as demonstrated by upregulation of Fas and FasL. Furthermore this mechanism is

dependent of ceramide production and reorganization of lipid rafts in ceramide grouped platforms which

are responsible for the oligomerization and activation of Fas receptors. Also, the event involves the

MAPK JNK and caspase 8 induction and PKB inhibition. Active caspase 8 initiates the apoptotic cascade

as demonstrated by an expressive activation of caspase 3 and downregulation of IAP1. The dominant

participation of extrinsic pathway induced by flavins was also indicated by the lower effect observed in

the caspase 9 activity in comparison to caspase 3 and increase in cell viability induced by the Fas

neutralising.

Chapter 4

86

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Acknowledgements. The authors are greatful to Jennie M. Pater (Academic Medical

Center, Amsterdam University) for the assistance with Facs analysis, Prof. Hernandes F.

Carvalho/Thaíse Machado Augusto (Department of Cell Biology, State University of

Campinas, Brasil) for western blotting analysis and Prof. Carlos Augusto Fernandes de

Oliveira (Zootecnia and Food Engineering Faculties, University of São Paulo) for

donating Fumonisin B1.

Summary

91

Summarizing discussion The principal aim of this thesis was to assess the MAPKs family status in

human leukaemia cells induced to undergo apoptosis and differentiation after drug

treatment, and to address the usefulness of targetting these pathways as a potential

avenue for combating disease. Special focus was given to natural products, as these may

these may be derived from the Amazon jungle. A knowledge-based industry dwelling

from such products may form the basis for sustainable economic exploitation of the

Amazon forest. To assess whether I have been able to make progress in addressing this

issue, let us review the subsequent chapters in this thesis:

Chapter 1 constitutes a brief introduction about the general overview on

Apoptosis and Mitogen-activated protein kinases (MAPKs). The former and the latter

emerge as important targets for drug intervention and will be the major cellular

pathways investigated in the experimental work and be the subject for discussion in

chapter 4. Together, the available literature is consistent with a role of both MAPKs and

apoptosis as targets for drugs in leukaemia. Evidence that they may act as thus in

practice was obtained in the following chapter:

In chapter 2, the effect of tetrahydroxyquinone (THQ), a highly redox active

molecule which can participate of a redox cycle with semiquinone radicals leading to

the formation of reactive oxygen species (ROS), was investigated in HL60 cells. THQ

caused substantial ROS formation followed by cytotoxicity that was sensitive to

glutathione and N-acetyl-L-cysteine. Furthermore, ROS production coincided with

HL60 cell apoptosis through the mitochondrial pathway and was followed by reduced

activity of various anti-apoptotic survival molecules, including the protein kinase B

pathway. Importantly, transfection of protein kinase B into HL60 cells leading to an

artificial increase in protein kinase B activity inhibited ROS-dependent cytotoxicity. In

a broader context this work shows that specific interference of signalling pathways for

clinically potentially beneficial effects is possible. Nevertheless, this work also shows

the limitation of the candidate approach when searching for mechanisms for drug

action. Hence, a more comprehensive approach is called for, and this will be the subject

of chapter 3.

Summary

92

In chapter 3, peptide arrays of 1176 different kinase consensus substrates

unambiguously identified the MAP kinase pathway as a major target for violacein, the

anti-leukemic purple-colored pigment produced by Chromobacterium violaceum from

the Amazon River that stimulates HL60 cells to differentiate into monocytes and

granulocytes . The results also indicate that kinome profiling using metabolic arrays is a

promising and suitable tool for studying the molecular mechanisms of drug action

without a priori assumptions as to the underlying molecular mechanism. Violacein,

however, has many side effects focussing our attention on other candidate drugs. This

prompted is a literature investigation into riboflavin as a potential drug in leukaemia,

and its investigation is discussed in the next chapter.

In chapter 4, it is shown that irradiated riboflavin-dependent cytotoxicity in

HL60 cells occured concomitantly with activation of caspase 8 caused by

overexpression of Fas and FasL and also stimulation of ceramide. The activation of this

cascade led to an increase of JNK activity and inhibition of survival mediators (PKB

and IAP1), as well as downregulation of cell cycle progression regulators. Taken

together, these results provide a molecular approach characterizing the riboflavin-

mediated apoptosis after photodynamic therapy.

Appendix 1 is a review article, originally published in Portuguese about

riboflavin, the subject of chapter 4 and a component of the vitamin B2 complex, that has

important roles in biochemistry, especially in redox reactions due to its ability to

participate in both one- and two-electron transfers, as well as acting as photosensitizer.

This article describes the peculiar and multifunctional behavior of riboflavin which

allows it to take part in several biochemical pathways both as nucleophile and

electrophile. Moreover, the association of this vitamin with different diseases, including

cancer and cardiovascular diseases, is also discussed. Its usefulness for combating

cancer is also addressed.

Together this work shows that investigation of the molecular mechanisms

mediating drug action in leukaemia is a fruitful approach which may greatly aid the

development of novel therapeutic avenues of disease. The evidence obtained was

consistent with the central hypothesis when the work was initiated: both MAPKs and

apoptosis are bona fide targets for drug intervention in leukaemia, at least as deduced

from these in vitro experiments. Further in vivo work is obviously necessary.

Summary (dutch)

93

Summary (dutch)

In dit proefschrift wordt het nut van natuurlijke componenten onderzocht als mogelijke

behandel wijze voor leukemie (oftewel bloedkanker). Met name heeft het onderzoek

zich gericht op de effecten van dergelijke substanties op MAP kinases (een familie van

essentiële regulatoren van de cellulaire fysiologie), op geprogrammeerde celdood en op

differentiatie (het verkrijgen van een gespecialiseerd niet-delend fenotype van de anders

wat onbestemde en snel profilerende leukemie cellen). Dat er in dit proefschrift gekozen

is voor het werken met natuurlijke componenten heeft alles te maken met de

aanwezigheid van het reusachtige Amazone oerwoud in Brazilië (de schrijver van dit

proefschrift is Braziliaan). Momenteel wordt dit gigantische bos voornamelijk gebruikt

voor laagwaardige economische activiteiten met een slecht lange termijnperspectief

zoals houtkap. Het oerwoud herbergt echter ook vele interessante biologisch actieve

stoffen. Indien in Brazilië een industrie zou ontstaan die zich specialiseert ion het

ontdekken van mogelijke klinische toepassingen van dergelijke stoffen zou dat een

belangrijke rol kunnen gaan spelen in de pogingen het oerwoud ook voor latere

generaties te bewaren. In het onderhavige proefschrift kijken we in dit kader naar

leukemie.

Na een introductie over moleculaire aspecten van de leukemische ziekte wordt er in

hoofdstuk twee gekeken naar de effect van tetrahydroxyquinon. Dit is een middel dat

reeds wordt gebruikt voor de behandeling van staar, maar in dit hoofdstuk laat ik zien

dat het ook celdood van een modelcel voor leukemie veroorzaakt. Het moleculaire

mechanisme van dit effect werd ontrafeld: iedere cel in ons lichaam heeft de neiging om

spontaan te sterven maar wordt in actief in level gehouden door zogenaamde “survival”

signalen. Het bleek tetrathydroxyquinon interfereert met deze laatste signalen en op

deze wijze de kankercel van haar lust tot leven beroofd. Het gevolg is dat zij zich zelf

ten gronde richt.

In hoofdstuk drie wordt een nieuwe methode gebruikt om de werking van mogelijke

geneesmiddelen te testen. Mijn vriend Sander Diks heeft onlangs zogenaamde peptide

arrays gesynthetiseerd welke substraten bevat voor alle kinases uit het menselijk

genoom. Ik heb deze arrays gebruikt om de werking van geneesmiddelen te testen en ik

Summary ( dutch)

94

laat in dit hoofdstuk een volledige beschrijving zien van de effecten van violacein op de

fysiologie van de leukemiecel. Violacien komt uit het oerwoud en wordt gebruikt door

de indianen om ontstekingsziekten te bestrijden. Het trekt nogal de aandacht vanwege

haar anti-leukemische werking. In dit hoofdstuk laat ik zien dat violaceine de

zogenaamde p42/p44 MAP kinase weg aanschakelt wat haar beurt dan weer leidt to

differentiatie van de cellen en exit uit de celcyclus.

In hoofdstuk vier en appendix 1 tenslotte wordt bestraald riboflavine onder de loupe

genomen. Riboflavine wordt gebruikt als een zogenaamde fotosensitizer bij het

bestrijden van leukemie. De gedachte was altijd dat zonlicht samen met riboflavine

zuurstofradicalen zou vrijstellen, die dan de tumorcel het leven zuur zouden maken. Het

blijkt echter dat voorbestraling van riboflavine een product oplevert dat de kankercel

dood. Het moleculaire mechanisme lijkt een activatie zijn van het zogenaamde Fas/FasL

geprogrammeerde celdood te zijn. Mogelijk zou dit op termijn betekenen dat patiënten

die behandelt worden met fotosensitizers makkelijker in de zon kunnen komen.

Samen laten deze studies dat natuurlijke producten inderdaad een aantrekkelijke kunnen

zijn voor het ontwerpen van nieuwe therapie voor leukemie. Tevens wordt ook duidelijk

dat het mogelijk is om het moleculaire mechanisme van werking te achterhalen.

Curriculum Vitae

95

Curriculum Vitae ALEXANDRE DONIZETI MARTINS CAVAGIS was born in April 22, 1974 in

Piracicaba, State of São Paulo, Brazil, where he spent his whole childhood. In 1993,

started the chemistry college at the State University of Campinas (UNICAMP) which is

one of the most distinguished Brazilian universities. In 1994, developed his first

research project, under supervision of Dr. Sérgio Gama, at the Physics Institute, using

the X-ray diffraction technique to study metallic policrystalline aggregates, especially

the behavior of iron-lanthanide (Fe-Nd) alloys under partial nitrogenation. In 1995,

worked at the Department of Physical Chemistry under supervision of Dr. Fernando

Galembeck in microscopic studies related to effects of electric fields on oil/water/rock

interfaces, applied to improve underwater petrol recovery. In 1996, started working at

the Department of Biochemistry with Dr. Hiroshi Aoyama, developing projects related

to plant acid phosphatases. The same year, he was employed to teach chemistry at the

secondary school "Colégio Luiz de Queiroz", where he holds the position of titular

teacher during the last 11 years. In 1997, received the "Merit Diploma" from the

Regional Council of Chemistry and the "Lavoisier Award" (medal) of the status of best

undergraduation student from 1994 to 1997 at the Chemistry Institute of the State

University of Campinas. After finished the chemistry college (1997), he was admitted in

the Master's Programme (1998) and developed a project on studies of conformational

changes and its repercussions on the activity of soybean seed acid phosphatases.

Thereafter, in 2003, started a research related to the apoptosis of human leukemia cells

at the Laboratory of Signal Transduction, under supervision of Prof. Dr. Carmen

Veríssima Ferreira and, the same year, went to the University of Amsterdam to test new

potential inductors of apoptosis in human leukemia, at the Laboratory of Internal

Medicine of the Academic Medical Center, under supervision of Dr. Maikel P.

Peppelenbosch. At present, he is collaborating with Dr. Carmen and Dr. Hiroshi at the

Department of Biochemistry of UNICAMP in research and teaching. Moreover, he is

professor of Chemistry at the Engineering School of Piracicaba (EEP/FUMEP),

teaching Chemistry Applied to Engineering and, since March 2006, holds the position

of professor of Biochemistry at the Methodist University of Piracicaba (UNIMEP).

Curriculum Vitae

96

Word of thanks

97

Word of thanks

Firstly, thanks God for granted strength to overcome this most important stage

in my life with health. To my family, especially to my daughter, mother and my wife for

the incentive during all the period of this doctorate;

To the University of Amsterdam, University of Groeningen and State

University of Campinas for the great academic background acquired, and for the

possibility to carry out both the experimental and theoretical parts of this work;

To my promoter Prof. Dr. Maikel P. Peppelenbosch for the opportunity to

come to the Netherlands, for the excellent reception provided to me at the Laboratory of

Internal Medicine of the Academic Medical Center, University of Amsterdam, as well

as for all the help and essential suggestions during the execution of the experiments and

also throughout the preparation of the manuscripts. For all his efforts in order to achieve

the publication of our articles in high standard journals.

To my co-promoter, Prof. Dr. Carmen Veríssima Ferreira, whom was crucial in

my evolution as researcher, since my undergraduation and for the privilege to share the

same laboratory with her. In spite of all dificulties we face in a country with limited

resources to research, I am sure we have much to celebrate.

To Prof. Dr. Hiroshi Aoyama for the incentive during my first steps in

biochemical research since I was still coursing the Chemical College, and for the

supervision throughout the Masters graduation in plant Enzimology;

Word of thanks

98

To all colleagues from the Laboratory of Internal Medicine, especially

Liudmila, Francesca, Sander and all the technicians, whom had a fundamental role in

the quality of the results here presented;

To all friends from the Laboratory of Enzimology and Laboratory of Signal

Transduction of the State University of Campinas for the pleasant times we had during

all this period;

To Ana Carolina and Karla for the great assistance on bibliography and also for

the orientations in the theoretical part of Cell Biology which is a little difficult for a

Chemist (sometimes I guess quantum mechanics easier than cell signaling…);

To Ms. Greetje Noppert for the great attention and all the people whom worked

in the printing of this thesis;

To all the the rest of people whom contributed direct or indirectly for the

realization of this work

My sincere gratitude.

Publications

99

Publications

Cavagis, A. D. M. ; Ferreira, C. V. ; Versteeg, H. H. ; Assis, C. F. ; Bos, C. L. ;

Bleuming, S. A. ; Diks, S. H. ; Aoyama, H. ; Peppelenbosch, M. P.

Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival

signaling. Experimental Hematology 34: 188-196, 2006.

Souza, A. C. S. ; Kodach, L. ; Gadelha, F. R. , Bos, C. L. ; Cavagis , A. D. M. ; Aoyama,

H. ; Peppelenbosch, M. P. ; Ferreira, C. V. A promising action of riboflavin as mediator

of leukaemia cells death. Apoptosis, article in press, 2006.

Ferreira , C. V. ; Diks, S. H. ; Milano, F. ; Bos, C. L. ; Cavagis , A. D. M. ; Justo, G. Z. ;

Duran, N. ; Peppelenbosch, M. P. Kinome profiling for studying the effects of violacein

in human leukaemia cells. Submitted, 2006.

Souza, A. C. S. ; Cavagis , A. D. M. ; Ferreira , C. V. ; Juca, M. B. ; Peppelenbosch, M.

P. ; Aoyama, H. Riboflavina: uma vitamina multifuncional. Química Nova 28: 887-891,

2005.

Cavagis , A. D. M. ; Granjeiro, P. A. ; Ferreira, C. V. ; Aoyama, H. Effect of chaotropic

agents on reversible unfolding of a soybean (Glycine max) seed acid phosphatase.

Phytochemistry 65: 831-836, 2004.

Granjeiro, P. A. ; Cavagis , A. D. M. ; Leite, L. C. ; Ferreira, C. V. ; Granjeiro, J. M. ;

Aoyama, H. The thermal stability of a castor bean seed acid phosphatase. Molecular and

Cellular Biochemistry 266: 11-15, 2004.

Granjeiro, P. A. ; Ferreira , C. V. ; Cavagis , A. D. M. ; Granjeiro, J. M. ; Aoyama, H.

Essential sulfhydryl groups in the active site of castor bean (Ricinus communis) seed

acid phosphatase. Plant Science 164: 629-633, 2003.

Publications

100

Ferreira , C. V. ; Cavagis , A. D. M. ; Taga, E. M. ; Aoyama, H. Endogenous lectin as a

possible regulator of the hydrolysis of physiological substrates by soybean seed acid

phosphatase. Phytochemistry 58: 221-225, 2001.

Cavagis , A. D. M. ; Ferreira , C. V. ; Milano, F. ; Bos, C. L. ; Justo, . Z. ; Duran, N. ;

Peppelenbosch, M. P. Natural product from Amazonian Region causes an expressive

myeloid leukemia cell differentiation. FEBS Journal 272: 438-438, 2005.

Ferreira, C. V. ; Cavagis , A. D. M. ; Taga, E. M. ; Aoyama, H. Characterization of

cytoplasmic soybean seeds acid phosphatases using inorganic pyrophosphate as

substrate. FASEB Journal, 11: 1709-1709, 1997.

Appendix 1 - Riboflavin: a multifunctional vitamin

101

Appendix 1

Riboflavin: a multifunctional vitamin

Ana Carolina Santos de Souza, Carmen Veríssima Ferreira,

Marilena Bezerra Jucá and Hiroshi Aoyama*

Departamento de Bioquímica, Instituto de Biologia, Universidade

Estadual de Campinas, CP 6109, 13.083-970, Campinas, SP, Brazil

Alexandre D. Martins Cavagis and Maikel P. Peppelenbosch

Department of Cell Biology, University of Groningen, Antonius

Deusinglaan 1, NL-9713 AV, Groningen, The Netherlands

*e-mail: [email protected]

Quimica Nova (2005) 28: 887-891.

**Article published in Portuguese


102

ABSTRACT Riboflavin, a component of the B2 vitaminic complex, plays important roles in

biochemistry, specially in redox reactions, due the ability to participate in both one- and

two-electron transfers as well as acting as photosensitizer. Accordingly, low intakes of

this vitamin have been associated to different diseases, including cancer and

cardiovascular diseases. Riboflavin is thought to contribute to oxidative stress through

its capacity to produce superoxide but, interestingly, it can also promote hydroperoxides

reduction. This peculiar and multifunctional behavior allows riboflavin to take part in

various biochemical pathways as nucleophile and electrophile, turning it into a versatile

and important biological compound.

Keywords: riboflavin,; electron transfer; oxidative-stress.

INTRODUCTION

The riboflavin was isolated (although not purified) for the first time in 1879 by

the English chemist A. Wynter Blyth. In his studies related to the composition of the

cow milk he reported the identification of a brilliant yellow pigment which he called

"lactochrome" and which nowadays we know as riboflavin. After that announcement,

almost fifty years passed before some significant new development had occurred with

the so recently discovered yellow-orange pigment. Then, in the late 1920s and early

1930s, a great scientific evolution took place and similar yellow pigments with a

greenish fluorescent shine were isolated from a wide variety of sources. The interest for

those compounds had eventually become even greater when the such yellow pigment

was recognized as a constituent of the vitamin B complex, whereas even the isolation of

the vitamin itself was helped enormously by the realization that the potency of the

vitamin was correlated with the green fluorescence. Some of the foremost chemists of

the time as Richard Kuhn in Heidelberg and Paul Karrer in Zurich engaged in a tough

race to determine the structure and prove it through chemical synthesis, and they both

succeeded almost concurrently. Several names were proposed for the so characterized

compound such as lactoflavin and ovoflavin in order to establish correlation with the


103

source through which the compound had been isolated. But the prefix ribo was

consecrated in the name because of the ribityl side chain and the characteristic yellow

colour originated from the π conjugated system in the isoalloxazine rings1-3.

RIBOFLAVIN AND ITS BIOLOGICAL FUNCTIONS

Riboflavin, 7,8-dimethyl-10-ribityl-isoalloxazine, is a water-soluble vitamin

belonging to the vitamin B2 comp lex, presents yellow color and is fluorescent. Besides

the milk which as mentioned was one of the first sources, riboflavin is also found in

meat, fish and especially in dark-green vegetables4. The riboflavin originating from the

diet is found in the form of the coenzymes flavin adenine dinucleotide (FAD) and flavin

mononucleotide (FMN), bound to proteins. However, when the nourishment reaches the

stomach, the gastric juice propitiates the release of these coenzymes. Afterwards, the

free coenzymes undergo dephosphorylation catalyzed by pyrophosphatases and

phosphatases present in the small intestine, thus releasing the riboflavin. As observed in

our laboratory, riboflavin is an important product of the reactions catalyzed by the low

molecular mass acid phosphatases (protein tyrosine phosphatases) purified from bovine

kidney5 and lung6, whereas FMN is a potential physiological substrate for these

enzymes. Once released, riboflavin can be absorbed via active or passive transport6,7,8 .

Riboflavin is a molecule of fundamental importance in aerobic organisms

being precursory of important coenzymes which take part in the electron-tranfer chain,

such as FAD and FMN4,7,8 . It also originates many of the flavins which are associated to

several enzymes that take part in the catalysis of a great number of important

biochemical processes such as, for instance, reactions related to DNA repair. The

structure of riboflavin is shown in Figure 1.


104

Figure 1. The structure of riboflavin (RF). The ribityl chain and the π conjugated isoalloxazine

ring system are outlined with dashed lines

The metabolism of lipids as well as the degradation of drugs and other

exogenous compounds (xenobiotics) via the microsomal hydroxylation system demand

riboflavin derivates. Riboflavin cofactors are required for the folic acid, pyridoxine and

niacin metabolisms and they are also associated to enzymes in erythrocytes such as

glutathione reductase, an important enzyme which participates of pathways related to

protection against reactive oxygen species (ROS) yield in the cells 9.

Interestingly, riboflavin may contribute both to increase and inhibit the

oxidative stress due to its dual ability to produce superoxide and, conversely, be

involved in hydroperoxide reduction1.

Finally, recent studies have been correlating the riboflavin to the signal

transduction mechanism of apoptotic cells and also to the regulation of the biological

clock 1, 10.

N

N

NH

N

O

O

O

OH OH

OH

H

1

2

3456

7

89 10

1' 2'3'

4'5'

Sistema de anéis Isoaloxazina

Cadeia RibitilRibityl Chain

Isoalloxazine Ring System


105

Consequences of alimentary deficiency

Currently, recommended riboflavin intakes vary from 0.4 mg (in the

childhood) to 1.3 mg/day for adults and, for pregnant women, a supplementary dose of

0.3 mg/day is recommended during the gestation and 0.5 mg /day during the nursing

period, since studies have been showing that during the third quarter of gestation there

is a progressive reduction in riboflavin levels 11,12.

As it participates of several important redox reactions of the metabolism in the

form of cofactors FMN and FAD which act as electron carriers, inadequate riboflavin

intakes might lead to disorders in the intermediary metabolism. In mice, riboflavin

deficiency was associated to a tissue-specific reduction in the succinate oxidorreductase

(succinate dehydrogenase) activity, an effect which might have implications in energy

production through the oxidative phosphorylation. Besides, the β-oxidation of fatty

acids is also dependent upon flavins as electron acceptors13.

In animal models, studies demonstrated that riboflavin deficiency is related to

abnormal developments in the fetus and, in humans, several studies correlate the

riboflavin deficiency with hematological pictures, mainly with those related to the

hematopoietic system. At the moment, the hematological influence of riboflavin

deficiency has been associated to its interference in iron metabolism, since the

mobilization of iron from the intracellular protein ferritin is a reductive process and

reduced flavins might act reducing the iron present in ferritin thus mobilizing it from

several tissues at physiologically relevant concentrations. Neurodegeneration-related

symptoms and neuropathy have been reported in several studies of riboflavin-deficient

diets in different species, although there be little information regarding the relevance of

those results in humans. Nevertheless, it is known that riboflavin has a function in the

metabolism of thyroxine so that riboflavin deficiency might contribute to the

physiopathology of some mental diseases14-16.

Researches on public health report the importance of riboflavin as a protection

factor against cardiovascular diseases and tumoral processes. Interesting works

published during the 1970s and 1980s indicated that riboflavin might present protective

effects against tissue damages caused by oxidation. Due to the non-toxicity, riboflavin

is a possible candidate as reducing agent of the iron from the heme group of proteins,

maybe acting as a protective agent of oxidative damages in certain tissues . However, the


106

therapeutic potential of this vitamin should still be thoroughly investigated, since most

of the studies to the present moment was accomplished in animal models 6.

With regard to the visual system, it is known that riboflavin is a normal

component of the human eye lenses and it presents a very strong photosensitizer activity

when exposed to light. Thus, radiations with wavelengths below 400 nm could lead to a

luminous excitation which might cause, in certain circumstances, damages to the

eyesight. Vascularization and opacity of the cornea and even cataract have been

reported in animals fed with diets poor in riboflavin. Riboflavin-dependent

photoreceptors (cryptochromes) are believed to play an important role in darkness

adaptation and, therefore, a riboflavin-rich diet could influence in the darkness

adaptation through these photoreceptors either by interaction with vitamin A or maybe

in a vitamin A independent fashion17,18.

Moreover, riboflavin deficiency might interfere in the metabolism of other

nutrients, especially of other vitamins B such as folate, cyanocobalamin (vitamin B12)

and pyridoxine (vitamin B6)13.

Riboflavin and cancer The riboflavin deficiency has been correlated as a risk factor for cancer, even

though this fact was not still satisfactorily established in humans, so that the influence

of riboflavin-deficient to the genesis and progression of the cancer has been a

controversial theme in the scientific milieu where there are reports proving, both its

stimulatory action in certain cases and inhibitory in others. In general, riboflavin

deficiency seems to be related to the decrease of the development of spontaneous

tumors in experimental models in vivo but also, paradoxically, to the increase of the

carcinogenesis provoked by determined agents6.

The correlation between riboflavin and cancer is quite complex and it may be

exemplified by the countless effects of this vitamin on the metabolism of different

drugs. Since riboflavin is a precursory molecule of flavocoenzymes of the microsomal

hydroxylation system of drugs, its deficiency might retard the inactivation of

carcinogens and, therefore, increase their distribution to susceptible tissues. On the other

hand, if the drug has lesser carcinogenic activity than its metabolites, riboflavin

deficiency possibly could have an inhibitory effect on carcinogenesis. We should also


107

remember the same reasoning can be applied to chemotherapeutics in situations in

which they might, not only have their metabolism affected by the availability of

riboflavin-derived cofactors, but also have their own availability and cellular captation

directly affected, whereas riboflavin might complex with certain substances and also act

as a regulatory factor of the drug entrance into the cell. Antifolate drugs may also have

their effects influenced by the riboflavin availability since flavin cofactors are related to

the folic acid metabolism. It has been difficult, however, to obtain evidences that

support or refute those hypotheses and the researches now change the focus in order to

get data on the real availability of riboflavin in neoplastic cells. The neoplastic cells

apparently lose certain mechanisms which regulate the riboflavin metabolism in normal

cells and are characterized by resis tance to riboflavin deficiency which leads to the

maintenance of high concentrations of FAD through mechanisms still not

determined9,19,20.

Throughout the 1990s, a new approach was given to the studies on cancer

treatment and, once again, riboflavin took on a prominent role due to its peculiar

characteristic of photosensitive compound. Besides, it was demonstrated that riboflavin

may present an important function in the transport and controlled release of drugs. This

new aspect of the riboflavin utilization came up to broaden and consolidate significantly

the biological and physiological importance of this compound21-24.

Some fundamental chemical properties of riboflavin

Riboflavin, as well as the other flavins in general, presents a very interesting

characteristic for its biological role which is the capacity to participate in both two- and

one-electron transfer reactions which automatically implies the existence of

semiquinone1.

In free solutions, i.e., absence of any enzyme, a chemical equilibrium between

the oxidized and reduced riboflavin sets up (auto-redox) and in this process a certain

amount of radical is formed:


108

Scheme 1. Semiquinone equilibria of riboflavin. RF = riboflavin; ox = oxidized; red = reduced

Just to be clearer, in Scheme 1 we presented the oxidation (1) and reduction (2)

semi -reactions of riboflavin in free aqueous solution, whereas equation 3 corresponds to

the global process. In these conditions, both the equilibria 4 and 5 are very much to the

left, so that starting from an equimolar mixture of oxidized and reduced riboflavin, at

pH 7.0, only 5% of radical is stabilized. Nevertheless, it is important to emphasize that

the presence of enzymes makes possible a wide variety of oxidation states for the

riboflavin, since the activation energies of several redox reactions involving riboflavin

is significantly reduced in enzymatic systems. This versatility of oxidation states

enables one to monitor events which happen in catalysis using riboflavin as a marker,

since its reactions are always characterized by semi-reactions which can be

accompanied separately.

In most cases in which riboflavin is reduced, the process may occur through

two different manners. The first could be via a two-electron tranfer by a substrate (S1)

which might undergo dehydrogenation leading to the formation of a reduced flavin

which, in turn, could be subsequently reoxidized through dehydrogenation by another

substrate (S2), as illustrated in Scheme 2.

RFox

RFred

RFox + 2 RF.

H2

H2RFred H

RFH.

RF.

+ H+

(1)

(2)

(3)

RF +

-RF +

+ + 2e

2e-

2 RF + 2 H +

2 H

+2

+2

+2

(4)

(5)

RFred H2 + RFox

+

+


109

Scheme 2. Reactions involving the riboflavin oxidized and reduced forms. R = ribityl chain

The second way might be through a single-electron transfer leading to an

intermediate semiquinone. In some cases, molecular oxygen is the physiological

substrate but, due to the non-specific reactivity between O2 and flavins in general, the

study of some reactions should be carried out under anaerobic conditions1.

The scheme 3 shows some chemical reactions which might occur between the

reduced riboflavin and oxygen and some alternative mechanisms.

S1H2 RFox

RFredH2S1

S2H2

S2

+2


110

Scheme 3. Reactions of reduced riboflavin with oxygen. R = ribityl chain

The initial reaction is a reduction of O2 which occurs through a single-electron

transfer from the reduced riboflavin (A) to yield a caged radical pair formed by the

neutral flavin and the superoxide anion (B). The radical pair formed now has several

alternative routes. It can collapse into a flavin peroxide, a nucleophile, which on

protonation becomes the electrophilic hydroperoxide. The peroxide species may

eliminate hydrogen peroxide to yield oxidized flavin, or there may be a second one-

electron transfer from the radical pair to give the same products and he riboflavin

peroxide species may be involved in hydroxylation reactions or Baeyer-Villiger

N

N

NH

N O

R

H O

O2

N

N

NH

N

R

H O

OH+_

N

N

NH

N O

R

O.O2

_ _O2

.

N

N

NH

N

R

H O

O

OO

N

N

NH

N

R

O

O

Reações de Baeyer-Villige

FlH ou Fl. -.

_

N

N

NH

N

R

H O

O

OOH

H+

H2O2

HO2

O2_

_

O2

_

A

B

C

D

E

F

Hidroxilações Aromáticas

HO2

_

Baeyer-Villiger reactions

Aromatic hydroxylations

or


111

reactions which are oxidations of substrates containing ketonic and aldehydic groups to

form, respectively, esters and carboxylic acids. The third alternative route is the

dissociation of the radical pair into its components, riboflavin radical and superoxide.

The superoxide produced can react with peroxide to form hydroxyl radicals and with

nitric oxide to form peroxynitrite. Production of hydroxyl radicals is commonly

believed to be one of the major sources of oxidative stress and tissue damage, largely by

reaction with lipids to produce lipid hydroperoxides. Recent works also suggests that

peroxynitrite is an active agent in apotosis 1,25.

Riboflavin: An efficient photosensitizer As we had already mentioned, riboflavin plays an important biological role as

photochemical sensitizer. It presents, as main characteristics, sensitivity to both UV and

visible radiations26,27 so that, once absorbing light, it reaches the excited triplet state

which in turn can either react with molecular oxygen yielding singlet oxygen (which is

called the type II mechanism ) or act directly on a substrate leading to its photooxidation

with consequent formation of intermediate radicals (called type I mechanism) and also

of reactive oxygen species such as superoxide anion, hydroxyl and hydrogen

peroxide20,28. This capacity can be useful to the organism, for instance, in the case of

enzymes which require visible light for their catalytic activities such as enzymes which

participate in photosynthesis and enzymes related to DNA repair, DNA liases, whose

prostetic groups function as substrates for the excited riboflavin triplet29.

The photosensitizer capacity of riboflavin in biological systems is due to the

high redox potential of the activated triplet. Although the redox potential of riboflavin30

is only -0.3V at pH 7.0, its irradiation and activation to triplet state considerably

increases the redox potential to 1.7V, which is much higher than the redox potential of

important biomolecules such as amino acids, proteins, lipids and nucleic acids which,

thus, can undergo photodegradation in the presence of riboflavin31,32. In this way, after

excitation, the electron transfer from the biomolecules mentioned above to yield the

respective radicals and reduced riboflavin becomes thermodynamically favorable.


112

Irradiated riboflavin reacts with the amino acids tryptophan (TrpH), tyrosine

(TyrOH) and, in lesser extension, with phenylalanine (PheH) - [in this text, in order to

make the indications of chemical reactions clearer, we will use an abbreviation for the

amino acids different from the international three- or one-letter standard]. As a result of

the photooxidation of the mentioned amino acids, there are production of reactive

oxygen species and other cytotoxic photoproducts. In proteins, tryptophan and tyrosine

residues have redox potentials of 1.5V and 0.93V, respectively, which makes them

susceptible substrates to photooxidation reactions in presence of irradiated

riboflavin33,34. In those processes , besides the yield of photoproducts, there is also

formation of photoadducts through the reaction of excited riboflavin and its substrate as,

for example, in the case of the reaction between riboflavin and TrpH through which the

yield of photoadducts indole-flavin and indole -indole, among others, is observed 35.

Scheme 4 summarizes some important photosensitization reactions which

possibly occur involving tryptophan, tyrosine and riboflavin:

Scheme 4. Some important reactions leading to tryptophan and tyrosine

photosensitization, illustrating the role of riboflavin as a photosensitizer.

products

RF

3RF*

1O2

O2_.

RF+. / RF(-H).

O2

O2

oxidation

(Type II) minor

reaction

Trp / TyrO. .

(Type I) main reaction

3RF*RF

_.

hν

RFH.

RFH2

O2._

O2 / H2O2

TrpH / TyrOH

TrpH / TyrOH

..OTrp / Tyr

.

TrpH / TyrOH

products


113

The presence of TrpH and riboflavin has been correlated to hepatic

dysfunctions produced by parenteral nutrition36 and the photoproducts have been

demonstraiting to exert lethal effects on animal cell cultures 35,37.

Several studies have been revealing that irradiation of tumoral cells with

visible light in presence of riboflavin induces cell death. When teratocarcinoma cells F9

and NSO/2 murines were irradiated in a culture medium supplemented with TrpH and

riboflavin, morphologic alterations similar to those described for apoptotic cells were

observed. The cytotoxic effect was attributed to the formation of reactive oxygen

species such an as superoxide anion and singlet oxygen, produced through a relatively

complex mechanism (scheme 5) which is a combination of type I and type II

mechanisms 17,22.

Besides the production of ROS, other cytotoxic photoproducts were originated

from reactions involving the riboflavin anion radical and the indole cation radical of

TrpH. Such photoproducts are constituted, basically, of aggregated forms of riboflavin,

indole products associated to flavins, indole products with molecular weight higher than

TrpH, as well as products from the photodecomposition of this amino acid28.


114

Scheme 5. Combination of type I and type II mechanisms in presence of molecular

oxygen (S represents a target compound for the irradiated riboflavin)

The effects of the irradiated riboflavin in presence of TrpH are also observed in

non-irradiated cell cultures which do not undergo the deleterious effects of the transient

reactive oxygen species produced during the irradiation of the medium. Such effects are

attributed to the other photoproducts formed whose cytotoxicities are even more evident

in experiments accomplished in anaerobic atmosphere, in which predominates the type I

mechanism22,38. Photoproducts derived from the 3-indoleacetic acid irradiated in the

RFIrradiação oumecanismoenzimático

3RF*

O2 S

RF + 1O2 RF-. + S+. Fotoprodutos/Fotoadutos

RF -.O2

RFox + RFredH2RFox + O2.-

O2

RF + H2O2

H2O2

OH + OH + O2. -

Irradiation orenzymatic

mechanism

Photoproducts/Photoadducts


115

presence of riboflavin demonstrated to cause severe damages in culture of human HL60

cells and in tumoral NOS/237 murines cells 37. Such effects were more accentuated than

the previously described for the tryptophan photoproducts. Moreover, they increase

proportionally to the photoproducts concentration22. Based on electronic microscopy

studies and flow cytometry analyses it was demonstrated that such photoproducts

induce cell death through an apoptotic mechanism. It has been postulated that the

mechanism of apoptosis activation is related to the photoproducts I3A-RF functioning as

a death signalling on the cell surface. Such hypothesis is based on the fact that a certain

amount of riboflavin added to the middle is incorporated by the cell which could happen

similarly to the photoproducts.

Another important aspect on riboflavin action is its capacity to generate

cytotoxic effects even when not exposed to the irradiation. The excited triplet state may

also be reached in the absence of light through an enzymatic mechanism mediated by

peroxidases39, which explains the cytotoxic effects observed for the non-irradiated

riboflavin22.

At present, riboflavin and other substances with photosensitizing ability have

been studied in combination with light irradiation for the treatment and prevention of

diseases. In the oncological field it stands out the so called Photodynamic Therapy, a

type of photochemotherapy in which a photosensitizer substance is accumulated in a

tumoral tissue which is specifically illuminated and, subsequently, removed22.

Moreover, recent works have been demonstrating both in vitro as in vivo, apoptosis

induction by Photodynamic Therapy 40-42.

ACKNOWLEDGEMENTS

This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de

São Paulo - FAPESP (Ana Carolina S. de Souza/ process nº 2002/12539-7). The authors

are grateful to Prof. Dr. José de Alencar Simoni from Institute of Chemistry

(UNICAMP) for his suggestions.


116

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# SEQUENCE PH_SITE KINASE TARGET PROTEIN

radioactive counts after

24 hr vehicle (average) error rank

radioactive counts after

24 hr violacein

(average) error rank delta rank (588 half point)Significance level

1 HEYIYVDPM Y-581 SWISS;P09619;PGDR_HUMAN 14825 1928 657 1293 899 542 115 0,892 KRPSVRAKA SWISS;P02687;MBP_BOVIN 20298 10 163 86 108 28 135 0,103 EDNEYTARQ Y-416 v-Src SWISS;P00526;SRC_RSVP 12909 2307 864 1586 1021 841 23 0,864 ENKLYGMSD Y-1062 SWISS;P07949;RET_HUMAN 13414 902 807 854 67 667 140 0,815 HRLLTLDPV T-556 PKC SWISS;P13504;IL1R_MOUSE 18428 6251 287 3269 4217 387 -100 0,706 LEKKYVRRD Y-706 c-Fms SWISS;P09581;KFMS_MOUSE 23458 8353 75 4214 5853 704 -629 0,05 �7 PYKFPSSPLRIPGZ na na na 19601 445 205 325 170 989 -784 0,218 HTRDSEAQR S-9 PKA SWISS;P27105;BAN7_HUMAN 12783 4318 871 2594 2437 798 73 0,989 MAEVSWKVL S-11 SWISS;P12798;KPBB_RABIT 16515 7065 466 3766 4666 320 146 0,98

10 EEGISQESS S-87 SWISS;P17095;HMGY_MOUSE 13327 4290 819 2555 2454 733 86 0,9711 EQQQTEDEL T-56 CKII SWISS;P02666;CASB_BOVIN 9135 517 1114 815 422 805 309 0,02 �12 IGEGTYGVV T-14 SWISS;P24941;CDK2_HUMAN 11213 832 1015 924 129 495 520 0,0513 MMTPYVVTR Y-185 SWISS;P45983;JNK1_HUMAN 13946 3673 758 2215 2061 140 618 0,04 �14 PYKFPSSPLRIPGZ na na na 16403 3951 481 2216 2454 175 306 0,6015 ILDTTGQEE T-59 transformingSWISS;P01117;RASK_MSVKI 12000 2353 946 1650 995 605 341 0,4316 NDSNYIVKG Y-809 SWISS;P07333;KFMS_HUMAN 12389 1136 906 1021 162 240 666 0,2817 EFLRTSAGS T-172 AMP-PK SWISS;P54645;AAK1_RAT 10580 5072 1050 3061 2844 428 622 0,3918 ERTNSLPPV S-1617 PKA SWISS;P07293;CIC1_RABIT 12642 1655 883 1269 546 278 605 0,2619 IRRASTIEM S-16 PKA SWISS;P07473;PPLA_PIG 11999 2437 947 1692 1053 304 643 0,5420 NFDDYMKSL Y-19 SWISS;P07483;FABH_RAT 16602 1084 452 768 447 182 270 0,2021 PYKFPSSPLRIPGZ na na na 12788 2098 870 1484 868 463 407 0,5922 KIQASFRGH S-34 PKC SWISS;P35722;NEUG_BOVIN 17529 1319 353 836 683 44 309 0,0623 NKGASQAGM S-215 SWISS;P26932;CLPO_CHICK 11393 1185 1002 1094 129 516 486 0,4424 EIVESLSSS S-30 SWISS;P02666;CASB_BOVIN 9640 237 1093 665 605 347 746 0,1625 ESSNYMAPY Y-771 SWISS;P09619;PGDR_HUMAN 12090 2620 941 1780 1187 490 451 0,3626 KRPSKRAKA SWISS;P02687;MBP_BOVIN 16406 5477 480 2979 3534 276 204 0,8927 NRAITARRQ T-11 PhK SWISS;P02643;TRIF_RABIT 15921 5809 526 3167 3736 520 6 0,8128 PYKFPSSPLRIPGZ na na na 16226 4363 495 2429 2735 573 -78 0,6329 GKRQTEREK T-199 PKC SWISS;P13789;TRT1_BOVIN 19310 5542 222 2882 3762 242 -20 0,7530 APATSPKAE S-96 SWISS;P07936;NEUM_RAT 23613 8638 70 4354 6059 22 48 0,7031 DEEESEQGA S-444 SWISS;P09652;TBB4_CHICK 15744 2237 542 1389 1198 174 368 0,2732 FFRRSKIAV S-683 PKC SWISS;P19491;GLR2_RAT 19928 4795 191 2493 3255 241 -50 0,6333 GMTEYVATR Y-182 SWISS;P16892;FUS3_YEAST 16343 2709 486 1597 1572 591 -105 0,4534 EAALYKNLL Y-703 SWISS;P10721;KKIT_HUMAN 18143 8333 302 4317 5678 1000 -698 0,43

35 ELILSPRSK S-24 MAPK SWISS;P16949;STHM_HUMAN 20633 5271 144 2708 3626 95 49 0,9036 GQEVYVKKT Y-992 SWISS;Q02763;TIE2_HUMAN 19199 2071 232 1152 1300 116 116 0,5037 AQETSGEEI T-194 SWISS;P08833;IBP1_HUMAN 14339 4255 720 2488 2500 916 -196 0,5438 DKAKSRPSL S-338 PKA SWISS;P19112;Q64594;F16P_RAT 18652 4987 274 2630 3332 354 -80 0,6039 FPVSYSSSG Y-687 SWISS;P07949;RET_HUMAN 13916 24 762 393 522 200 562 0,5640 GRLSSMAMI S-491 CaM-II SWISS;P11799;KMLC_CHICK 15074 1083 626 854 323 414 212 0,6741 EEDLSDENI S-720 CKII SWISS;P14164;BAF1_YEAST 14236 2292 732 1512 1103 685 47 0,8342 EPGPYAQPS Y-221 c-Abl SWISS;P46108;CRK_HUMAN 12759 1446 873 1160 405 485 388 0,3943 GRTGRRNSI S-8 PEPTIDE DERIVED FROM PKI (14-22) 13797 530 775 652 174 308 467 0,03 �44 ATPIYLDIL Y-859 SWISS;Q03351;TRKC_RAT 12721 399 877 638 338 462 415 0,4245 DMRQTVAVG T-432 PKC SWISS;P04720;EF11_HUMAN 9513 4269 1101 2685 2240 596 505 0,3946 GDRFTDEEV T-134 CKII SWISS;P02612;MLRM_CHICK 13170 2155 838 1497 931 603 235 0,7747 GSGSSVTSR S-38 PKC SWISS;P02542;DESM_CHICK 11798 2399 965 1682 1014 645 320 0,7648 EEPQYEEIP Y-324 c-Src SWISS;P03079;TAMI_POVHA 12108 2454 935 1695 1074 760 175 0,8049 ERRLSLVPD S-737 PKA SWISS;P13569;CFTR_HUMAN 11150 3159 1018 2088 1514 218 800 0,1450 GSRPSESNG S-243 PKA SWISS;P06624;MIP_BOVIN 10302 3852 1063 2457 1972 758 305 0,5151 DAHKSKRQH S-624 SWISS;P14164;BAF1_YEAST 12714 3320 878 2099 1727 570 308 0,6352 DSLIYDDGL Y-1029 SWISS;P07949;RET_HUMAN 12003 2421 945 1683 1044 373 572 0,2453 GGRDSRSGS S-160 PKA SWISS;P02687;MBP_BOVIN 13025 3618 853 2236 1955 620 233 0,7854 GVERSVRPT S-972 PhK SWISS;P18688;KPB1_RABIT 14249 5940 731 3336 3684 787 -56 0,7955 EGVKSDQAE S-536 SWISS;P12839;NFM_RAT 13972 5416 755 3086 3296 724 31 0,8856 ESLSSSEES S-33 CKI SWISS;P02666;CASB_BOVIN 14554 6363 690 3526 4011 659 31 0,8757 RRAVSEQDA S-19 CaM-II SWISS;P04177;TY3H_RAT 13542 5904 794 3349 3614 285 509 0,5658 SEDNSEDEI S-29 CKII SWISS;P11497;P97902;COAC_RAT 15421 2769 587 1678 1543 483 104 0,8459 PASLSRAKA S-352 SWISS;P19836;CTPT_RAT 15492 4649 574 2612 2882 723 -149 0,6360 RRASL SWISS;P12928;P04763;Q64618;KPYR_RAT15201 5185 613 2899 3233 1035 -422 0,4061 AKKMSTYNV S-315 myosin SWISS;P19706;MYSB_ACACA 20362 2893 159 1526 1934 238 -79 0,4462 DDINSYEAW S-114 SWISS;P00740;FA9_HUMAN 24144 550 63 307 344 670 -607 0,1363 ETRFTDTRK T-57 CaM-III SWISS;P13639;EF2_HUMAN 22654 1030 91 560 664 187 -96 0,4264 RRRASQLKV S-434 PKA SWISS;P18505;Q16166;GAB1_HUMAN 16526 1971 464 1217 1065 918 -454 0,1365 SGADYPDEL Y-154 SWISS;P07477;TRY1_HUMAN 13023 283 854 568 404 821 33 0,2166 KQISVR S-15 SWISS;P11217;PHS2_HUMAN 26527 1479 27 753 1027 38 -11 0,7767 SLKDH S-1 PKC SWISS;P00340;LDHM_CHICK 13764 3403 779 2091 1856 717 62 0,8968 APRTAGGRR SWISS;P02687;MBP_BOVIN 16174 5701 500 3101 3678 964 -464 0,4469 DGHEYIYVD Y-579 SWISS;P09619;PGDR_HUMAN 13740 2133 781 1457 956 818 -37 0,5570 FKRSYEEHI Y-1355 INSR SWISS;P06213;INSR_HUMAN 14114 925 742 833 129 577 165 0,9271 RRSSSRPIR S-8 PKA SWISS;P02337;PRT3_CLUPA 14265 1940 729 1334 856 707 22 0,8372 SKIGSTENL S-262 GSK3 PIR;2144820 12126 950 932 941 12 731 201 0,4673 NDMTSL S-283 tropomyosinSWISS;P02558;TPMA_RABIT 9655 2599 1092 1845 1065 582 510 0,2474 AARLSLTDP S-191 PKA SWISS;P13224;GPBB_HUMAN 10058 2135 1080 1607 746 861 219 0,5675 ARRSTTDAG T-117 PKA SWISS;P01233;CGHB_HUMAN 9021 405 1120 763 505 1130 -10 0,92

76 DLKDTKYKL T-1823 MHCK SWISS;P08799;MYS2_DICDI 16575 1301 456 879 598 102 354 0,0877 GAFSTVKGV T-489 RhK SWISS;P28327;RK_BOVIN 15931 1414 524 969 629 152 372 0,1278 RVRMSADAM S-117 PKA SWISS;P02643;TRIF_RABIT 7053 2056 1163 1609 631 960 203 0,4579 SNPTYSVMR Y-250 c-Src SWISS;P12906;TAMI_POVMC 10318 980 1061 1021 57 393 668 0,0980 SSKRAK S-2 PKC SWISS;P02612;MLRM_CHICK 14487 2491 707 1599 1262 429 278 0,7181 AGDGSDEEV S-1944 SWISS;P35579;MYSN_HUMAN 13013 4669 855 2762 2697 507 348 0,7082 AVDRYIAIT Y-132 SWISS;P04274;B2AR_MESAU 14543 4393 695 2544 2615 406 289 0,8183 DPLLTYRFP T-480 PKC SWISS;P02545;LAMA_HUMAN 14855 8280 654 4467 5392 470 184 0,9884 GENIYIRHS Y-627 EGFR SWISS;P11171;41_HUMAN 16536 5847 462 3155 3808 275 187 0,9185 QLSTSEENS S-146 CKI SWISS;P02663;CAS2_BOVIN 12839 5134 869 3001 3016 310 559 0,6286 SPRKSPKKS S-18 sperm-specificSWISS;P02256;H1_PARAN 15682 7760 550 4155 5099 43 507 0,3187 RRKASGP S-35 SWISS;P02253;H11_BOVIN 12982 4127 858 2492 2311 345 513 0,6788 RQLRSPRRT S-199 p34cdc2 SWISS;P20338;RB4A_HUMAN 15596 2162 562 1362 1131 1006 -444 0,1589 SAVASNMRD S-294 GRK SWISS;P08172;ACM2_HUMAN 13355 1484 812 1148 475 1149 -337 0,0790 RTPPPSG SWISS;P02687;MBP_BOVIN 14887 1276 649 962 443 1173 -524 0,1691 LRRAS S-5 PKA BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)18613 4028 279 2154 2651 1151 -872 0,1192 REARSRAST S-44 GSK3 SWISS;P00515;KAP2_BOVIN 14890 6655 648 3651 4247 1107 -459 0,4293 DRVYVHPF ANGIOTENSIN II 17241 4996 388 2692 3259 538 -150 0,6194 VRRSDAA SWISS;P02646;TRIC_RABIT 13145 3290 842 2066 1731 1079 -237 0,3595 RRKMSRGLP S-687 PKA SWISS;P07293;CIC1_RABIT 15068 2414 628 1521 1263 254 374 0,3696 SEVPYREVQ Y-547 SWISS;P00528;SRC1_DROME 12366 151 909 530 536 845 64 0,8997 PRRASATSS S-116 PKA SWISS;P13834;UGS1_RABIT 11834 410 962 686 390 837 125 0,3298 RRLSI S-1018 PKA SWISS;P18688;KPB1_RABIT 12975 1410 859 1134 390 940 -81 0,4599 RGRASSHSS S-403 PKC SWISS;P02545;LAMA_HUMAN 11303 499 1009 754 361 811 198 0,55

100 RKRSRAEF SWISS;P02278;H2B_HUMAN 14544 3536 693 2114 2010 619 74 0,85101 LRAASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)9249 7686 1109 4397 4650 963 146 0,77102 RRRQSVLNL S-768 PKA SWISS;P13569;CFTR_HUMAN 13206 1283 832 1057 319 217 615 0,08103 SIDEYFSEQ Y-481 p135tyk2 SWISS;P17181;INR1_HUMAN 11630 607 984 796 266 695 289 0,63104 EESESD S-514 CKII SWISS;Q01827;VMT2_RAT 10597 1913 1049 1481 611 709 340 0,26105 RRRS SWISS;P02646;TRIC_RABIT 8118 921 1142 1032 156 714 428 0,40106 RLSPSPTSQ S-392 cdc2 SWISS;P11516;LAMC_MOUSE 5593 2188 1173 1681 718 979 194 0,30107 RKESYSV S-36 SWISS;P02278;H2B_HUMAN 11517 897 993 945 68 377 616 0,19108 PLSRTLS S-3 CKI SWISS;P13834;UGS1_RABIT 8936 1215 1124 1169 64 702 422 0,47109 RSRASTPPA S-47 GSK3 SWISS;P00515;KAP2_BOVIN 11627 2456 985 1721 1040 663 322 0,59110 SLSSSEESI S-34 CKI SWISS;P02666;CASB_BOVIN 13760 2951 780 1865 1535 418 362 0,76111 LRRASLAG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)13877 4349 765 2557 2534 852 -87 0,76112 ADSESEDEE S-76 CKII SWISS;P00515;KAP2_BOVIN 14444 6298 711 3505 3951 346 365 0,82113 SSTGSIDMV S-416 PKA PIR;2144820 12687 10189 880 5534 6582 53 827 0,32114 TLASSFKRR S-890 PKC SWISS;Q05586;P35437;NMZ1_HUMAN 16907 7103 414 3758 4730 77 337 0,46115 VGAFSTVKG S-488 RhK SWISS;P28327;RK_BOVIN 13321 7681 822 4251 4850 326 496 0,70116 YSGHSMSDP S-289 SWISS;P26267;ODPA_ASCSU 12288 7430 919 4175 4604 1098 -179 0,68

117 SPGEYVNIE Y-895 INSR SWISS;P35570;IRS1_RAT 13348 3081 814 1947 1603 1136 -322 0,22118 RKRSAKE SWISS;P02278;H2B_HUMAN 15661 2176 554 1365 1147 365 189 0,90119 VINETSQHH T-457 p37 SWISS;P20152;VIME_MOUSE 13820 730 773 751 31 755 18 0,71120 STSLSPFYL S-45 crystalline SWISS;P02510;CRAB_BOVIN 16092 1637 510 1073 797 498 12 0,46121 TRDIYETDY Y-1185 INSR SWISS;P06213;INSR_HUMAN 17382 2685 365 1525 1640 466 -101 0,44122 VPTLSTFRT S-56 PKC SWISS;P02542;DESM_CHICK 14079 8367 748 4558 5388 889 -141 0,78123 RASTSKSES S-242 SWISS;P10660;P08227;RS6_HUMAN 14690 5611 672 3141 3492 759 -87 0,74124 SPSSSPTHE S-323 SWISS;P19836;CTPT_RAT 13525 3077 796 1937 1613 1062 -266 0,56125 STNDSLL S-411 GRK5 SWISS;P07550;B2AR_HUMAN 15628 676 560 618 82 669 -109 0,64126 VRTFTHEVV T-160 CAK SWISS;P23437;CDK2_XENLA 14212 3137 735 1936 1699 540 195 0,94127 TAYGTRRHL T-184 PKC SWISS;P26932;CLPO_CHICK 13931 2400 759 1579 1160 385 374 0,68128 TRRISQTSQ S-2809 CaM-II SWISS;P30957;RYNC_RABIT 7504 1315 1155 1235 113 1140 15 0,73129 VTPRTPPPS T-97 ERT SWISS;P02687;MBP_BOVIN 6953 5449 1165 3307 3029 1122 43 0,71130 REVSSLKSK S-1915 PKC SWISS;P14105;MYSN_CHICK 14821 2795 659 1727 1511 135 524 0,19131 PEETQTQD T-4 ds-DNA SWISS;P07900;HS9A_HUMAN 13096 340 844 592 356 608 236 0,83132 KKQISVR SWISS;P11217;PHS2_HUMAN 23402 657 77 367 410 10 67 0,32133 WLTKSPDGN S-491 p34cdc2 SWISS;Q62736;CALD_RAT 8301 4015 1137 2576 2035 568 569 0,28134 THERSPSPS S-329 SWISS;P19836;CTPT_RAT 7069 4053 1162 2608 2044 962 200 0,36135 TVTRSYRSV S-625 PKC SWISS;P02545;LAMA_HUMAN 10787 67 1040 554 688 154 886 0,12136 YETDYYRKG Y-1189 INSR SWISS;P06213;INSR_HUMAN 14648 897 680 789 154 94 586 0,35137 RKQISVRGL S-15 SWISS;P11217;PHS2_HUMAN 16927 2589 412 1500 1539 70 342 0,13138 AGTTYAL T-3 myosin I heavy chain kinasemyosin heavy chain IA (fragment) 16059 3207 513 1860 1905 440 73 0,80139 LRRASVA S-43 SWISS;P12928;P04763;Q64618;KPYR_RAT14803 1532 660 1096 617 235 425 0,47140 YRGYSLGNW S-42 PKA SWISS;P00698;Q90884;LYC_CHICK 16965 6796 407 3602 4518 48 359 0,45141 SPVVSGDTS S-391 SWISS;P19332;TAU_RAT 13392 12293 809 6551 8120 449 360 0,87142 LRRFSLATM S-128 PKA SWISS;P00176;CPB1_RAT 16585 8106 455 4280 5410 218 237 0,83143 PRPASVPPS S-59 GSK3 SWISS;P13834;UGS1_RABIT 11372 7810 1004 4407 4813 848 156 0,90144 SSRPSSNRS S-24 PKA SWISS;P20152;VIME_MOUSE 11583 5508 989 3249 3196 1175 -186 0,35145 TKFASDDEH S-366 CKII SWISS;P05455;LA_HUMAN 12105 4896 936 2916 2800 1127 -191 0,51146 LSDDSFIED S-1408 CKII SWISS;P06786;TOP2_YEAST 12964 2641 861 1751 1259 595 266 0,88147 PSPKTPPGS T-172 SWISS;P19332;TAU_RAT 11237 1679 1014 1346 470 793 221 0,74148 SRKMSVQEY S-73 PKA SWISS;P19632;PHOS_BOVIN 14014 875 752 814 87 125 627 0,19149 LSGLSFKRN S-103 PKC SWISS;P28667;MRP_MOUSE 17604 4207 348 2277 2728 80 268 0,35150 PTGTTPQRK T-927 SWISS;P52732;EG5_HUMAN 12548 4911 894 2902 2840 903 -9 0,87151 STLASSFKR S-889 PKC SWISS;Q05586;P35437;NMZ1_HUMAN 16798 313 425 369 79 149 276 0,40152 TPPKSPSSA S-235 GSK3 PIR;2144820 15677 5332 551 2941 3380 160 391 0,64153 PAAVSEHGD S-16 PKC SWISS;P06685;ATN1_RAT 10870 1138 1035 1087 73 27 1008 0,54154 PVSPSLVQG S-234 GRK SWISS;P08172;ACM2_HUMAN 11681 2308 979 1643 940 303 676 0,05 �155 SRRSSLGSL S-1854 PKA SWISS;P07293;CIC1_RABIT 12110 3572 934 2253 1866 592 342 0,57156 PETVYEVAG Y-483 v-Src SWISS;Q01406;SRC8_CHICK 11678 1623 980 1301 455 701 279 0,35157 QEPGSGPPE S-351 SWISS;P30936;SSR3_RAT 6854 6648 1167 3907 3875 1113 54 0,76

158 SVSSSPIKE S-664 CDC28-dependentSWISS;P08153;SWI5_YEAST 10004 2732 1081 1907 1168 674 407 0,28159 TRLHSLRER S-10 SWISS;P04712;SUS1_MAIZE 10395 3469 1055 2262 1707 392 663 0,28160 PKEVYDVML Y-825 SWISS;Q03351;TRKC_RAT 10070 2278 1079 1678 848 420 659 0,20161 PRTPGGRR SWISS;P02687;MBP_BOVIN 6634 2351 1169 1760 836 1043 126 0,26162 SSLKSRKRA S-39 SWISS;P22613;STP1_SHEEP 14550 633 692 662 42 183 509 0,12163 PMRRSVSEA S-8 PKA SWISS;P16386;LIPS_BOVIN 9149 1421 1113 1267 218 893 220 0,20164 RSKRSGSV SWISS;P12798;KPBB_RABIT 14540 2141 696 1419 1022 141 555 0,12165 TEGQYQQQP Y-503 SWISS;P42683;LCK_CHICK 15596 920 562 741 253 291 271 0,64166 TTPLSPTRL S-22 cdc2 SWISS;P20700;LAM1_HUMAN 17535 5617 352 2985 3723 653 -301 0,51167 PQPEYVNQP Y-1139 SWISS;P04626;ERB2_HUMAN 19313 3123 219 1671 2054 269 -50 0,65168 VKRGISGL SWISS;P02304;P02305;H4_HUMAN 22424 3992 96 2044 2755 81 15 0,96169 KDIGSESTE S-61 CKII SWISS;P02662;CAS1_BOVIN 12641 9586 884 5235 6153 660 224 0,91170 KRRGSVPIL S-247 PKA SWISS;P16452;42_HUMAN 17212 7570 392 3981 5075 136 256 0,71171 KRAKAKTAKKR SWISS;P02612;MLRM_CHICK 30936 13463 10 6736 9512 5 5 0,70172 QSYSSSQRV S-6 cdc2 SWISS;P02542;DESM_CHICK 18860 3285 259 1772 2139 857 -598 0,05 �173 KGTGYIKTE Y-701 SWISS;P42224;STA1_HUMAN 12935 3540 863 2201 1893 588 275 0,73174 KRSLSEMEI S-103 SWISS;P11831;SRF_HUMAN 11207 506 1016 761 361 514 502 0,55175 KRKQGSVRGL SWISS;P11217;PHS2_HUMAN 16836 281 423 352 100 72 351 0,42176 KKDASDDLD S-67 CKII SWISS;P41035;IF2B_RABIT 12758 977 874 925 72 293 581 0,70177 KSRPSLPLP S-341 PKA SWISS;P19112;Q64594;F16P_RAT 13495 815 799 807 11 272 527 0,38178 LSVSSLPGL S-10 CKI SWISS;P13834;UGS1_RABIT 11882 637 957 797 226 610 347 0,12179 SRHSSPHQS S-71 GSK3 SWISS;P13834;UGS1_RABIT 11975 731 950 841 155 535 415 0,57180 KKPPTPPPE T-701 SWISS;P03070;TALA_SV40 11740 1192 971 1082 156 36 935 0,45181 KVPQTPLHT T-300 MAPK SWISS;P49139;MKK2_RABIT 15454 99 585 342 344 7 578 0,04 �182 PRRSSIRNA S-304 SWISS;P14598;NCF1_HUMAN 9581 4332 1100 2716 2285 202 898 0,63183 KLSPSPSSR S-392 cdc2 SWISS;P20700;LAM1_HUMAN 12561 3352 892 2122 1739 213 679 0,20184 LEKKYVRRD Y-706 c-Fms SWISS;P09581;KFMS_MOUSE 17584 3752 349 2050 2406 124 225 0,47185 QASSTPLSP T-19 cdc2 SWISS;P02545;LAMA_HUMAN 8725 3526 1127 2326 1696 1023 104 0,61186 SRRDSLFVP S-610 PKA SWISS;P04775;CIN2_RAT 11994 4037 948 2492 2184 735 213 0,76187 KQPIYIVME Y-424 v-Fps SWISS;P00541;FPS_AVISP 13073 4313 848 2580 2450 283 565 0,40188 LLQDSVDFS S-82 CaM-II SWISS;P20152;VIME_MOUSE 9607 6376 1096 3736 3734 899 197 0,71189 QLSTSEENS S-146 CKI SWISS;P02663;CAS2_BOVIN 6231 4667 1171 2919 2472 1019 152 0,42190 KRKQISVRG S-15 SWISS;P11217;PHS2_HUMAN 14745 210 667 439 323 107 560 0,09191 LPVPSTHIG S-50 PKG SWISS;P00516;KGPA_BOVIN 12640 74 885 480 573 706 179 0,58192 QRRHSLEPP S-17 PKA SWISS;P00526;SRC_RSVP 14339 1827 720 1274 783 924 -204 0,59193 SRTLSVSSL S-7 AMP-PK SWISS;P13834;UGS1_RABIT 21071 2135 132 1133 1416 329 -197 0,18194 KRPSERAKA SWISS;P02687;MBP_BOVIN 24371 1049 58 553 700 93 -35 0,49195 LRGRSFMNN S-376 PKA SWISS;P00511;K6PF_RABIT 24184 747 61 404 485 338 -277 0,12196 QRVSSYRRT S-12 PKC SWISS;P02542;DESM_CHICK 20513 1419 148 784 899 169 -21 0,55197 HDLSSEMFN S-64 SWISS;P01239;PRL_BOVIN 10381 6577 1056 3816 3904 1063 -7 0,95198 KRPSRRAKA SWISS;P02687;MBP_BOVIN 15214 1324 612 968 503 150 462 0,26

199 EDNEYTARP Y-413 SWISS;P31693;SRC_RSVPA 11244 5413 1013 3213 3111 835 178 0,85200 ENAPSSTSS S-1266 CKII SWISS;P06786;TOP2_YEAST 13998 129 753 441 441 1041 -288 0,41201 HMRSSMSGL S-77 PKA SWISS;P11497;P97902;COAC_RAT 11567 2861 990 1925 1323 1168 -178 0,18202 LDRSSHAQR S-1333 insulin-sensitiveSWISS;P06213;INSR_HUMAN 7070 930 1161 1045 163 1011 150 0,11203 PYKFPSSPLRIPGZ na na na 10264 3415 1064 2240 1663 1038 26 1,00204 HSSQSQGGG S-409 SWISS;P11516;LAMC_MOUSE 10111 4374 1074 2724 2334 1176 -102 0,39205 MAEAYSEIG Y-122 Lck/Fyn SWISS;P20963;CD3Z_HUMAN 7520 2927 1154 2040 1253 684 470 0,22206 EEESSYSYE S-117 SWISS;P20338;RRPP_HRSVL 11248 2700 1012 1856 1194 361 651 0,21207 EQPGSDDED S-303 CKII SWISS;P00860;Q61997;DCOR_MOUSE 9371 6584 1104 3844 3875 370 734 0,37208 IEQFSTVKG S-484 GRK5 SWISS;P43249;GRK5_BOVIN 12347 4904 913 2908 2822 277 636 0,51209 MLDHSESTK S-226 SWISS;P27573;MYP0_MOUSE 12180 2695 930 1812 1248 456 474 0,75210 PYKFPSSPLRIPGZ na na na 11472 1694 996 1345 494 762 234 0,86211 IHQRSRKRL S-315 SWISS;P14598;NCF1_HUMAN 14652 3978 679 2329 2333 378 301 0,81212 NDALSGSGN S-11 CKI SWISS;P38486;LEG3_CANFA 9601 994 1097 1046 73 1116 -19 0,82213 EETQTQDQP T-6 ds-DNA SWISS;P07901;HS9A_MOUSE 11190 2481 1017 1749 1035 897 120 0,82214 ERSQSRKDS S-400 PKC SWISS;P15823;A1AB_RAT 11992 3456 949 2202 1772 894 55 0,97215 IRQASQAGP S-603 CaM-II SWISS;P09951;SYN1_RAT 9019 6013 1121 3567 3459 814 307 0,56216 NFDDYMKEV Y-19 INSR SWISS;P04117;FABA_MOUSE 11309 2772 1008 1890 1247 430 578 0,45217 PYKFPSSPLRIPGZ na na na 11289 4835 1010 2923 2705 471 539 0,47218 KGGSYSQAA Y-344 SWISS;P01889;1B02_HUMAN 13725 3417 784 2101 1862 575 209 0,87219 NIYISPLKS S-807 p34cdc2 SWISS;P06400;RB_HUMAN 16473 3504 473 1989 2144 348 125 0,94220 EIRVSINEK S-338 PKA SWISS;P00517;KAPA_BOVIN 15253 2802 607 1704 1552 828 -221 0,37221 ESRISLPLP S-411 autophosphorylation-dependentSWISS;P20152;VIME_MOUSE 20418 1783 154 969 1152 720 -566 0,18222 KRPSIRAKA SWISS;P02687;MBP_BOVIN 30556 7969 13 3991 5626 21 -8 0,94223 NPGFYVEAN Y-783 EGFR SWISS;P08487;PIP4_BOVIN 21029 2338 133 1236 1559 305 -172 0,23224 PYKFPSSPLRIPGZ na na na 19258 225 225 225 0 411 -186 0,19225 GKEIYNTIR Y-460 PDGFR SWISS;P20936;GTPA_HUMAN 11389 8390 1003 4696 5223 389 614 0,60226 APATPGGRR SWISS;P02687;MBP_BOVIN 10649 7066 1046 4056 4257 764 282 0,76227 DEEESEEAK S-111 CKII SWISS;P34826;EF1B_RABIT 10082 4461 1076 2769 2394 1159 -83 0,54228 FFKKSKIST S-697 PKA SWISS;Q13002;GLR6_HUMAN 16499 4897 469 2683 3131 139 330 0,62229 GMGTSVERA S-232 pyruvate SWISS;P08559;ODPA_HUMAN 9619 4278 1095 2686 2251 1146 -51 0,67230 DYDSSDIED S-468 CKII SWISS;Q03017;CACT_DROME 9024 3565 1119 2342 1730 1119 0 0,97231 EKRASGQAF S-15 SWISS;P16949;STHM_HUMAN 6939 2490 1166 1828 936 1083 83 0,50232 GPRTTRAQG T-58 PKG SWISS;P00516;KGPA_BOVIN 6975 8076 1164 4620 4887 1167 -3 0,93233 AQDTYLVLD Y-368 SWISS;P19235;EPOR_HUMAN 4741 2289 1175 1732 788 970 205 0,11234 DIPESQMEE S-29 CK SWISS;P02730;B3AT_HUMAN 11771 6073 969 3521 3609 522 447 0,63235 FPRASFGSR S-29 PKA SWISS;P02542;DESM_CHICK 10907 4581 1032 2806 2509 655 377 0,55236 GRKASGSSP S-504 CaM-II SWISS;P11799;KMLC_CHICK 11084 5683 1021 3352 3297 1008 13 0,95237 EDVGSDEEE S-262 CKII SWISS;P07900;HS9A_HUMAN 12419 5964 903 3434 3579 1007 -104 0,80238 EPAVSPLLP S-569 p34cdc2 SWISS;P00520;ABL_MOUSE 11860 2430 960 1695 1039 799 161 0,82239 GRRQSLIQD S-39 CaM-II SWISS;P17289;TY3H_BOVIN 10168 5616 1069 3342 3215 984 85 0,86

240 ASFEYTILD Y-426 SWISS;P19235;EPOR_HUMAN 10355 776 1059 918 200 995 64 0,64241 DLSTYASIN Y-1222 INSR SWISS;P35570;IRS1_RAT 11004 3972 1025 2498 2084 1088 -63 0,69242 GDNDYIIPL Y-1021 SWISS;P09619;PGDR_HUMAN 14222 3451 734 2093 1921 795 -61 0,65243 GSESTEDQA T-64 CKII SWISS;P02662;CAS1_BOVIN 7688 5286 1152 3219 2923 1058 94 0,65244 EENVSVDDT S-15 CKII SWISS;P07260;IF4E_YEAST 6675 4588 1168 2878 2418 627 541 0,26245 ERRKSKSGA S-10 PIR;A33361 10780 637 1041 839 286 480 561 0,22246 GSRGSGSSV S-35 PKA SWISS;P02542;DESM_CHICK 8986 149 1122 636 688 751 371 0,30247 DAGASPVEK S-99 SWISS;P12624;MACS_BOVIN 11935 4 952 478 671 447 505 0,01 �248 DRRVSVAAE S-95 PKA SWISS;P00515;KAP2_BOVIN 13237 2418 830 1624 1123 1001 -171 0,63249 GGRASDYKS S-131 PKA SWISS;P02687;MBP_BOVIN 25618 402 35 219 260 115 -80 0,01 �250 GVDTYVEMR Y-723 SWISS;P07333;KFMS_HUMAN 25484 1695 38 866 1172 267 -229 0,09251 EGTHSTKRG S-618 PKC SWISS;P02671;FIBA_HUMAN 20061 4455 182 2319 3022 287 -105 0,50252 ESLESYEIN S-28 SWISS;P07507;MGP_BOVIN 17499 2485 355 1420 1506 786 -431 0,22253 RRAVSELDA S-18 CaM-II SWISS;P17289;TY3H_BOVIN 9791 7459 1089 4274 4504 1131 -42 0,87254 SDGGYMDMS Y-740 SWISS;P09619;PGDR_HUMAN 9339 5746 1106 3426 3281 895 211 0,67255 NWHMTPPRK T-316 cdc2 SWISS;P13681;PP11_SCHPO 12401 4055 905 2480 2227 130 775 0,20256 RRASI SWISS;P12928;P04763;Q64618;KPYR_RAT12514 2997 897 1947 1485 1123 -226 0,42257 AKKGSEQES S-10 SWISS;P00517;KAPA_BOVIN 8885 2922 1125 2023 1271 1145 -20 0,79258 DDEMTGYVA T-180 SWISS;P47811;MP38_MOUSE 6299 1406 1170 1288 167 1148 22 0,41259 ETDYYRKGG Y-1190 INSR SWISS;P06213;INSR_HUMAN 10601 3285 1047 2166 1583 600 447 0,43260 RRRASQLKI S-434 PKC SWISS;P47870;GAB2_HUMAN 9187 4774 1112 2943 2589 662 450 0,49261 SFTTTAERE T-203 AFK SWISS;P02576;ACTA_PHYPO 6069 3198 1172 2185 1433 1169 3 0,96262 PLSRTL S-3 CKI glycogen (starch) synthase, muscle 8037 5816 1146 3481 3302 1153 -7 0,93263 SGRGK S-1 H4-PK-II SWISS;P02304;P02305;H4_HUMAN 10343 5387 1060 3223 3059 836 224 0,70264 APRSPGGRR SWISS;P02687;MBP_BOVIN 10953 6705 1028 3866 4014 1105 -77 0,78265 DGERYDEDE Y-93 SWISS;P13834;UGS1_RABIT 12369 5561 908 3235 3290 938 -30 0,87266 FKRPTLRRV T-144 PKC SWISS;P08057;TRIC_BOVIN 19017 1893 246 1069 1165 113 133 0,43267 RRSRSRSRS S-78 RS SWISS;P23913;LBR_CHICK 12387 5593 907 3250 3314 511 396 0,66268 SKIGSLDNI S-356 PKA PIR;2144820 7805 2032 1149 1590 624 800 349 0,12269 NDITSL S-283 tropomyosinSWISS;P02560;TPMB_RABIT 10166 3371 1071 2221 1626 954 117 0,71270 AARGSFDAS S-21 RhK SWISS;P28327;RK_BOVIN 8599 4402 1130 2766 2314 1037 93 0,70271 ARNDSVTVA S-62 PKA SWISS;P15336;CREP_HUMAN 8717 2750 1128 1939 1147 1044 84 0,59272 DLFGSDEED S-89 CKII SWISS;P12262;EF1B_ARTSA 9125 114 1115 614 708 646 469 0,24273 FSSRSYTSG S-23 SWISS;P05787;K2C8_HUMAN 14868 5664 651 3157 3545 618 33 0,86274 RVRKTKGKY T-710 PKC SWISS;P19490;GLR1_RAT 20428 107 153 130 32 34 119 0,47275 SNPEYLSAS Y-999 SWISS;P06213;INSR_HUMAN 11952 97 951 524 604 420 531 0,10276 RRSTVA T-21 SWISS;P02322;PRT2_THUTH 12847 178 868 523 488 951 -83 0,49277 AEPGSPTAA S-116 SWISS;P12624;MACS_BOVIN 16439 3010 477 1744 1791 986 -509 0,34278 AVDGYVKPQ Y-694 SWISS;P42229;STA5_HUMAN 22517 2174 93 1134 1472 525 -432 0,12279 DPGVSYRTR S-300 pyruvate SWISS;P08559;ODPA_HUMAN 20050 4282 183 2232 2898 623 -440 0,26280 GEINTEDDD T-381 CKII SWISS;P31235;CAQS_RABIT 18464 11 284 147 193 715 -431 0,10

281 YVTTSTRTY S-33 PKC SWISS;P20152;VIME_MOUSE 10167 8602 1070 4836 5326 530 540 0,60282 SPQPSRRGS S-12 GSK3 PIR;S04004 8461 6759 1133 3946 3978 862 271 0,59283 RRAASVA SWISS;P12928;P04763;Q64618;KPYR_RAT7287 6662 1158 3910 3892 1086 72 0,77284 RPSESNGQP S-245 SWISS;P06624;MIP_BOVIN 8293 6413 1138 3776 3730 1115 23 0,94285 SASTTPVKK T-46 SWISS;P10156;H1_TETTH 10081 3566 1077 2321 1760 687 390 0,41286 NSYGSRRGN S-711 PKC SWISS;Q63270;IREB_RAT 8057 3331 1145 2238 1546 469 676 0,35287 LRRASL S-5 BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)7206 5136 1160 3148 2811 697 463 0,48288 REAEYEPET Y-477 v-Src SWISS;Q01406;SRC8_CHICK 7306 5071 1157 3114 2768 607 550 0,52289 DRVYIHPF Y-4 ANGIOTENSIN II 11621 202 986 594 555 601 385 0,67290 VRRISGL SWISS;P02304;P02305;H4_HUMAN 8456 2064 1134 1599 658 1027 107 0,59291 RRKGTDVNV T-215 PKA SWISS;P04083;ANX1_HUMAN 12497 7280 899 4090 4512 854 45 0,96292 SETKTEEEE T-1258 CKII SWISS;P06786;TOP2_YEAST 13090 11105 846 5976 7254 1162 -316 0,59293 PQRATSNVF T-18 SWISS;P24844;MLRN_HUMAN 13913 6834 763 3799 4293 1080 -317 0,53294 RRGSV SWISS;P12928;P04763;Q64618;KPYR_RAT12292 7623 916 4270 4743 1097 -181 0,69295 RGKSSSYSK S-577 PKC SWISS;P02671;FIBA_HUMAN 17939 2070 316 1193 1240 104 212 0,39296 RKRSRAEA SWISS;P02278;H2B_HUMAN 8784 759 1126 942 260 982 144 0,27297 LKRASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)8036 1546 1147 1346 282 901 246 0,19298 RRRPTPATL T-35 PKA SWISS;P01099;IPP1_RABIT 5306 372 1174 773 567 1018 156 0,14299 SIADTFVGT S-363 SWISS;P06784;STE7_YEAST 11052 1120 1023 1072 69 534 489 0,35300 LRRNSI SWISS;P13569;CFTR_HUMAN 9115 1849 1116 1483 518 941 175 0,44301 RRRRASVA SWISS;P12928;P04763;Q64618;KPYR_RAT10900 4044 1033 2538 2129 957 76 0,91302 RLSISTESQ S-1020 AMP-PK SWISS;P18688;KPB1_RABIT 10560 1022 1051 1036 21 802 249 0,39303 RKEISVR SWISS;P11217;PHS2_HUMAN 28787 12801 16 6409 9041 156 -140 0,49304 PGSPQKR S-3 sperm-specificSWISS;P02256;H1_PARAN 13238 1942 829 1385 787 1004 -175 0,63305 RSGYSSPGS S-189 SWISS;P19332;TAU_RAT 17333 2348 371 1359 1398 757 -386 0,33306 SLRASTSKS S-240 SWISS;P10660;P08227;RS6_HUMAN 27152 2490 23 1257 1745 68 -45 0,24307 VRKISGL SWISS;P02304;P02305;H4_HUMAN 22147 1631 102 866 1081 537 -435 0,05308 ADGVYAASG Y-368 v-Fes SWISS;P00543;FES_FSVST 16611 1895 448 1172 1023 566 -118 0,32309 SSTDSADSG S-231 SWISS;P03255;E1A_ADE05 9812 8531 1088 4809 5263 1056 32 0,98310 TKSGSTTKN S-209 SWISS;P06730;IF4E_HUMAN 9600 428 1098 763 474 261 837 0,01 �311 VETTYADFI Y-7 EGFR SWISS;P04541;IPKI_HUMAN 8974 8441 1123 4782 5175 1117 6 1,00312 YSFTTTAER T-202 AFK SWISS;P02576;ACTA_PHYPO 7789 4854 1150 3002 2619 939 211 0,49313 SPFKYQSLL Y-141 SWISS;P04274;B2AR_MESAU 10203 8593 1067 4830 5322 712 355 0,72314 RKRSAAE SWISS;P02278;H2B_HUMAN 7314 3644 1156 2400 1760 740 416 0,56315 VIKRSPRKR S-646 CDC28-dependentSWISS;P08153;SWI5_YEAST 15462 3157 580 1868 1822 66 514 0,26316 STSKSESSQ S-244 SWISS;P10660;P08227;RS6_HUMAN 9236 3933 1111 2522 1995 840 271 0,69317 TRAPSRTAS S-450 MFPK SWISS;P16638;ACLY_RAT 7661 1049 1153 1101 73 1085 68 0,76318 VPRTPGGRR SWISS;P02687;MBP_BOVIN 11736 2942 972 1957 1393 972 0 0,85319 RASSSRSVR S-405 PKC SWISS;P20700;LAM1_HUMAN 10912 4229 1030 2630 2262 1073 -43 0,75320 SPSSSPASL S-347 SWISS;P19836;CTPT_RAT 10409 7499 1054 4276 4557 1172 -118 0,57321 RVYVHPF ANGIOTENSIN II 15165 4161 615 2388 2507 197 418 0,46

322 VRRVSDDVR S-153 PKA SWISS;P04177;TY3H_RAT 10543 4543 1052 2798 2469 837 215 0,72323 TATDYHTTS Y-46 SWISS;P02730;B3AT_HUMAN 9922 2748 1084 1916 1176 996 88 0,76324 TRRASRPVR S-9 PKA SWISS;P02335;PRT1_CLUPA 8504 291 1132 712 594 958 174 0,38325 VSSSSYRRM S-9 PKC SWISS;P20152;VIME_MOUSE 12588 2462 889 1676 1112 583 306 0,53326 REVSSLKNK S-1917 PKC SWISS;P35579;MYSN_HUMAN 11439 161 1000 580 594 700 300 0,11327 LSYRRYSL S-7 PEPTIDE 14501 5738 706 3222 3558 460 246 0,90328 KKKASVA SWISS;P12928;P04763;Q64618;KPYR_RAT18716 3229 269 1749 2093 91 178 0,66329 WKRTSMKLL S-1627 PKA SWISS;P15381;CICC_RABIT 22407 651 98 375 391 25 73 0,35330 TGFLTEYVA T-183 SWISS;P27703;ERK2_MOUSE 14662 977 675 826 214 435 240 0,81331 TVSTSLGHS S-311 GRK SWISS;P08172;ACM2_HUMAN 13071 2433 849 1641 1120 934 -85 0,54332 YDKEYYSVH Y-1234 SWISS;P08581;MET_HUMAN 16886 296 418 357 86 477 -59 0,21333 RKLKSQGTR S-8 0 SWISS;P22613;STP1_SHEEP 19603 1038 204 621 590 539 -335 0,04 �334 NRKPSKDKD S-366 PKC SWISS;P18507;GAC2_HUMAN 21377 1490 122 806 967 258 -136 0,24335 LRRASPG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)16117 1319 505 912 575 930 -425 0,14336 YMAPYDNYV Y-775 SWISS;P09619;PGDR_HUMAN 18030 3698 307 2003 2398 596 -289 0,36337 SPVKSPEAK S-506 SWISS;P12839;NFM_RAT 11098 8011 1019 4515 4944 775 244 0,81338 LRRASVAQL S-43 PKA SWISS;P12928;P04763;Q64618;KPYR_RAT10753 8106 1042 4574 4995 487 555 0,60339 PRMPSLSVP S-1776 PKA SWISS;P02719;CINA_ELEEL 7752 6497 1151 3824 3780 1040 111 0,68340 SSPVYQDAV Y-430 v-Src SWISS;Q01406;SRC8_CHICK 9687 8066 1091 4578 4932 824 267 0,73341 TKDTYDALH Y-153 SWISS;P24161;CD3Z_MOUSE 9261 2240 1108 1674 801 833 275 0,39342 LRSPSWEPF S-15 PKA SWISS;P14602;HS27_MOUSE 15471 7186 578 3882 4672 298 280 0,83343 PSLPTPPTR T-208 SWISS;P19332;TAU_RAT 24962 26101 46 13073 18423 956 -910 0,59344 SRKMSIQEY S-73 PKA SWISS;P20942;PHOS_RAT 13925 5086 760 2923 3059 417 343 0,78345 LSGFSFKKS S-162 PKC SWISS;P30009;MACS_RAT 17870 5446 322 2884 3623 146 176 0,75346 PSSTSSSSI S-1269 CKII SWISS;P06786;TOP2_YEAST 17467 9237 359 4798 6278 943 -584 0,52347 STGIYEALE Y-43 v-Src SWISS;P06733;ENOA_HUMAN 14610 2351 684 1518 1179 774 -90 0,48348 TPPKSPSAS S-226 SWISS;P19332;TAU_RAT 11673 3959 981 2470 2106 1066 -85 0,64349 LYSSSPGGA S-55 cdc2 SWISS;P20152;VIME_MOUSE 11776 4215 967 2591 2297 1081 -114 0,59350 PVPKSPVEE S-608 SWISS;P12839;NFM_RAT 11289 5115 1010 3063 2903 1095 -85 0,70351 SRRQSVLVK S-715 PKA SWISS;Q13002;GLR6_HUMAN 23425 4009 76 2042 2781 45 31 0,73352 PENDYEDVE Y-378 SWISS;P14317;HS1_HUMAN 11028 542 1024 783 341 955 69 0,69353 QEGLYNELQ Y-110 Lck/Fyn SWISS;P20963;CD3Z_HUMAN 12251 883 925 904 30 1034 -109 0,15354 SVPPSPSLS S-63 GSK3 SWISS;P13834;UGS1_RABIT 9430 3982 1103 2543 2036 1126 -23 0,88355 TRKVSLAPQ S-795 PKA SWISS;P13569;CFTR_HUMAN 9877 4410 1085 2748 2351 1053 32 0,96356 PKDPSQRRR S-11 PKC SWISS;P00523;Q91345;Q92013;SRC_CHICK14083 4961 747 2854 2980 974 -227 0,56357 KRKQISVR S-15 SWISS;P11217;PHS2_HUMAN 24125 361 64 213 210 39 25 0,09358 SSKAYGNGY Y-350 INSR SWISS;P04274;B2AR_MESAU 15111 3625 622 2123 2123 844 -222 0,46359 PLTPSGEAP S-695 SWISS;P00533;P06268;EGFR_HUMAN 12241 3562 926 2244 1864 1068 -142 0,51360 RRRRPTPA SWISS;P12928;P04763;Q64618;KPYR_RAT15742 1415 543 979 617 1074 -531 0,08361 TEGQYQPQP Y-504 SWISS;P06239;LCK_HUMAN 18462 336 285 310 36 953 -668 0,04 �362 TSSSSIFDI S-1272 CKII SWISS;P06786;TOP2_YEAST 21210 6848 128 3488 4752 716 -588 0,33

363 PPSPSLSRH S-65 CKI SWISS;P13834;UGS1_RABIT 18721 387 268 328 84 494 -226 0,42364 SSKRAKAK S-1 SWISS;P02612;MLRM_CHICK 19983 3869 187 2028 2603 166 21 0,88365 KASASPRRK S-29 sperm-specificSWISS;P02256;H1_PARAN 16992 2648 406 1527 1585 82 324 0,22366 KRRDYLDLA Y-1015 SWISS;P07949;RET_HUMAN 12291 2019 917 1468 780 350 567 0,22367 PAPAVRASDRA SWISS;P02646;TRIC_RABIT 10798 3286 1039 2162 1589 1093 -54 0,68368 QSRASDKQT S-148 PKC SWISS;P09693;CD3G_HUMAN 11699 2053 977 1515 761 1003 -26 0,73369 KGQESFKKQ S-227 PKC SWISS;P06748;NPM_HUMAN 14442 2242 712 1477 1082 673 39 0,73370 KRSGSVYEP S-26 PKA SWISS;P12798;KPBB_RABIT 17904 186 318 252 93 419 -101 0,02 �371 KRAQISVRGL SWISS;P11217;PHS2_HUMAN 24172 85 62 73 16 100 -38 0,61372 KKASFKAKK S-4 PKC peptide KKASFKAKK 16180 3254 499 1877 1948 163 336 0,63373 KSPAKTPVK SWISS;P12957;CALD_CHICK 18028 1066 308 687 536 318 -10 0,85374 PLSKTLSVSS SWISS;P13834;UGS1_RABIT 18932 8564 255 4410 5876 980 -725 0,42375 SRGKSSSYS S-576 PKC SWISS;P02671;FIBA_HUMAN 18501 1668 283 976 979 427 -144 0,23376 KKLGSKKPQ S-1506 PKC SWISS;P04775;CIN2_RAT 24976 9406 45 4725 6619 251 -206 0,46377 KTTASTRKV S-790 PKC SWISS;P13569;CFTR_HUMAN 21936 3187 110 1648 2176 75 35 0,90378 PRRRTRRAS T-5 PKA SWISS;P02335;PRT1_CLUPA 11425 5510 1001 3256 3189 1075 -74 0,72379 KLRRSSSVG S-381 PKA SWISS;P02718;ACHD_TORCA 16782 334 428 381 66 118 310 0,25380 LDPLSEPED S-12 CKII SWISS;P04625;THA_CHICK 14753 1706 665 1185 736 879 -214 0,24381 QAGMTAPGT T-220 SWISS;P26932;CLPO_CHICK 12450 2582 901 1741 1188 1142 -241 0,22382 SRRASRPVR S-10 PKA SWISS;P02336;PRT2_CLUPA 16529 2456 463 1459 1409 1163 -700 0,06383 KPGFSPQPS S-8 GSK3 PIR;S04004 13303 138 823 480 484 853 -30 0,44384 LLPMSPEEF S-727 SWISS;P42224;STA1_HUMAN 13726 623 783 703 113 807 -24 0,23385 QLNDSSEEE S-31 CKII SWISS;P03129;VE7_HPV16 13162 4159 840 2500 2347 779 61 0,93386 KRKNSILNP S-700 PKA SWISS;P13569;CFTR_HUMAN 18800 2064 263 1164 1274 106 157 0,60387 LNRMSFASN S-1200 AMP-PK SWISS;P11497;P97902;COAC_RAT 14799 4 661 332 465 563 98 0,39388 QRHGSKYLA S-12 PKA SWISS;P02686;MBP_HUMAN 17830 276 329 302 38 532 -203 0,11389 SRTASFSES S-454 PKA SWISS;P16638;ACLY_RAT 18968 3854 252 2053 2547 692 -440 0,27390 KRPSDRAKA SWISS;P02687;MBP_BOVIN 27666 12765 20 6393 9012 99 -79 0,60391 LRGPSWDPF S-15 MAPKAPK2SWISS;P04792;HS27_HUMAN 16842 356 421 388 46 244 177 0,41392 QRSTSTPNV S-259 AMP-PK SWISS;P04049;KRAF_HUMAN 13513 2230 797 1513 1013 766 31 0,86393 HDALSGSGN S-11 CKI SWISS;P17931;LEG3_HUMAN 20131 3388 174 1781 2273 137 37 0,98394 KRPSQRAKY SWISS;P02687;MBP_BOVIN 25855 4190 31 2110 2941 12 19 0,37395 EDNEYTARE Y-411 c-Hck SWISS;P08631;HCK_HUMAN 22443 1067 95 581 687 738 -643 0,02 �396 ENAFSPSRS S-249 p34cdc2 SWISS;Q62736;CALD_RAT 19455 2797 214 1505 1826 352 -138 0,34397 HKSGYLSSE Y-145 EGFR SWISS;P15311;P23714;EZR1_HUMAN 19271 2934 223 1578 1917 1118 -895 0,12398 LAYESHESM S-22 SWISS;P08494;MGP_RAT 17490 1551 357 954 845 1106 -749 0,04 �399 PYKFPSSPLRIPGZ na na na 21570 10958 117 5538 7666 865 -748 0,44400 HSIYSSDDD S-11 CKII SWISS;P01103;MYB_CHICK 9858 3135 1086 2111 1449 1060 26 0,97401 LVMQTAAGT T-166 SWISS;P25323;KMLC_DICDI 11692 1645 978 1312 472 1033 -55 0,45402 EEELYLEPL Y-918 SWISS;P18475;TOR_DROME 11777 515 966 740 319 1091 -125 0,28403 EQLSTSEEN T-145 CKII SWISS;P02663;CAS2_BOVIN 12095 846 940 893 67 1160 -220 0,04 �

404 IDMESQERI S-249 DNA-PK SWISS;P05412;AP1_HUMAN 15489 2284 575 1429 1208 1152 -577 0,08405 MILLSELSR S-48 haem-controlledSWISS;P12962;IF43_YEAST 13199 687 834 760 104 1051 -217 0,20406 PYKFPSSPLRIPGZ na na na 16012 3781 518 2149 2307 1096 -578 0,23407 IGSVSEDNS S-25 CaM-II SWISS;P11497;P97902;COAC_RAT 14314 6581 722 3652 4143 1137 -415 0,41408 NAPVSALGE S-346 GSK3 SWISS;P01103;MYB_CHICK 14129 4597 740 2668 2727 1164 -424 0,27409 EETPYSYPT Y-31 v-Src SWISS;P49023;PAXI_HUMAN 16727 5865 435 3150 3840 749 -314 0,54410 ERSPSPSFR S-331 SWISS;P19836;CTPT_RAT 15588 250 564 407 222 765 -201 0,31411 IRKYTMRRL T-686 SWISS;P04626;ERB2_HUMAN 22013 593 107 350 343 270 -163 0,02 �412 NEEESSYSY S-116 SWISS;P20338;RRPP_HRSVL 20272 1018 164 591 604 290 -126 0,12413 PYKFPSSPLRIPGZ na na na 14086 3543 746 2144 1977 581 165 0,97414 IVYKSPVVS S-396 PIR;2144820 10601 638 1047 842 289 964 83 0,78415 NGYISAAEL S-101 CKII SWISS;P02593;CALM_HUMAN 12361 1593 910 1252 483 932 -22 0,61416 EILNSPEKA S-185 SWISS;P29358;143B_BOVIN 15109 1038 623 831 293 909 -286 0,17417 ESPESTEIT S-126 SWISS;P08833;IBP1_HUMAN 17366 1507 368 937 805 919 -551 0,09418 KRPSHRAKA SWISS;P02687;MBP_BOVIN 22100 6620 103 3361 4608 479 -376 0,37419 NNYVYIDPT Y-570 SWISS;P10721;KKIT_HUMAN 15809 2688 534 1611 1523 929 -395 0,23420 PYKFPSSPLRIPGZ na na na 17030 981 403 692 409 500 -97 0,13421 GHQGTVPSD T-393 GRK5 SWISS;P07550;B2AR_HUMAN 16912 218 413 316 138 402 11 0,58422 ANDEYFIRK Y-379 SWISS;P02712;ACHB_TORCA 28268 13593 18 6806 9599 493 -475 0,39423 DEASTTVSK T-335 PKC SWISS;P02699;OPSD_BOVIN 17272 3001 381 1691 1853 578 -197 0,56424 FEARYQQPF Y-1254 EGFR SWISS;P19174;PIP4_HUMAN 16148 1523 503 1013 721 315 188 0,70425 GLLRSWNDP S-120 SWISS;P01239;PRL_BOVIN 13251 602 828 715 159 937 -109 0,64426 DTVTSPQRA S-71 p34cdc2 SWISS;P00523;Q91345;Q92013;SRC_CHICK13354 3405 813 2109 1833 1054 -241 0,38427 EKHHSIDAQ S-15 SWISS;P04711;CAP1_MAIZE 14941 4785 645 2715 2927 1032 -387 0,40428 GPAASPAAA S-80 SWISS;P12624;MACS_BOVIN 10893 734 1034 884 212 1165 -131 0,15429 AQAASPAKG S-386 cdc2 SWISS;P18031;PTN1_HUMAN 10659 395 1045 720 460 971 74 0,62430 DHSRSTKAA S-228 SWISS;P27573;MYP0_MOUSE 11586 1142 988 1065 109 698 290 0,26431 FPFHSPSRL S-19 crystalline SWISS;P02510;CRAB_BOVIN 18681 1836 272 1054 1106 926 -654 0,08432 GRILTLPRS T-1375 PKC SWISS;P06213;INSR_HUMAN 18683 1307 271 789 733 1138 -867 0,12433 EDVGSDEED S-254 CKII SWISS;P08238;HS9B_HUMAN 19913 5889 194 3042 4027 1069 -875 0,20434 ENTVSTSLG S-309 GRK SWISS;P08172;ACM2_HUMAN 18626 812 276 544 379 1101 -825 0,23435 GRRQSLIED S-40 PKA SWISS;P04177;TY3H_RAT 16727 40 434 237 279 1121 -687 0,17436 ASATSSSGG S-119 CKI SWISS;P13834;UGS1_RABIT 13469 3287 801 2044 1758 773 28 0,84437 DLPMSPRTL S-727 SWISS;P42227;STA3_MOUSE 14432 2040 714 1377 938 1039 -325 0,16438 GDLQSAEFH S-103 SWISS;P01038;CYT_CHICK 15147 1150 618 884 376 1077 -459 0,16439 GSEEYMNMD Y-939 INSR SWISS;P35570;IRS1_RAT 15131 1602 620 1111 694 849 -229 0,25440 EENTYDEYE Y-499 v-Src SWISS;Q01406;SRC8_CHICK 15306 2391 601 1496 1266 777 -176 0,37441 ERRKSHEAE S-62 PKA SWISS;P54227;STHM_MOUSE 11449 1996 997 1497 707 721 276 0,42442 GSPRTPRRG T-252 p34cdc2 SWISS;P06400;RB_HUMAN 10832 2947 1036 1991 1351 961 75 0,86443 DADEYLIPQ Y-1016 EGFR SWISS;P00533;P06268;EGFR_HUMAN 15918 3123 528 1826 1835 936 -408 0,33444 DRLVSARSV S-985 PKC SWISS;P08581;MET_HUMAN 19108 3261 237 1749 2138 1014 -777 0,15

445 GGLTSPGLS S-431 MAPK SWISS;P05787;K2C8_HUMAN 14680 1778 674 1226 781 946 -272 0,20446 GTVPSDNID S-396 GRK2 SWISS;P07550;B2AR_HUMAN 15506 3187 572 1879 1849 887 -315 0,49447 EGSAYEEVP Y-472 EGFR SWISS;P08487;PIP4_BOVIN 15013 1540 634 1087 641 1012 -378 0,11448 ESIISQETY S-31 CKI SWISS;P02663;CAS2_BOVIN 17740 1293 338 815 675 639 -301 0,43449 RRASTIEMP T-17 CaM-II SWISS;P07473;PPLA_PIG 16536 6391 461 3426 4193 437 24 0,84450 SDEESNDDS S-401 CKII SWISS;P12637;CAQC_CANFA 19099 1368 238 803 799 732 -494 0,06451 NVFSSPGGT S-682 p34cdc2 SWISS;P12957;CALD_CHICK 17726 7785 340 4062 5264 923 -583 0,47452 RRASF SWISS;P12928;P04763;Q64618;KPYR_RAT16687 5767 439 3103 3768 214 225 0,76453 AKGGTVKAA T-6 PKC SWISS;P08132;ANX4_PIG 20130 4478 175 2327 3043 369 -194 0,42454 DDEITQDEN T-302 GRK SWISS;P08172;ACM2_HUMAN 14871 1752 650 1201 779 945 -295 0,17455 ETDDYAEII Y-397 SWISS;P34152;FAK_MOUSE 13382 1317 810 1063 358 856 -46 0,41456 RRQHSYDTF S-1303 CaM-II SWISS;Q00960;NME2_RAT 14249 124 730 427 428 1072 -342 0,18457 SFMMTPYVV T-183 SWISS;P45983;JNK1_HUMAN 13190 1610 835 1223 548 875 -40 0,57458 PGTESFVNA S-391 beta-ARK SWISS;P04274;B2AR_MESAU 9852 1006 1087 1047 57 1129 -42 0,34459 SDEEV S-1 PhK SWISS;P02641;P19349;P19350;TRT3_RABIT14550 1834 691 1263 808 1139 -448 0,20460 APQTPGGRR MAPK SWISS;P02687;MBP_BOVIN 17430 506 363 435 101 1049 -686 0,04 �461 DFPLSPPKK S-37 p34cdc2 SWISS;P54227;STHM_MOUSE 26630 1616 25 820 1125 881 -856 0,01 �462 FKAFSPKGS S-597 p34cdc2 SWISS;P12957;CALD_CHICK 20251 38 165 102 90 809 -644 0,01 �463 RRRVTSATR T-7 PKA SWISS;Q28115;GFAP_BOVIN 20544 1884 146 1015 1229 871 -725 0,10464 SKDSSKRGR S-210 SWISS;P27573;MYP0_MOUSE 21634 2251 115 1183 1511 513 -398 0,14465 MSVEEV S-2 CKII SWISS;P07260;IF4E_YEAST 15657 3924 555 2239 2382 1065 -510 0,26466 AALESEDED S-154 CKII SWISS;P12962;IF43_YEAST 12026 515 944 729 304 908 36 0,88467 ARKSTRRSI T-114 PKC SWISS;P08567;P47_HUMAN 16385 2459 484 1471 1396 201 283 0,40468 DLFGSDDEE S-105 CKII SWISS;P34826;EF1B_RABIT 12104 190 937 564 528 788 149 0,24469 FRRLSISTE S-1018 AMP-PK SWISS;P18688;KPB1_RABIT 10148 220 1073 646 603 686 387 0,34470 RVRKSKGKY S-717 PKC SWISS;P19491;GLR2_RAT 23307 2840 78 1459 1953 17 61 0,17471 SNDSTSVSA T-287 GRK SWISS;P08172;ACM2_HUMAN 13455 4202 802 2502 2404 863 -61 0,75472 RRPTVA SWISS;P01099;IPP1_RABIT 14962 1090 642 866 317 933 -291 0,10473 AEPDYGALY Y-771 EGFR SWISS;P19174;PIP4_HUMAN 17526 2949 354 1651 1835 572 -218 0,36474 AVASSPSKA S-26 SWISS;P12624;MACS_BOVIN 19021 1976 245 1110 1224 282 -37 0,41475 DPGTSYRTR S-296 SWISS;P26267;ODPA_ASCSU 16890 1807 417 1112 983 212 205 0,72476 GEGTYGVVY Y-15 SWISS;P13863;CC2_CHICK 18984 2972 249 1610 1925 222 27 0,88477 YTTNSPSKI S-522 CDC28-dependentSWISS;P08153;SWI5_YEAST 23649 3921 69 1995 2724 50 19 0,78478 SPLKSPYKI S-811 p34cdc2 SWISS;P06400;RB_HUMAN 20032 3302 185 1744 2204 114 71 0,84479 RKRTRKE SWISS;P02278;H2B_HUMAN 16605 3480 450 1965 2142 128 322 0,36480 RPPGFTPFR BRADYKININ 15081 2407 625 1516 1260 143 482 0,16481 SASGTPNKE T-468 p34cdc2 SWISS;Q62736;CALD_RAT 15398 2208 590 1399 1144 564 26 0,65482 NSVDTSSLS T-132 CKI SWISS;P13834;UGS1_RABIT 19401 10726 216 5471 7432 547 -331 0,63483 LRANSI SWISS;P13569;CFTR_HUMAN 16781 3672 429 2051 2293 914 -485 0,26484 RDSNYISKG Y-857 PDGFR SWISS;P09619;PGDR_HUMAN 18851 3043 261 1652 1967 424 -163 0,43485 DDEESESD S-512 CKII SWISS;Q01827;VMT2_RAT 13738 1013 782 898 164 1067 -285 0,48

486 VKRISGL S-47 SWISS;P02304;P02305;H4_HUMAN 15244 3176 608 1892 1816 1009 -401 0,29487 RRKDYPALH SITE PKA G-SUBSTRATE (FRAGMENTS) 24488 4376 55 2216 3056 882 -827 0,10488 SEKKSKGLG S-237 SWISS;P27573;MYP0_MOUSE 23575 3115 71 1593 2152 826 -755 0,07489 PPSAYGSVK Y-23 v-Src SWISS;P07355;ANX2_HUMAN 24485 5969 57 3013 4180 527 -470 0,21490 RRFSV SWISS;P12928;P04763;Q64618;KPYR_RAT16606 3678 449 2064 2284 891 -442 0,45491 RGAISAEVY S-99 PKG SWISS;P00514;KAP0_BOVIN 16901 2423 416 1419 1419 1094 -678 0,08492 RKISASEF S-92 GENBANK;L16545 16953 1841 409 1125 1013 869 -460 0,16493 LHRASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)11912 3345 954 2150 1691 917 37 0,95494 RRRPTPAML T-34 PKA SWISS;P07516;IPPD_BOVIN 10538 3202 1053 2128 1520 831 222 0,59495 SGYSSPGSP S-199 PIR;2144820 10199 1042 1068 1055 19 1047 21 0,99496 LGEGTP T-113 SWISS;P06702;S109_HUMAN 10165 1752 1072 1412 480 1029 43 0,90497 RRLSSLRA S-235 p90RSK SWISS;P10660;P08227;RS6_HUMAN 9715 559 1090 824 376 403 687 0,03 �498 RLRLSPSPT S-390 cdc2 SWISS;P11516;LAMC_MOUSE 9984 776 1082 929 216 967 115 0,52499 RKASRKE SWISS;P02278;H2B_HUMAN 15263 536 604 570 48 753 -149 0,09500 LRRWSLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)15189 3046 614 1830 1719 855 -241 0,39501 RRVTSATRR S-8 PKA SWISS;Q28115;GFAP_BOVIN 15828 3332 532 1932 1980 858 -326 0,36502 SLQASIVTD S-113 SWISS;P16051;GBA2_DICDI 15413 1235 588 912 458 772 -184 0,26503 RRATPA SWISS;P12928;P04763;Q64618;KPYR_RAT17367 5380 367 2873 3544 554 -187 0,61504 ADGIYAASG Y-500 v-Fes SWISS;P00542;FES_FSVGA 16931 619 411 515 147 785 -374 0,33505 SSSSSPSRR S-71 cdc2 SWISS;P23913;LBR_CHICK 19193 793 233 513 396 172 61 0,99506 TKSASFLKG S-262 calcium-dependentSWISS;P08995;NO26_SOYBN 20041 3160 184 1672 2104 67 117 0,53507 VESLSSSEE S-32 SWISS;P02666;CASB_BOVIN 19206 4632 229 2430 3113 210 19 0,89508 YRRNSVRFL S-328 SWISS;P14598;NCF1_HUMAN 22665 4685 90 2387 3249 98 -8 0,76509 SPALTGDEA T-502 CKII SWISS;P35570;IRS1_RAT 20243 5885 166 3026 4044 562 -396 0,49510 RKQITVR SWISS;P11217;PHS2_HUMAN 30640 9771 12 4891 6901 47 -35 0,55511 VHNRSKINL S-871 AMP-PK SWISS;P51639;HMDH_RAT 25805 6963 33 3498 4900 64 -31 0,72512 STRRSVRGS S-671 PKC SWISS;P08183;Q12755;MDR1_HUMAN 15558 4349 566 2458 2675 476 90 0,92513 TQSTSGRRR S-340 PKC SWISS;P15336;CREP_HUMAN 16475 1000 471 735 374 407 64 0,81514 VNELSKDIG S-56 CKI SWISS;P02662;CAS1_BOVIN 20099 1008 177 593 588 710 -533 0,21515 RAGETRFTD T-54 CaM-III SWISS;P13639;EF2_HUMAN 17048 342 402 372 43 1147 -745 0,11516 SPSPSFRWP S-333 PKC SWISS;P19836;CTPT_RAT 16269 3839 492 2165 2367 650 -158 0,51517 RVSGSRR S-572 PKC SWISS;P11516;LAMC_MOUSE 16307 439 488 464 35 1082 -594 0,02 �518 VRLRSSVPG S-71 autophosphorylation-dependentSWISS;P20152;VIME_MOUSE 15443 2826 586 1706 1584 1022 -436 0,20519 TAESSQAEE S-204 CKII SWISS;P06836;NEUM_BOVIN 13180 3227 836 2031 1690 944 -108 0,59520 TRRASFSAQ S-230 yeast SWISS;P07248;ADR1_YEAST 11098 4727 1019 2873 2622 915 104 0,90521 VSRTSAVPT S-50 PKA SWISS;P02542;DESM_CHICK 8348 5861 1136 3498 3341 1157 -21 0,87522 REQLSTSEE S-144 CKII SWISS;P02663;CAS2_BOVIN 9347 3152 1105 2129 1448 1156 -51 0,60523 LSYRGYSL S-2 PhK PEPTIDE 11705 647 976 812 233 922 54 0,93524 KASGSSP S-3 PKA PIR;A25335 9077 4387 1117 2752 2312 991 126 0,67525 VVGGSLRGA S-378 PKC SWISS;P18031;PTN1_HUMAN 10908 726 1031 879 216 1048 -17 0,67526 TGDTYTAHA Y-393 c-Abl SWISS;P00520;ABL_MOUSE 8273 356 1140 748 555 1070 70 0,18

527 TVSKTETSQ T-340 RhK SWISS;P02699;OPSD_BOVIN 11750 2966 970 1968 1412 708 262 0,64528 WTSDTQGDE T-232 CKI SWISS;P29361;143Z_SHEEP 14409 528 717 623 133 1109 -392 0,16529 RKFSSARPE S-7 PKA SWISS;P02719;CINA_ELEEL 15391 3145 592 1868 1805 850 -258 0,37530 KKKKASVA SWISS;P12928;P04763;Q64618;KPYR_RAT24767 3865 50 1958 2698 223 -173 0,21531 LRRASLG S-5 BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)16459 1497 476 986 722 644 -168 0,18532 YKNDYYRKR Y-327 SWISS;P08941;KROS_CHICK 24566 2579 53 1316 1786 59 -6 0,77533 SPVHSIADE S-356 PKA SWISS;P19112;Q64594;F16P_RAT 18454 885 286 586 424 119 167 0,31534 LRRASLGAF BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)20345 1126 160 643 683 61 99 0,16535 PRKGSPRKG S-18 sperm-specificSWISS;P06146;H2B2_LYTPI 18976 1443 251 847 843 62 189 0,19536 SSPGSPGTP S-193 SWISS;P19332;TAU_RAT 17696 1625 344 984 906 192 152 0,79537 TKAASEKKT S-204 PKC SWISS;P10522;MYP0_BOVIN 18614 6266 278 3272 4234 195 83 0,98538 LRRSSSVGY S-382 PKA SWISS;P02718;ACHD_TORCA 18977 2874 250 1562 1856 191 59 0,97539 PSEKSEEIT S-116 PKC SWISS;Q62048;PE15_MOUSE 18588 9839 280 5060 6760 97 183 0,78540 SRKLSDFGQ S-16 PKA SWISS;P04176;PH4H_RAT 15058 3191 631 1911 1810 76 555 0,37541 LSGFSFKKN S-169 PKC SWISS;P29966;MACS_HUMAN 18857 2698 260 1479 1724 57 203 0,31542 PSRRSRSRS S-76 RS SWISS;P23913;LBR_CHICK 17301 4322 374 2348 2792 453 -79 0,61543 STDYYREGP Y-710 SWISS;Q03351;TRKC_RAT 17288 1548 379 963 827 776 -397 0,11544 TPAISPSKR S-11 SWISS;P33316;DUT_HUMAN 21961 2353 109 1231 1587 294 -185 0,19545 LVVASAGPT S-156 SWISS;P20338;RRPP_HRSVL 15293 4538 603 2571 2783 1128 -525 0,28546 PVPEYINQS Y-1092 EGFR SWISS;P00533;P06268;EGFR_HUMAN 14422 5431 715 3073 3334 1143 -428 0,31547 SRRPSYRKI S-133 PKA SWISS;P16220;P21934;CREB_HUMAN 17750 7401 336 3869 4996 306 30 0,89548 PEGDYEEVL Y-397 SWISS;P14317;HS1_HUMAN 17902 9148 319 4733 6243 680 -361 0,62549 QEGDTDAGL T-39 CKII PIR;2144820 13432 8488 804 4646 5433 1111 -307 0,60550 SVFSSPSAS S-462 p34cdc2 SWISS;Q62736;CALD_RAT 11524 5766 992 3379 3376 843 149 0,89551 TRKISQTAQ S-2843 PKA SWISS;P11716;RYNR_RABIT 10975 1348 1027 1188 227 415 612 0,07552 PINGSPRTP S-249 p34cdc2 SWISS;P06400;RB_HUMAN 8081 2909 1143 2026 1249 1150 -7 0,91553 KQISVRGL S-15 SWISS;P11217;PHS2_HUMAN 17493 436 356 396 57 179 177 0,11554 SSEITTKDL T-12 CKII SWISS;P01252;THYA_BOVIN 14101 134 743 438 431 882 -139 0,53555 PLSRTLSVS S-7 PKC/CAMIIglycogen (starch) synthase, muscle14702 1974 670 1322 922 791 -121 0,40556 LRRASLRG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)14991 424 639 532 152 1052 -413 0,27557 TEGQYELQP Y-494 SWISS;P16277;BLK_MOUSE 14501 4575 705 2640 2736 771 -66 0,72558 TSPSSSPAS S-346 SWISS;P19836;CTPT_RAT 14544 2003 693 1348 926 864 -171 0,42559 PPSAYGSVK Y-23 v-Src SWISS;P07355;ANX2_HUMAN 17789 2460 332 1396 1505 302 30 0,69560 RTKRSGSV S-26 PKC phosphorylase b kinase beta regulatory chain22866 3748 87 1917 2588 147 -60 0,45561 KAQEYFNIK Y-393 SWISS;P02718;ACHD_TORCA 17995 537 309 423 161 31 278 0,26562 KRQSSTSNA S-700 SWISS;P12798;KPBB_RABIT 15011 1010 636 823 264 35 601 0,27563 QKRPSQRSKYL S-7 SWISS;P02687;MBP_BOVIN 19259 2640 224 1432 1709 3 221 0,32564 QSPSSSPTH S-322 SWISS;P19836;CTPT_RAT 12402 3772 904 2338 2028 73 831 0,13565 KGHEYTNIK Y-542 SWISS;Q06124;PTNB_HUMAN 15244 1546 608 1077 663 79 529 0,09566 KRRSSSYHV S-687 PKA SWISS;P04775;CIN2_RAT 24261 2372 60 1216 1635 20 40 0,46567 KAKQISVRGL SWISS;P11217;PHS2_HUMAN 29961 7403 14 3709 5225 13 1 0,71

568 KKAESPVKE S-666 SWISS;P12839;NFM_RAT 16162 28 501 264 335 228 273 0,47569 KSLNYIDLD Y-1172 INSR SWISS;P35570;IRS1_RAT 18728 1015 267 641 529 337 -70 0,33570 KRKQISVRGL S-15 SWISS;P11217;PHS2_HUMAN 20159 1173 172 673 708 16 156 0,53571 SRGDYMTMQ Y-987 INSR SWISS;P35570;IRS1_RAT 17253 2337 383 1360 1382 745 -362 0,31572 KKKGSGEDD S-91 CKII SWISS;P12962;IF43_YEAST 15231 1742 611 1177 800 827 -216 0,31573 KTSPSSSPA S-345 SWISS;P19836;CTPT_RAT 14311 5377 723 3050 3291 1055 -332 0,46574 PRRRSSFGI S-370 PKA SWISS;P02714;ACHG_TORCA 14900 7618 647 4132 4929 1158 -511 0,39575 KLINSIADT S-359 SWISS;P06784;STE7_YEAST 15240 7268 610 3939 4708 949 -339 0,59576 LDDQYTSSS Y-518 SWISS;P24604;TEC_MOUSE 16064 8391 512 4451 5571 743 -231 0,70577 PYDNYVPSA Y-778 SWISS;P09619;PGDR_HUMAN 15530 4668 567 2618 2900 1108 -541 0,28578 SRQLSSGVS S-82 MAPKAPK2SWISS;P04792;HS27_HUMAN 12279 5209 921 3065 3032 1089 -168 0,61579 KNIVTPRTP T-94 MAPK1 SWISS;P02687;MBP_BOVIN 15341 639 597 618 29 523 74 0,65580 LKLASPELE S-73 p34cdc2 SWISS;P05412;AP1_HUMAN 9628 1377 1094 1235 200 912 182 0,33581 QLIDSMANS S-217 c-Raf SWISS;Q02750;MPK1_HUMAN 12037 3409 943 2176 1744 1120 -177 0,41582 KRFGSKAHM S-374 PKA SWISS;P29476;NOS1_RAT 18203 4285 298 2292 2819 108 190 0,58583 LNDSSEEED S-32 CKII SWISS;P03129;VE7_HPV16 13965 4146 756 2451 2397 966 -210 0,52584 QRATSNVFA S-19 SWISS;P24844;MLRN_HUMAN 18300 1481 294 888 840 561 -267 0,11585 SRSRTPSLP T-212 GSK3 PIR;2144820 15518 3306 571 1939 1934 880 -309 0,36586 KRPSARAKA SWISS;P02687;MBP_BOVIN 20484 811 152 482 466 30 122 0,08587 LRAPSWIDT S-59 SWISS;P02510;CRAB_BOVIN 17580 1061 350 705 503 196 154 0,77588 QRRTSVSGE S-144 yeast SWISS;P07278;KAPR_YEAST 15738 1838 545 1192 915 790 -245 0,24589 HATPSPPVD S-357 GSK3 SWISS;P01103;MYB_CHICK 13541 1112 795 954 224 88 707 0,03 �590 KRPSQRAKA SWISS;P02687;MBP_BOVIN 15306 3893 600 2247 2329 6 594 0,32591 EDAESEDEE S-125 SWISS;P06748;NPM_HUMAN 15155 4019 617 2318 2406 54 563 0,17592 ENAEYLRVA Y-1197 EGFR SWISS;P00533;P06268;EGFR_HUMAN 12124 4639 933 2786 2620 89 844 0,28593 HKRKSSQAL S-14 PKA SWISS;P03373;ERBA_AVIER 27255 20543 22 10282 14510 83 -61 0,77594 LAYESHESL S-22 SWISS;P07507;MGP_BOVIN 20624 3393 145 1769 2296 376 -231 0,26595 PYKFPSSPLRIPGZ na na na 18351 105 291 198 131 193 98 0,89596 HRTPSRSFG S-47 SWISS;P00523;Q91345;Q92013;SRC_CHICK16649 2956 443 1700 1777 446 -3 0,63597 LSGFSFKKN S-169 PKC SWISS;P29966;MACS_HUMAN 23550 3992 73 2033 2771 56 17 0,88598 EEEEYMPME Y-315 c-Src SWISS;P12906;TAMI_POVMC 15049 1018 633 826 272 780 -147 0,47599 EQFSTVKGV T-485 GRK5 SWISS;P43249;GRK5_BOVIN 20940 7699 137 3918 5347 999 -862 0,28600 IDKISRIGF S-419 PKC SWISS;P07727;GRA1_RAT 18076 4218 306 2262 2766 519 -213 0,50601 MHRQETVDA T-286 SWISS;P11275;KCCA_RAT 16754 3569 432 2000 2218 1030 -598 0,19602 PYKFPSSPLRIPGZ na na na 15758 9619 540 5079 6420 1046 -506 0,56603 IGSESTEDQ S-63 CKII SWISS;P02662;CAS1_BOVIN 12285 6251 920 3585 3769 1155 -235 0,47604 MRRNSFTPL S-466 PKA SWISS;P26285;F26H_BOVIN 15164 5841 616 3229 3695 737 -121 0,71605 EEQEYVQTV Y-20 EGFR SWISS;P04083;ANX1_HUMAN 16663 4573 442 2507 2921 1010 -568 0,29606 ERRVSNAGG S-157 PKA SWISS;P50552;VASP_HUMAN 16182 5448 498 2973 3500 1064 -566 0,33607 IREESPPHS S-343 SWISS;P09258;VGLI_VZVD 13031 2546 852 1699 1198 981 -129 0,60608 NDSVYANWM Y-1096 SWISS;P07949;RET_HUMAN 18078 1351 305 828 740 643 -338 0,14

609 PYKFPSSPLRIPGZ na na na 17145 4675 396 2536 3026 590 -194 0,53610 ISTESQPNG S-1023 SWISS;P18688;KPB1_RABIT 14866 3584 652 2118 2073 1141 -489 0,19611 NGNNYVYID Y-567 SWISS;P05532;KKIT_MOUSE 15651 1008 557 783 319 1059 -502 0,12612 EIKKSWSRW S-467 PKA SWISS;P25107;PTRR_DIMDA 21094 1706 131 919 1114 206 -75 0,26613 ESPASDEAE S-89 SWISS;P02316;HG14_BOVIN 18309 455 293 374 115 536 -243 0,02 �614 KRKSSQALV S-15 PKA SWISS;P03373;ERBA_AVIER 25303 811 40 426 545 33 7 0,58615 NNMPSSDDG S-250 GRK SWISS;P08172;ACM2_HUMAN 15807 1539 536 1037 709 473 63 0,72616 PYKFPSSPLRIPGZ na na na 19207 625 228 427 281 683 -455 0,02 �617 GGVDYKNIH Y-699 SWISS;P07333;KFMS_HUMAN 16556 3118 458 1788 1881 123 335 0,66618 ALRPSTSRS S-46 PKA SWISS;P20152;VIME_MOUSE 12736 1541 876 1208 470 78 798 0,13619 DEAATKTQT T-1833 MHCK SWISS;P08799;MYS2_DICDI 13002 3724 856 2290 2028 178 678 0,23620 FARKSTRRS S-113 PKC SWISS;P08567;P47_HUMAN 13785 5705 776 3240 3485 131 645 0,34621 GLGESRKDK S-214 PKC SWISS;P10522;MYP0_BOVIN 13331 593 816 705 157 126 690 0,14622 DTHRTPSRS T-45 p34cdc2 SWISS;P00523;Q91345;Q92013;SRC_CHICK16564 4044 457 2250 2536 356 101 0,90623 EKESSNDST S-283 GRK SWISS;P08172;ACM2_HUMAN 15808 4106 535 2321 2525 506 29 0,78624 GNHTYQEIA v-Src SWISS;P49023;PAXI_HUMAN 19135 1485 234 859 885 363 -129 0,65625 APVASPAAP S-62 MAPK SWISS;P17599;SYN1_BOVIN 14748 33 666 349 448 464 202 0,03 �626 DGNNSDEES S-397 CKII SWISS;P12637;CAQC_CANFA 16147 2727 504 1616 1572 747 -243 0,42627 FMTEYVVTR Y-215 SWISS;Q07176;MSK7_MEDSA 21980 3696 108 1902 2537 167 -59 0,51628 GRGLSLSRF S-109 PKA SWISS;P02687;MBP_BOVIN 20676 6660 141 3400 4609 452 -311 0,44629 EDTLSDSDD S-261 CKII SWISS;P04198;MYCN_HUMAN 18946 5467 254 2860 3686 928 -674 0,27630 ENQASEEED S-102 CKII SWISS;P07516;IPPD_BOVIN 17689 7963 345 4154 5387 1061 -716 0,40631 GRRESLTSF S-1755 PKA SWISS;P29994;IP3R_RAT 17567 7606 351 3978 5130 728 -377 0,56632 ARVFSVLRE S-411 CaM-II SWISS;P48452;P2BA_BOVIN 16765 4408 431 2420 2812 458 -27 0,70633 DLPLSPSAF S-220 MAPK2 SWISS;P22893;TTP_MOUSE 16784 3045 427 1736 1851 585 -158 0,61634 GDKKSKKAK S-23 PKC SWISS;P06685;ATN1_RAT 15567 5175 565 2870 3259 910 -345 0,50635 GSDVSFNEE S-1423 CKII SWISS;P06786;TOP2_YEAST 13586 113 789 451 478 1015 -226 0,21636 EEKGSPLNA S-352 SWISS;P18031;PTN1_HUMAN 13870 1921 766 1343 816 1090 -324 0,13637 ERQKTQTKL T-289 MLCK SWISS;P25323;KMLC_DICDI 19010 4906 247 2576 3294 651 -404 0,36638 GSPGTPGSR T-205 p34cdc2-p58cyclinPIR;2144820 15766 4627 539 2583 2891 1092 -553 0,28639 AVVRTPPKS T-231 GSK3 PIR;2144820 19241 1354 227 791 797 408 -181 0,19640 DRKLSTKEA S-683 SWISS;P08183;Q12755;MDR1_HUMAN 20370 1945 157 1051 1264 994 -837 0,04 �641 GGIRSLNVA S-113 isocitrate SWISS;P08200;IDH_ECOLI 19091 2603 239 1421 1672 884 -645 0,10642 GTRLSLARM S-34 PKA SWISS;Q28115;GFAP_BOVIN 19120 1877 236 1057 1161 334 -98 0,28643 EGNKSPAPK S-717 p34cdc2 SWISS;P12957;CALD_CHICK 20342 1316 161 738 817 589 -428 0,04 �644 ESHESMESY S-25 SWISS;P08493;MGP_HUMAN 18878 1398 257 827 807 839 -582 0,06645 RRAISGDLT S-1700 PKA SWISS;P15381;CICC_RABIT 19120 11510 235 5873 7973 147 88 0,95646 SAYRSVDEV S-343 branched SWISS;P11960;ODBA_RAT 12542 1843 895 1369 671 176 719 0,14647 NTVSTSLGH T-310 GRK SWISS;P08172;ACM2_HUMAN 13280 1467 827 1147 453 333 494 0,20648 RASLG S-5 BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)12975 5822 859 3341 3510 584 275 0,82649 AKDASKRGR S-181 PKC SWISS;P10522;MYP0_BOVIN 14395 1078 718 898 255 162 556 0,07

650 DDEASTTVS S-334 PKC SWISS;P02699;OPSD_BOVIN 12570 5612 891 3252 3338 526 365 0,70651 ETAESSQAE S-203 CKII SWISS;P06836;NEUM_BOVIN 14984 6270 640 3455 3981 1042 -402 0,47652 RRPTSPVSR S-219 SWISS;P03255;E1A_ADE05 14714 4373 668 2520 2620 784 -116 0,79653 SFMDSSGLG S-57 SWISS;P10727;SP21_BACSU 15665 3301 552 1927 1944 567 -15 0,67654 QKRPSQRSK S-7 PKA SWISS;P02687;MBP_BOVIN 21771 3397 114 1755 2321 208 -94 0,39655 SDEEH S-1 SWISS;P23301;IF52_YEAST 17290 643 377 510 188 988 -611 0,20656 APLTPGGRR SWISS;P02687;MBP_BOVIN 20946 4577 136 2357 3140 959 -823 0,16657 DEPSTPYHS T-72 GSK3 SWISS;P11845;IPP2_RABIT 17018 7880 404 4142 5286 823 -419 0,56658 FGSRSLYGL S-59 SWISS;P48668;K2CE_HUMAN 20084 7942 178 4060 5490 668 -490 0,45659 RRRSSKDTS S-897 PKC SWISS;Q05586;P35437;NMZ1_HUMAN 16196 6450 497 3474 4210 475 22 0,84660 SKAGSLGNI S-324 PKA PIR;2144820 18090 6151 304 3228 4135 444 -140 0,63661 MSGDEM S-2 CKII SWISS;P41035;IF2B_RABIT 12581 6754 890 3822 4147 1025 -135 0,74662 AAATTPAAE T-172 SWISS;P07936;NEUM_RAT 12255 6915 923 3919 4237 1166 -243 0,52663 ARKKSSAQL S-179 PKA SWISS;P09526;RAPB_HUMAN 11802 5045 964 3005 2886 888 76 0,98664 DKVTSPTKV S-527 p34cdc2 SWISS;Q62736;CALD_RAT 11712 2246 974 1610 899 859 115 0,89665 FRRFTPDSL T-17 PKA SWISS;P02719;CINA_ELEEL 15398 5463 589 3026 3447 987 -398 0,45666 RVRISADAM S-146 PKA SWISS;P02646;TRIC_RABIT 14371 3468 719 2093 1944 815 -96 0,58667 SNDDSDDDD S-405 CKII SWISS;P12637;CAQC_CANFA 15889 4146 530 2338 2557 905 -375 0,38668 RRPTPA T-35 SWISS;P01099;IPP1_RABIT 17749 4516 337 2426 2955 1103 -766 0,18669 AEGSSNVFS S-13 MLCK SWISS;P02609;MLRS_CHICK 19037 955 244 600 503 874 -630 0,05670 AVADSESED S-74 CKII SWISS;P00515;KAP2_BOVIN 20951 2337 135 1236 1557 425 -290 0,13671 DNPDYQQDF Y-1172 EGFR SWISS;P00533;P06268;EGFR_HUMAN 18873 4294 258 2276 2854 657 -399 0,32672 GDVKYADIE Y-763 SWISS;P09619;PGDR_HUMAN 20384 3024 156 1590 2028 689 -533 0,14673 YTRFSLARQ S-24 PKC SWISS;P02786;TRSR_HUMAN 11345 2801 1006 1903 1269 296 710 0,17674 SPKQSPSSS S-319 SWISS;P19836;CTPT_RAT 12264 28 922 475 632 355 567 0,39675 RKRSRKE S-32 SWISS;P02278;H2B_HUMAN 11441 1889 998 1444 630 548 450 0,23676 RPPGFSPFR S-6 BRADYKININ 11864 2785 959 1872 1291 207 752 0,15677 SARVYENVG Y-580 SWISS;Q06124;PTNB_HUMAN 9475 1478 1102 1290 266 769 333 0,16678 NRSASEPSL S-621 AMP-PK SWISS;P04049;KRAF_HUMAN 7845 1340 1148 1244 136 110 1038 0,23679 KGYSLG SWISS;P00698;Q90884;LYC_CHICK 13844 2431 769 1600 1175 111 658 0,40680 RDPVTENAV T-271 GRK SWISS;P08172;ACM2_HUMAN 14654 1320 677 999 455 892 -215 0,19681 AVRRSDRA S-20 SWISS;P02646;TRIC_RABIT 17169 6217 395 3306 4117 736 -341 0,53682 VKRGSGL SWISS;P02304;P02305;H4_HUMAN 16591 3219 454 1836 1955 804 -350 0,31683 RRKDTPALH SITE PKA G-SUBSTRATE (FRAGMENTS) 17278 1806 380 1093 1008 693 -313 0,16684 SEITTKDLK T-13 CKII SWISS;P01252;THYA_BOVIN 18741 2660 266 1463 1693 252 14 0,64685 PNVSYIASR Y-447 SWISS;P23646;KSGM_DROME 17289 6523 378 3451 4345 394 -16 0,79686 RRDSV SWISS;P12928;P04763;Q64618;KPYR_RAT18629 12889 275 6582 8920 1071 -796 0,51687 RFTDTRKDE T-59 CaM-III SWISS;P13639;EF2_HUMAN 16685 7791 440 4115 5198 796 -356 0,60688 RKISASEA GENBANK;L16545 13801 5689 774 3232 3476 948 -174 0,66689 LARASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)13598 3558 788 2173 1959 816 -28 0,73690 RRRLSSLRA S-235 S6K SWISS;P10660;P08227;RS6_HUMAN 12554 6821 893 3857 4192 1005 -112 0,77

691 SGYISSLEY S-206 CKII SWISS;P00736;C1R_HUMAN 13923 5498 761 3130 3350 661 100 0,95692 LARNSI SWISS;P13569;CFTR_HUMAN 11909 2503 955 1729 1095 935 20 0,87693 TIAVG T-1 SWISS;P06005;PSBD_SPIOL 12358 5502 911 3206 3246 801 110 0,96694 RLQDYEEKT Y-353 EGFR SWISS;P15311;P23714;EZR1_HUMAN 14027 6399 751 3575 3993 890 -139 0,72695 FKKSFKL S-162 SWISS;P29966;MACS_HUMAN 16642 4307 445 2376 2731 973 -528 0,29696 LRRPSLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)19924 3942 193 2068 2651 906 -713 0,19697 RRVRSQEPG S-346 SWISS;P30936;SSR3_RAT 19509 3154 210 1682 2082 1112 -902 0,07698 SLDDSGSAM S-408 GRK SWISS;P15823;A1AB_RAT 22419 886 97 492 558 927 -830 0,02 �699 RRASVA S-43 SWISS;P12928;P04763;Q64618;KPYR_RAT19856 2198 195 1197 1417 808 -613 0,08700 ADDEYAPKQ Y-434 c-Src SWISS;P00528;SRC1_DROME 21845 161 112 137 35 756 -644 0,00 �701 SSSSSPKAE S-133 SWISS;P12624;MACS_BOVIN 12295 3490 915 2202 1820 188 727 0,18702 TKKTSFVNF S-218 PKA SWISS;P41035;IF2B_RABIT 14200 2479 736 1608 1233 161 575 0,20703 VEPLTPSGE T-693 ERT SWISS;P00533;P06268;EGFR_HUMAN 10309 751 1062 906 220 624 438 0,47704 YRLPSNVDQ S-122 crystalline SWISS;P02470;CRAA_BOVIN 8078 694 1144 919 318 524 620 0,37705 SPAISIHEI S-985 PhK SWISS;P18688;KPB1_RABIT 10361 4037 1058 2547 2106 616 442 0,55706 RKQISVR S-15 SWISS;P11217;PHS2_HUMAN 8528 3270 1131 2200 1512 92 1039 0,40707 VGWPTVRER T-15 PKC SWISS;P03406;NEF_HV1BR 10259 728 1066 897 239 504 562 0,24708 STRRSIRLP S-117 PKC SWISS;P08567;P47_HUMAN 14464 1100 709 905 277 439 270 0,48709 TPQVSDTMR S-43 PKA SWISS;P13280;GLYG_RABIT 14506 1454 704 1079 530 557 147 0,93710 VNATYVNVK Y-1356 SWISS;P08581;MET_HUMAN 15987 2459 522 1490 1369 741 -219 0,47711 RAAHSIKGG S-48 SWISS;P07363;CHEA_ECOLI 18620 28 277 152 176 280 -3 0,08712 SPSLSRHSS S-67 GSK3 SWISS;P13834;UGS1_RABIT 17294 4381 376 2379 2832 170 206 0,63713 RTPPPSG SWISS;P02687;MBP_BOVIN 13328 6791 818 3805 4224 1026 -208 0,66714 VRKRTLRRL T-678 PKC SWISS;P00533;P06268;EGFR_HUMAN 13764 5460 778 3119 3311 925 -147 0,68715 TADISEDEE S-362 CKII SWISS;P19836;CTPT_RAT 17008 8362 405 4384 5627 931 -526 0,52716 TRQTSVSGQ S-568 CaM-II SWISS;P17599;SYN1_BOVIN 14076 5397 749 3073 3287 1084 -335 0,44717 VSEEYLDLR Y-754 SWISS;P22455;FGR4_HUMAN 16026 4253 517 2385 2642 870 -353 0,40718 REQESSGEE S-120 CKII SWISS;P11845;IPP2_RABIT 13496 5837 798 3318 3563 896 -98 0,76719 GTKRSGSV SWISS;P12798;KPBB_RABIT 14416 5300 716 3008 3241 330 386 0,68720 NRLQTMKEE T-199 PKC SWISS;P11516;LAMC_MOUSE 11710 2346 975 1661 970 810 165 0,71721 VVELSGESD S-1353 CKII SWISS;P06786;TOP2_YEAST 15521 820 570 695 176 851 -281 0,07722 TESQYQQQP Y-522 SWISS;P08631;HCK_HUMAN 14941 1420 644 1032 549 729 -85 0,71723 TVKSSKGGP S-27 PKC SWISS;P04083;ANX1_HUMAN 16475 2851 471 1661 1683 611 -140 0,43724 WTSDSAGEE S-232 CKI SWISS;P27348;143T_HUMAN 19490 3415 212 1814 2265 1021 -809 0,17725 RKAASVIAK S-43 PKC SWISS;Q06766;DPOB_RAT 28427 2761 17 1389 1940 58 -41 0,21726 LRRASLDG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)19486 2650 213 1431 1723 822 -609 0,12727 LRRASGG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)19203 345 231 288 81 699 -468 0,15728 YHTTSHPGT S-50 CK SWISS;P02730;B3AT_HUMAN 18928 426 256 341 120 313 -57 0,26729 QTYRSFHDL S-72 PKG SWISS;P00516;KGPA_BOVIN 18654 12533 273 6403 8669 71 202 0,71730 LRRASLGAA BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)10717 2893 1043 1968 1308 398 645 0,25731 PRKGSPKRG S-23 sperm-specificSWISS;P06146;H2B2_LYTPI 11355 15 1005 510 700 678 327 0,00 �

732 SSPASLSRA S-350 SWISS;P19836;CTPT_RAT 3713 462 1176 819 505 976 200 0,27733 TKAASEKKS S-233 SWISS;P27573;MYP0_MOUSE 8139 1340 1141 1240 141 209 932 0,26734 LRRPSDQEV S-266 PKA SWISS;P16236;REL_CHICK 10697 426 1044 735 437 968 76 0,55735 PSAPSPQPK S-8 p34cdc2-p58cyclinSWISS;P04177;TY3H_RAT 8353 1302 1135 1218 118 713 422 0,12736 SRKGSGFGH S-233 PKA SWISS;P08033;CXB1_RAT 16409 1385 479 932 641 181 298 0,37737 LSGESDLEI S-1356 CKII SWISS;P06786;TOP2_YEAST 13612 744 787 765 30 656 131 0,96738 PSQRSKYLA S-10 PKA SWISS;P02687;MBP_BOVIN 15992 1324 520 922 568 806 -286 0,20739 STDEYLDLS Y-760 SWISS;P22607;FGR3_HUMAN 17686 51 346 198 209 379 -33 0,13740 TNHIYSNLA Y-936 SWISS;P10721;KKIT_HUMAN 16870 3742 419 2081 2350 658 -239 0,44741 LSRHSSPHQ S-70 CKI SWISS;P13834;UGS1_RABIT 18409 895 288 592 429 1020 -732 0,01 �742 PTRHSRVAE S-50 PKA SWISS;P08928;LAM0_DROME 16246 5399 494 2946 3468 820 -326 0,50743 SRRPSRATW S-397 PKA SWISS;P04004;VTNC_HUMAN 19629 5263 202 2732 3579 718 -516 0,32744 PASQTPNKT T-33 p34cdc2 SWISS;P00523;Q91345;Q92013;SRC_CHICK17609 1223 347 785 620 1002 -655 0,03 �745 QDPVSPSLV S-232 GRK SWISS;P08172;ACM2_HUMAN 16464 7734 474 4104 5133 792 -318 0,62746 STTVSKTET S-338 SWISS;P02700;OPSD_SHEEP 14267 7125 727 3926 4524 544 183 0,97747 TRKISASEF S-92 PKA GENBANK;L16545 18711 4886 270 2578 3264 649 -379 0,36748 PGPQSPGSP S-345 SWISS;P14598;NCF1_HUMAN 9960 2147 1083 1615 753 612 471 0,19749 KQGSGRGL SWISS;P11217;PHS2_HUMAN 17794 3875 331 2103 2506 730 -399 0,31750 SSEESITRI S-37 CKI SWISS;P02666;CASB_BOVIN 19558 1114 208 661 641 797 -589 0,03 �751 PLAGSPVIA S-370 cdc2 SWISS;P19138;P20426;KC21_HUMAN 17942 3073 315 1694 1950 312 3 0,85752 LGSPLRRR SWISS;P20152;VIME_MOUSE 21372 5370 124 2747 3710 574 -450 0,28753 TEDQYSLVE Y-607 c-Src SWISS;P27986;P85A_HUMAN 19355 3338 217 1777 2207 467 -250 0,31754 TRTYSLGSA S-38 CaM-II SWISS;P20152;VIME_MOUSE 21460 3013 120 1566 2046 484 -364 0,16755 PPSAYATVK Y-23 p60-Src SWISS;P17785;ANX2_CHICK 23532 1349 74 712 902 413 -339 0,04 �756 RTKRSGSV S-26 PKC phosphorylase b kinase beta regulatory chain24866 4412 48 2230 3086 153 -105 0,33757 KAKVTGRWK T-280 PKC SWISS;P13789;TRT1_BOVIN 19781 1313 197 755 789 14 183 0,13758 KRPTQRAKY SWISS;P02687;MBP_BOVIN 16386 1368 483 926 626 19 464 0,28759 PLSRTLSVRSL SWISS;P13834;UGS1_RABIT 11497 2987 995 1991 1408 780 215 0,86760 QSPGSPLEE S-348 SWISS;P14598;NCF1_HUMAN 7244 2889 1159 2024 1223 1024 135 0,42761 KGGSYSQAA Y-344 SWISS;P01889;1B02_HUMAN 9586 1022 1099 1061 54 829 270 0,14762 KRRRSSKDT S-896 PKC SWISS;Q05586;P35437;NMZ1_HUMAN 19754 6119 198 3159 4187 386 -188 0,52763 KAAQISVRGL SWISS;P11217;PHS2_HUMAN 18165 3016 301 1658 1920 247 54 0,84764 KISITSRKA T-36 ERA SWISS;P06616;ERA_ECOLI 20396 3419 155 1787 2308 52 103 0,34765 KSKISASRK S-43 PKC SWISS;P08057;TRIC_BOVIN 17837 449 328 389 86 41 287 0,22766 KRKQISVGGL SWISS;P11217;PHS2_HUMAN 18560 99 281 190 129 159 122 0,48767 SRALSRQLS S-78 MAPKAPK2SWISS;P04792;HS27_HUMAN 19204 5351 230 2790 3621 401 -171 0,51768 KKKFSFKKP S-92 PKC SWISS;P28667;MRP_MOUSE 29406 2568 15 1292 1805 29 -14 0,50769 KTRSSRAGL S-19 PKA SWISS;P02261;H2A1_HUMAN 25584 1995 36 1015 1385 301 -265 0,04 �770 PAPGSPEPP S-89 SWISS;P03255;E1A_ADE05 17977 2125 310 1217 1283 1134 -824 0,14771 KKSWSRWTL S-469 PKA SWISS;P25107;PTRR_DIMDA 25811 2801 32 1416 1958 84 -52 0,36772 LASSSKEEN S-23 SWISS;P01036;CYTS_HUMAN 16388 6422 482 3452 4200 458 24 0,83

773 PLRRTLSVA S-7 PKC/CAMIIglycogen (starch) synthase, muscle18782 7426 264 3845 5064 794 -530 0,44774 SRPSSNRSY S-25 autophosphorylation-dependentSWISS;P20152;VIME_MOUSE 17108 5636 398 3017 3704 383 15 0,79775 KNDYYRKRG Y-328 SWISS;P08941;KROS_CHICK 17245 5395 385 2890 3543 133 252 0,59776 LIEDAEYTA SWISS;P00523;Q91345;Q92013;SRC_CHICK13415 2308 806 1557 1062 734 72 0,90777 QKAQTERKS T-190 PKC SWISS;P13789;TRT1_BOVIN 14607 2927 685 1806 1586 1045 -360 0,28778 KREASLDNQ S-598 PKA SWISS;P06593;PHY3_AVESA 15372 5068 596 2832 3162 860 -264 0,53779 LMDKYHVDN Y-195 SWISS;P03264;DNB2_ADE02 19244 2562 226 1394 1652 664 -438 0,19780 QQGMTVYGL T-259 SWISS;P26932;CLPO_CHICK 17804 4748 330 2539 3124 711 -381 0,39781 SRSRSRSRS S-80 RS SWISS;P23913;LBR_CHICK 21140 4473 129 2301 3072 438 -309 0,31782 KRNSSPPPS S-1178 PKA SWISS;P20020;ATCP_HUMAN 20645 5073 142 2607 3487 665 -523 0,30783 LQRYSSDPT S-1070 SWISS;P00533;P06268;EGFR_HUMAN 22998 3469 84 1777 2394 633 -549 0,11784 QRRTSLTGS S-1757 PKA SWISS;P07293;CIC1_RABIT 26352 12276 28 6152 8661 689 -661 0,36785 GVRQSRASD S-145 SWISS;P09693;CD3G_HUMAN 11654 3299 983 2141 1637 911 72 1,00786 KRPSNRAKA SWISS;P02687;MBP_BOVIN 16088 1710 511 1111 848 647 -136 0,39787 EAVTSPRFI S-31 SWISS;P04177;TY3H_RAT 13126 1776 843 1310 660 783 60 0,95788 EMTGYVATR Y-182 SWISS;P47811;MP38_MOUSE 11586 843 987 915 102 465 522 0,62789 HKIKSGAEA S-54 PKC SWISS;Q06766;DPOB_RAT 9288 23 1107 565 766 1076 31 0,64790 LARRSTTDA S-116 PKA SWISS;P01233;CGHB_HUMAN 10800 3136 1038 2087 1484 1161 -123 0,64791 PYKFPSSPLRIPGZ na na na 12662 1608 882 1245 513 744 138 0,89792 HRQETVEAL T-287 CaM-II SWISS;P08413;KCCB_RAT 13066 141 850 495 502 878 -28 0,82793 LRRFSLATM S-128 PKA SWISS;P00176;CPB1_RAT 13409 1209 808 1009 284 621 187 0,88794 EEEAYGWMD Y-87 v-Src SWISS;P01350;GAST_HUMAN 18217 1913 296 1105 1144 819 -523 0,19795 EQESSGEED S-121 CKII SWISS;P11845;IPP2_RABIT 16841 392 422 407 21 985 -563 0,15796 IAADSEAEQ S-99 CKII SWISS;P35570;IRS1_RAT 13169 1236 839 1038 281 1133 -294 0,15797 MGEASGAQL S-369 beta-ARK SWISS;P04274;B2AR_MESAU 12503 2896 898 1897 1413 1100 -202 0,34798 PYKFPSSPLRIPGZ na na na 16273 5042 490 2766 3219 877 -387 0,44799 IGRFSEPHA S-139 SWISS;P00517;KAPA_BOVIN 18277 5295 295 2795 3535 969 -674 0,29800 MQLKSEIKQ S-1007 PhK SWISS;P18688;KPB1_RABIT 19502 5830 211 3021 3973 227 -16 0,78801 EEQEYIKTV Y-9 EGFR SWISS;P19619;ANX1_PIG 18357 3046 290 1668 1948 531 -241 0,31802 ERRPSNVSQ S-623 PKA SWISS;P04775;CIN2_RAT 16035 6446 515 3480 4194 703 -188 0,67803 IPPHTPVRT T-373 p34cdc2 SWISS;P06400;RB_HUMAN 13356 3621 811 2216 1987 947 -136 0,59804 NDSTSVSAV S-288 GRK SWISS;P08172;ACM2_HUMAN 12473 2386 900 1643 1051 599 301 0,60805 PYKFPSSPLRIPGZ na na na 14034 4341 750 2545 2539 396 354 0,70806 ISQESSEEE S-90 SWISS;P17095;HMGY_MOUSE 15254 6295 606 3450 4022 950 -344 0,54807 NGDASPAAA S-45 SWISS;P12624;MACS_BOVIN 17252 7196 384 3790 4816 913 -529 0,47808 EHVSSSEES S-24 CKI SWISS;P02663;CAS2_BOVIN 14518 7500 702 4101 4807 579 123 0,96809 ESMESYEVS S-28 SWISS;P08494;MGP_RAT 20369 7675 158 3916 5315 543 -385 0,47810 KQLASFEIY S-119 SWISS;P01036;CYTS_HUMAN 22897 1423 85 754 946 803 -718 0,05811 NMPSSDDGL S-251 GRK SWISS;P08172;ACM2_HUMAN 20196 3562 168 1865 2400 426 -258 0,28812 PYKFPSSPLRIPGZ na na na 23565 6814 72 3443 4768 671 -599 0,25813 GGTGTPNKE T-688 p34cdc2 SWISS;P12957;CALD_CHICK 13583 1392 791 1091 425 872 -81 0,69

814 ALGISYGRK S-46 PKC SWISS;P04326;TAT_HV112 19796 1942 196 1069 1234 90 106 0,79815 DDSGSAMSG S-410 GRK SWISS;P15823;A1AB_RAT 14612 371 683 527 221 640 43 0,93816 EYVQTVKSS T-23 PKC SWISS;P04083;ANX1_HUMAN 16150 5074 502 2788 3233 998 -496 0,55817 GKTDYMGEA Y-364 INSR SWISS;P04274;B2AR_MESAU 15494 7586 573 4079 4959 1104 -531 0,44818 DSTYYKASK Y-577 SWISS;P34152;FAK_MOUSE 19643 5861 201 3031 4002 239 -38 0,74819 EKEISDDEA S-225 CKII SWISS;P08238;HS9B_HUMAN 10940 2862 1029 1945 1296 722 307 0,46820 GNGDYMPMS Y-628 INSR SWISS;P35570;IRS1_RAT 13480 5485 800 3143 3313 992 -192 0,66821 APRYPGGRR SWISS;P02687;MBP_BOVIN 18765 381 265 323 82 830 -565 0,21822 DGNKSPAPK S-497 p34cdc2 SWISS;Q62736;CALD_RAT 18091 1601 303 952 918 846 -543 0,10823 FMTEYVATR Y-185 SWISS;P14681;KSS1_YEAST 16905 2605 415 1510 1549 789 -374 0,33824 GRASSHSSQ S-404 S6K SWISS;P11516;LAMC_MOUSE 12859 2290 866 1578 1007 983 -117 0,64825 EDSTYYKAS Y-576 SWISS;P34152;FAK_MOUSE 17958 5747 312 3029 3843 725 -413 0,45826 ENPQYFRQG Y-516 SWISS;Q03351;TRKC_RAT 19039 5956 243 3099 4040 876 -633 0,34827 GRPITPPRN T-320 cdc2 SWISS;P08129;P22802;P20653;PP1A_HUMAN17342 3690 370 2030 2348 919 -549 0,23828 ARTKRSGSV S-26 SWISS;P12798;KPBB_RABIT 18958 5766 253 3010 3899 340 -87 0,63829 DLPGTEDFV T-384 GRK2 SWISS;P07550;B2AR_HUMAN 17924 5315 317 2816 3534 264 53 0,90830 GALYSGSEG S-77 SWISS;P11831;SRF_HUMAN 17183 6548 393 3470 4352 613 -220 0,62831 GRVLTLPRS T-1376 PKC SWISS;P15127;P97681;INSR_RAT 14842 8599 655 4627 5617 388 267 0,91832 EEKESSNDS S-282 GRK SWISS;P08172;ACM2_HUMAN 12601 4611 887 2749 2633 410 477 0,54833 ERNLSFEIK S-435 PKA SWISS;Q05209;PTNC_HUMAN 13883 1997 764 1380 872 885 -121 0,61834 GSLKSRKRA S-39 SWISS;P17306;STP1_PIG 20501 2208 149 1179 1456 177 -28 0,55835 AVRRSDRAY S-20 PKA SWISS;P02646;TRIC_RABIT 19621 2227 203 1215 1431 553 -350 0,14836 DPTMSKKKK S-13 PKC SWISS;P41035;IF2B_RABIT 17454 3304 360 1832 2081 489 -129 0,46837 GGGTSPVFP S-22 cdc2 SWISS;P02542;DESM_CHICK 19313 6206 218 3212 4234 328 -110 0,66838 GSTSTPAPS T-446 MFPK SWISS;P16638;ACLY_RAT 20821 2283 140 1211 1515 726 -586 0,11839 EGGRTVGAG T-394 SWISS;P07157;EFTU_THETH 23190 937 81 509 605 158 -77 0,66840 ESHESLESY S-25 SWISS;P07507;MGP_BOVIN 25573 1020 37 528 695 364 -327 0,17841 RRADSLQKN S-138 PKC SWISS;Q63270;IREB_RAT 15607 3187 561 1874 1857 766 -205 0,49842 SAYGSVKAY S-25 PKC SWISS;P07355;ANX2_HUMAN 15091 1080 624 852 323 391 233 0,75843 NTSSSPQPK S-315 p34cdc2 SWISS;P04637;P53_HUMAN 15902 4334 529 2431 2690 632 -103 0,64844 GSRRR S-53 SWISS;P15340;HSP_CHICK 16251 4596 493 2545 2901 1154 -661 0,18845 AKAKTTKKR T-9 SWISS;P02612;MLRM_CHICK 25064 569 43 306 372 15 28 0,13846 DDAYSDTET S-86 CKI SWISS;P11845;IPP2_RABIT 16733 2829 433 1631 1694 873 -440 0,25847 ESVDYVPML Y-751 PDGFR SWISS;P09619;PGDR_HUMAN 14148 4602 739 2670 2731 951 -212 0,55848 RRPTPATVA SWISS;P01099;IPP1_RABIT 16630 2546 447 1497 1484 993 -546 0,14849 SFKLSGFSF S-166 PKC SWISS;P29966;MACS_HUMAN 19449 514 215 365 211 1012 -797 0,22850 QEKESERLA S-379 beta-ARK SWISS;P04274;B2AR_MESAU 23209 2813 80 1446 1932 1132 -1052 0,08851 RRVSV SWISS;P12928;P04763;Q64618;KPYR_RAT17344 3698 369 2034 2354 975 -606 0,28852 APFTPGGRR SWISS;P02687;MBP_BOVIN 15758 5458 540 2999 3478 1031 -491 0,39853 DEKLSEILG S-143 SWISS;P20338;RRPP_HRSVL 17231 1915 389 1152 1079 886 -497 0,16854 FGHNTIDAV T-189 SWISS;P55013;NKC1_SQUAC 14558 3035 688 1861 1660 672 16 0,73

855 RRRRSRRAS S-6 PKA SWISS;P02336;PRT2_CLUPA 13841 579 770 675 135 145 625 0,49856 SIYSSDDDE S-12 CKII SWISS;P01103;MYB_CHICK 14226 830 733 782 69 292 441 0,23857 HGYSLG SWISS;P00698;Q90884;LYC_CHICK 15456 1426 583 1004 596 322 261 0,47858 AAASFKAKR S-4 PKC peptide AAASFKAKR 17715 5924 342 3133 3947 32 310 0,43859 ARKFSSARP S-6 PKA SWISS;P02719;CINA_ELEEL 16602 3181 451 1816 1930 309 142 0,96860 DKEYYSVHN Y-1235 c-Met SWISS;P08581;MET_HUMAN 15002 871 637 754 166 399 238 0,51861 FRKLSFTES S-576 PKA PIR;I52824 16953 5166 409 2788 3364 512 -103 0,66862 RVLESFRAA S-103 PKC SWISS;P15383;MINK_RAT 15377 5187 595 2891 3247 545 50 0,85863 SMANSFVGT S-221 p74Raf-1 SWISS;P29678;MPK1_RABIT 11441 322 998 660 478 560 438 0,05 �864 RRPSPA SWISS;P12928;P04763;Q64618;KPYR_RAT15061 3980 630 2305 2369 834 -204 0,51865 ADSFSLNDA S-5 CKI SWISS;P38486;LEG3_CANFA 19644 6788 200 3494 4659 679 -479 0,42866 ATSASPPQK S-312 SWISS;P02340;P53_MOUSE 22445 1263 94 678 827 594 -500 0,03 �867 DNLYYWDQD Y-1222 SWISS;P04626;ERB2_HUMAN 24067 244 65 154 127 380 -315 0,04 �868 GDSSYKNIH Y-697 c-Fms SWISS;P09581;KFMS_MOUSE 27353 1868 21 945 1306 314 -293 0,04 �869 YSTDYYREG Y-709 SWISS;Q03351;TRKC_RAT 14510 380 703 541 229 688 15 0,75870 SPKKSPRKA S-22 sperm-specificSWISS;P02256;H1_PARAN 17138 2442 397 1419 1446 103 294 0,54871 RKRSRKA SWISS;P02278;H2B_HUMAN 19077 4447 240 2343 2975 648 -408 0,37872 RPPASPSPQ S-551 p34cdc2-p58cyclinSWISS;P17599;SYN1_BOVIN 15390 1926 594 1260 942 1174 -580 0,13873 SARLSAKPA S-24 PKG SWISS;P02316;HG14_BOVIN 16462 52 475 263 299 1124 -649 0,23874 NRQSSQARV S-17 PKA SWISS;P06593;PHY3_AVESA 15391 3443 592 2017 2016 1170 -578 0,11875 KEAKSD S-99 SWISS;P02316;HG14_BOVIN 13624 1610 786 1198 583 904 -118 0,33876 RDEEYGYEA Y-411 SWISS;P21548;GAC2_CHICK 15072 1626 627 1126 706 629 -2 0,47877 AVRRSDAA SWISS;P02646;TRIC_RABIT 18533 7212 282 3747 4901 1016 -734 0,48878 TKRSGSV S-26 SWISS;P12798;KPBB_RABIT 17844 3524 327 1926 2261 1017 -690 0,20879 RRKATQVGE T-278 PKA SWISS;P50552;VASP_HUMAN 14994 2954 638 1796 1637 1036 -398 0,25880 SEGDSESGE S-83 SWISS;P11831;SRF_HUMAN 14584 3623 687 2155 2076 921 -234 0,45881 PLDRSSHAQ S-1332 insulin-sensitiveSWISS;P06213;INSR_HUMAN 16111 5796 506 3151 3741 1028 -522 0,39882 RRASS SWISS;P12928;P04763;Q64618;KPYR_RAT13850 805 768 786 26 1078 -310 0,48883 RFHKSSKDS S-205 SWISS;P27573;MYP0_MOUSE 12593 3329 888 2109 1726 505 383 0,52884 RAKRSGSV SWISS;P12798;KPBB_RABIT 14438 700 713 707 9 127 586 0,29885 KSESSQK S-247 SWISS;P10660;P08227;RS6_HUMAN 14153 37 738 388 495 105 633 0,10886 RRRLSDSNF S-9 PKA SWISS;P09951;SYN1_RAT 11823 1911 963 1437 671 351 612 0,38887 SGKTSPSSS S-343 SWISS;P19836;CTPT_RAT 10379 1087 1057 1072 21 677 380 0,26888 KSRRTI T-271 PKC SWISS;P01589;IL2A_HUMAN 15063 1286 629 957 464 231 398 0,55889 TAILE T-1 SWISS;P02955;PSBA_SPIOL 15139 7821 619 4220 5093 395 224 0,94890 RKRLSQDAY S-320 SWISS;P14598;NCF1_HUMAN 17705 5361 343 2852 3548 482 -139 0,60891 ASGSFKL S-103 PKC SWISS;P02253;H11_BOVIN 19312 6496 220 3358 4438 780 -560 0,38892 LRRGSLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)18185 3461 299 1880 2236 372 -73 0,52893 RRSVSEAAL S-10 AMP-PK SWISS;P16386;LIPS_BOVIN 20241 5844 167 3005 4014 638 -471 0,36894 SLAMSPRQR S-67 SWISS;P01099;IPP1_RABIT 19683 6675 199 3437 4579 634 -435 0,43895 RRASLG S-5 BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)22174 560 101 331 325 602 -501 0,10

896 AAVDTSSEI T-7 CKII SWISS;P01252;THYA_BOVIN 24952 3372 47 1710 2351 336 -289 0,13897 SSSNTIRRP T-475 PKC SWISS;P26285;F26H_BOVIN 18360 1797 289 1043 1067 900 -611 0,22898 TKKQSFKQT S-686 PKC SWISS;P13569;CFTR_HUMAN 21120 4553 130 2342 3128 171 -41 0,67899 VDSAYEVIK Y-238 gag-fps SWISS;P00340;LDHM_CHICK 17856 2742 324 1533 1710 942 -618 0,17900 YRKSSLKSR S-36 SWISS;P22613;STP1_SHEEP 22090 1037 104 570 659 157 -53 0,47901 SNVSSTGSI S-413 GSK3 PIR;2144820 16104 1909 507 1208 991 1114 -607 0,07902 RARSRKE SWISS;P02278;H2B_HUMAN 15013 2048 635 1342 999 719 -84 0,43903 VGPGYLGSG Y-826 SWISS;P07949;RET_HUMAN 14453 2593 710 1651 1331 978 -268 0,32904 STPLSPTRI S-22 cdc2 SWISS;P02545;LAMA_HUMAN 16272 976 491 733 343 1135 -644 0,01 �905 TPPLSPSRR S-62 ERT SWISS;P01106;P01107;MYC_HUMAN 19926 7338 192 3765 5053 770 -578 0,41906 VKRISGLIY S-47 H4-PK-I SWISS;P02304;P02305;H4_HUMAN 20841 4733 139 2436 3248 39 100 0,38907 RAAASRARQ S-367 PKC SWISS;P15336;CREP_HUMAN 17845 4928 326 2627 3254 541 -215 0,51908 SPRTPGGRR SWISS;P02687;MBP_BOVIN 15053 3515 632 2074 2039 1099 -467 0,24909 RRRRSVA SWISS;P12928;P04763;Q64618;KPYR_RAT17324 5781 372 3076 3824 1057 -685 0,32910 VRFESIRLP S-86 PKA SWISS;P01233;CGHB_HUMAN 17719 3534 341 1938 2258 752 -411 0,31911 SYPLSPLSD S-505 MAPK SWISS;P47712;PA2Y_HUMAN 23219 2054 79 1067 1397 165 -86 0,22912 TRQASQAGP S-605 CaM-II SWISS;P17599;SYN1_BOVIN 10812 578 1037 807 325 492 545 0,15913 VRVYTHEVV T-161 SWISS;P24033;CC22_XENLA 13338 374 815 595 312 236 579 0,08914 RENEYMPMA Y-298 c-Src SWISS;P03079;TAMI_POVHA 12708 1273 879 1076 279 324 555 0,35915 GRGLSLSR S-136 SWISS;P02686;MBP_HUMAN 13079 233 847 540 434 416 431 0,04 �916 YIYGSFK Y-3 p60c-src peptide YIYGSFK 16095 588 509 548 56 190 319 0,27917 VTRSSAVRL S-65 autophosphorylation-dependentSWISS;P20152;VIME_MOUSE 15457 108 582 345 335 499 83 0,03 �918 TESQSLTLT S-139 PKA SWISS;P02666;CASB_BOVIN 21920 5090 111 2600 3521 528 -417 0,26919 TTRVTPLRT T-64 cdc2 SWISS;P02542;DESM_CHICK 17781 1726 335 1031 984 676 -341 0,12920 WTADSGEGD S-22 SWISS;P02671;FIBA_HUMAN 15780 4243 538 2390 2620 727 -189 0,55921 RHRDTGILD T-33 PKA SWISS;P02687;MBP_BOVIN 18343 5314 292 2803 3551 742 -450 0,38922 NRIYTHQVV T-176 SWISS;P20911;CDK7_XENLA 22672 3903 89 1996 2697 164 -75 0,46923 LRKASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)25138 2817 42 1430 1962 349 -307 0,14924 YHGHSMSDP S-293 pyruvate SWISS;P08559;ODPA_HUMAN 28164 1715 19 867 1199 509 -490 0,07925 QTVKSSKGG S-26 PKC SWISS;P04083;ANX1_HUMAN 21431 272 121 196 106 614 -493 0,01 �926 LRMFSFKAP S-382 PKC SWISS;P22300;GAC2_BOVIN 31029 15557 9 7783 10994 279 -270 0,43927 PRHLSNVSS S-409 PKA PIR;2144820 14946 185 643 414 324 1125 -482 0,17928 SSNEYMDMK Y-719 PI3-KINASESWISS;P05532;KKIT_MOUSE 14526 6827 699 3763 4333 902 -203 0,67929 THYGSLPQK S-69 CaM-II SWISS;P02687;MBP_BOVIN 17846 6817 325 3571 4590 748 -423 0,50930 LRRPSDQAV S-275 PKA SWISS;P01126;REL_AVIRE 13324 6469 821 3645 3994 1171 -350 0,35931 PRRNSRASL S-573 PKA SWISS;P04775;CIN2_RAT 12633 15 886 450 616 1110 -224 0,11932 SRKESYSVY S-36 PKA SWISS;P02278;H2B_HUMAN 17734 1529 339 934 842 977 -638 0,16933 LSELSRRRI S-51 ds-RNA SWISS;P12962;IF43_YEAST 17786 1112 334 723 550 812 -478 0,04 �934 PSPSSRVTV S-395 PKC SWISS;P20700;LAM1_HUMAN 17244 3524 386 1955 2219 907 -521 0,26935 SSVPTPSPL T-360 cdc2 SWISS;P19138;P20426;KC21_HUMAN 15464 5167 579 2873 3244 696 -117 0,68936 TNEEYLDLS Y-769 SWISS;P21802;FGR2_HUMAN 15782 7072 537 3804 4621 817 -280 0,63

937 LSRFSWGAE S-114 PKC SWISS;P02687;MBP_BOVIN 17297 5717 375 3046 3777 615 -240 0,56938 PTKRSPTKR S-10 sperm-specificSWISS;P06145;H2B1_STRPU 17381 2614 366 1490 1590 457 -91 0,54939 SRRLSQETG S-813 PKA SWISS;P13569;CFTR_HUMAN 15652 5385 556 2970 3414 750 -194 0,68940 PASPSPQRQ S-553 Cdk5-p23 SWISS;P17599;SYN1_BOVIN 17093 3539 400 1970 2220 580 -180 0,49941 PWRITDNEL T-1114 SWISS;P55013;NKC1_SQUAC 13579 505 792 648 203 763 29 0,83942 STSVSAVAS S-290 GRK SWISS;P08172;ACM2_HUMAN 10078 1331 1078 1204 179 497 581 0,12943 TRIPSAKKY S-104 PKC SWISS;Q62048;PE15_MOUSE 20493 3939 150 2044 2679 37 113 0,30944 PGGSTPVSS T-344 cdc2 SWISS;P19138;P20426;KC21_HUMAN 11053 2628 1022 1825 1136 609 413 0,33945 QGTLSKIFK S-150 PKC SWISS;P02687;MBP_BOVIN 17170 4182 394 2288 2678 220 174 0,80946 SSEESIISQ S-28 CKI SWISS;P02663;CAS2_BOVIN 16375 3871 485 2178 2395 558 -73 0,60947 PKRGSGKDG S-54 PKA SWISS;P02687;MBP_BOVIN 17410 5697 364 3031 3771 754 -390 0,47948 LGSALRRR SWISS;P20152;VIME_MOUSE 17947 5194 313 2753 3451 357 -44 0,71949 TDEDSDNEI S-462 SWISS;P18139;PP65_HCMVT 20637 9588 143 4866 6679 358 -215 0,62950 TRSVSSSSY S-6 PKA SWISS;P20152;VIME_MOUSE 21817 7119 113 3616 4954 189 -76 0,66951 PPRRSSIRN S-303 SWISS;P14598;NCF1_HUMAN 24804 6766 49 3408 4750 565 -516 0,26952 RTKGSGSV SWISS;P12798;KPBB_RABIT 34520 7083 5 3544 5005 268 -263 0,18953 KAKTTKKRP T-10 SWISS;P02612;MLRM_CHICK 34536 15306 4 7655 10820 8 -4 0,98954 KRPSGRAKA SWISS;P02687;MBP_BOVIN 22877 3161 86 1624 2175 26 60 0,43955 PLSKTLSVSSL SWISS;P13834;UGS1_RABIT 15257 2785 605 1695 1542 286 319 0,53956 QSGMTEYVA T-180 SWISS;P16892;FUS3_YEAST 16785 4139 426 2282 2625 868 -442 0,34957 KGATSDEED S-1087 CKII SWISS;P06786;TOP2_YEAST 17319 7083 373 3728 4745 1102 -729 0,35958 KRRNSEFEI S-58 PKA SWISS;P09810;TR5H_RAT 18840 6157 262 3209 4168 778 -516 0,38959 LTRRASFSAQ S-230 SWISS;P07248;ADR1_YEAST 13296 1488 824 1156 469 825 -1 0,56960 KISASRKLQ S-45 PKC SWISS;P08057;TRIC_BOVIN 15631 107 559 333 319 273 286 0,55961 KSFGSPNRI S-170 SWISS;P20911;CDK7_XENLA 16667 1476 441 959 732 245 196 0,59962 KRKQISVAGL SWISS;P11217;PHS2_HUMAN 20529 3673 147 1910 2493 63 84 0,45963 SQHSTPPKK T-124 p34cdc2 SWISS;P03070;TALA_SV40 15740 6693 544 3619 4348 768 -224 0,66964 KKIDSFASN S-95 PKC SWISS;P11497;P97902;COAC_RAT 17945 8141 314 4227 5534 300 14 0,89965 KTETSQVAP S-343 RhK SWISS;P02699;OPSD_BOVIN 15317 5340 599 2970 3352 24 575 0,60966 PAAPSPGSS S-67 MAPK SWISS;P17599;SYN1_BOVIN 14833 5720 656 3188 3581 635 21 0,84967 KKRLSVERI S-29 PKC SWISS;P11388;TOPA_HUMAN 25026 5890 44 2967 4134 51 -7 0,93968 KYRKSSLKS S-35 SWISS;P22613;STP1_SHEEP 20171 4098 170 2134 2777 46 124 0,35969 PLSRRLSVA S-7 PKC glycogen (starch) synthase, muscle15327 2324 598 1461 1221 317 281 0,56970 SRLHSVRER S-15 SWISS;P49036;SUS2_MAIZE 14538 4451 697 2574 2655 694 3 0,81971 KNDKSKTWQ S-53 endogenousSWISS;P06730;IF4E_HUMAN 15458 4861 581 2721 3026 316 265 0,78972 LGGGTFDIS T-198 DnaK SWISS;P04475;DNAK_ECOLI 12768 7702 872 4287 4829 443 429 0,72973 QHLKSVMLQ S-19 PKA SWISS;P02643;TRIF_RABIT 15483 6525 576 3551 4207 266 310 0,76974 KRAASPRKS S-10 sperm-specificSWISS;P02256;H1_PARAN 18208 5308 297 2803 3543 134 163 0,71975 LMAPSEEDH T-144 SWISS;P08833;IBP1_HUMAN 15984 6626 523 3575 4316 274 249 0,86976 QNPVYHNQP Y-1110 EGFR SWISS;P00533;P06268;EGFR_HUMAN 17262 7708 382 4045 5180 215 167 0,88977 SRSRSRSPG S-82 RS SWISS;P23913;LBR_CHICK 20162 7881 171 4026 5452 408 -237 0,56

978 KRKVSSAEG S-6 SWISS;P02316;HG14_BOVIN 22371 9178 99 4638 6419 366 -267 0,50979 LQDDYEDMM Y-8 SWISS;P02730;B3AT_HUMAN 25772 6679 34 3357 4699 332 -298 0,30980 QRRSSEGST S-1772 PKA SWISS;P07293;CIC1_RABIT 32020 2731 8 1369 1925 284 -276 0,03 �981 GVLRRASVA SWISS;P12928;P04763;Q64618;KPYR_RAT21622 458 116 287 242 533 -417 0,14982 KRPSLRAKA SWISS;P02687;MBP_BOVIN 25211 3723 41 1882 2604 42 -1 1,00983 EASTTVSKT T-336 SWISS;P02700;OPSD_SHEEP 15477 3024 577 1800 1730 496 81 0,81984 ELSNYIAMG Y-460 INSR SWISS;P35570;IRS1_RAT 16644 4824 444 2634 3097 990 -546 0,33985 HHHATPSPP T-355 GSK3 SWISS;P01103;MYB_CHICK 12229 5869 927 3398 3495 997 -70 0,80986 KTETSQVAP S-343 RhK SWISS;P02699;OPSD_BOVIN 12070 2307 942 1624 965 1144 -202 0,19987 PYKFPSSPLRIPGZ na na na 13156 115 841 478 514 521 320 0,30988 HRQETVDAL T-286 CaM-II SWISS;P11275;KCCA_RAT 9036 1559 1118 1339 312 866 252 0,17989 LNRMSFASN S-1200 AMP-PK SWISS;P11497;P97902;COAC_RAT 13330 3012 817 1914 1552 434 383 0,64990 EEDTYTMPS Y-407 SWISS;P34152;FAK_MOUSE 14660 3473 676 2075 1978 23 653 0,54991 EQEEYEDPD Y-21 SWISS;P02730;B3AT_HUMAN 15820 2420 533 1476 1334 341 192 0,82992 HVSSSEESI S-25 CKI SWISS;P02663;CAS2_BOVIN 16802 4351 424 2387 2777 898 -474 0,33993 METPSQRRA S-5 PKC SWISS;P11516;LAMC_MOUSE 16964 6426 408 3417 4256 362 46 0,87994 PYKFPSSPLRIPGZ na na na 16490 7852 470 4161 5220 331 139 0,99995 IGHHSTSDD S-333 branched SWISS;P11960;ODBA_RAT 16555 7464 459 3961 4953 604 -145 0,72996 MPGETPPLS T-239 GSK3 SWISS;P05412;AP1_HUMAN 15124 5312 621 2967 3317 617 4 0,81997 EEPVYEAEP Y-360 SWISS;P14317;HS1_HUMAN 12101 4998 939 2968 2870 550 389 0,64998 ERRNSILTE S-660 PKA SWISS;P13569;CFTR_HUMAN 19072 8269 241 4255 5677 705 -464 0,51999 INETSQHHD S-458 p37 SWISS;P20152;VIME_MOUSE 15296 5998 602 3300 3815 682 -80 0,75

1000 NDSNYVVKG Y-821 SWISS;P05532;KKIT_MOUSE 13974 6658 754 3706 4175 250 504 0,591001 PYKFPSSPLRIPGZ na na na 16333 4461 487 2474 2810 488 -1 0,731002 ISITSRKAQ S-37 ERA SWISS;P06616;ERA_ECOLI 21561 7155 118 3636 4976 233 -115 0,611003 NFLKTSAGS T-210 CCD SWISS;P06782;SNF1_YEAST 15869 4735 531 2633 2973 288 243 0,801004 EHIPYTHMN Y-1361 INSR SWISS;P06213;INSR_HUMAN 16845 3556 420 1988 2218 552 -132 0,511005 ESMESYELN S-28 SWISS;P08493;MGP_HUMAN 19970 7510 188 3849 5178 359 -171 0,591006 KKRFSFKKS S-157 PKC SWISS;P12624;MACS_BOVIN 40524 16896 2 8449 11946 4 -2 0,871007 NKQGYKARQ Y-187 SWISS;Q05655;KPCD_HUMAN 24705 11157 52 5604 7852 151 -99 0,641008 PYKFPSSPLRIPGZ na na na 32104 425 7 216 296 185 -178 0,00 �1009 GGSVTKKRK T-416 PKC SWISS;P11516;LAMC_MOUSE 24557 6271 54 3162 4396 49 5 0,991010 AKRISGKMA S-277 SWISS;P13863;CC2_CHICK 20111 4940 176 2558 3369 121 55 0,921011 DDPSYVNVQ Y-317 SWISS;P29353;SHC_HUMAN 14764 8642 663 4652 5642 141 522 0,571012 EVEKSPVKS S-502 SWISS;P12839;NFM_RAT 14821 4158 658 2408 2475 550 108 0,931013 GKSSSYSKQ S-578 PKC SWISS;P02671;FIBA_HUMAN 15527 9076 568 4822 6016 299 269 0,871014 DSRSSLIRK S-661 PKC SWISS;P08183;Q12755;MDR1_HUMAN 14626 2633 681 1657 1380 203 478 0,241015 EKAKSPVPK S-603 SWISS;P12839;NFM_RAT 11332 88 1007 548 650 631 376 0,04 �1016 GNFNYVEFT Y-155 SWISS;P02612;MLRM_CHICK 11672 1705 982 1343 511 666 316 0,381017 APRTPGGRR SWISS;P02687;MBP_BOVIN 13440 2754 803 1779 1380 382 421 0,481018 DGNGYISAA Y-99 INSR SWISS;P02593;CALM_HUMAN 15920 5291 527 2909 3368 344 183 0,94

1019 FLTEYVATR Y-190 SWISS;P26696;ERK2_XENLA 16723 2877 436 1656 1726 168 268 0,421020 GRALSTRAQ S-72 PhK SWISS;P02646;TRIC_RABIT 14483 5844 708 3276 3632 442 266 0,881021 EDRMSLVNS S-132 SWISS;P01036;CYTS_HUMAN 16004 7338 519 3928 4822 622 -103 0,761022 ENPEYLGLD Y-1248 SWISS;P04626;ERB2_HUMAN 16100 5711 508 3109 3679 371 137 0,971023 GRNASTNDS S-407 GRK2 SWISS;P07550;B2AR_HUMAN 14783 1511 662 1087 600 842 -180 0,591024 ARSGSSTYS S-485 PKA SWISS;P25107;PTRR_DIMDA 13703 465 785 625 227 586 199 0,591025 DLLTSPDVG S-63 p34cdc2 SWISS;P05412;AP1_HUMAN 12103 1862 938 1400 653 556 382 0,331026 GAGNSLRTA S-525 SWISS;P11516;LAMC_MOUSE 14757 4153 664 2408 2467 681 -17 0,751027 GRTWTLAGT T-197 PKA SWISS;P00517;KAPA_BOVIN 15727 3688 546 2117 2221 481 65 0,831028 EEHVYSFPN Y-118 FAK SWISS;P49024;PAXI_CHICK 14281 1925 726 1326 848 431 295 0,591029 ERHHSIDAQ S-8 SWISS;P15804;CAP3_SORVU 13418 2476 805 1640 1181 593 212 0,871030 GSGTSSRPS S-20 PKC SWISS;P20152;VIME_MOUSE 13091 1534 845 1189 487 847 -2 0,781031 AVMVSHYIH S-56 SWISS;P01239;PRL_BOVIN 20071 4485 179 2332 3045 112 67 0,871032 DPPGTESFV T-389 beta-ARK SWISS;P04274;B2AR_MESAU 15925 5974 525 3250 3853 353 172 0,971033 GFKRSYEEH S-1354 SWISS;P06213;INSR_HUMAN 19939 5534 190 2862 3779 221 -31 0,731034 GSRRRRRRY SWISS;P15340;HSP_CHICK 22571 7748 92 3920 5414 248 -156 0,521035 EFPLSPPKK S-37 cdc2 SWISS;P16949;STHM_HUMAN 26589 6572 26 3299 4629 18 8 0,741036 ESEKTKTKE T-2029 MHCK SWISS;P08799;MYS2_DICDI 25412 5139 39 2589 3606 205 -166 0,281037 RQRKSRRTI S-268 PKC SWISS;P01589;IL2A_HUMAN 21299 9226 126 4676 6434 739 -613 0,431038 SAYATVKAY T-25 PKC SWISS;P17785;ANX2_CHICK 17086 5679 401 3040 3732 587 -186 0,601039 NTDGSTDYG S-68 PKA SWISS;P00698;Q90884;LYC_CHICK 15698 5571 549 3060 3551 637 -88 0,791040 KRTLR T-678 SWISS;P00533;P06268;EGFR_HUMAN 24741 10994 51 5523 7738 119 -68 0,691041 AGPTSARDG S-161 SWISS;P20338;RRPP_HRSVL 12904 5911 865 3388 3568 642 223 0,881042 DAPDTPELL T-547 p34cdc2 SWISS;P00520;ABL_MOUSE 10984 564 1026 795 327 343 683 0,141043 ESSYSYEEI S-119 SWISS;P20338;RRPP_HRSVL 11840 2206 961 1584 880 259 702 0,311044 RRLSSLRAS S-236 S6K SWISS;P10660;P08227;RS6_HUMAN 12326 90 914 502 583 397 517 0,601045 SFKKSFKLS S-161 PKC SWISS;P12624;MACS_BOVIN 21370 3772 125 1948 2579 60 65 0,521046 PSKKYAIKG Y-12 SWISS;P16406;AMPE_MOUSE 16516 4105 465 2285 2574 117 348 0,391047 RRPSV SWISS;P12928;P04763;Q64618;KPYR_RAT14126 3872 741 2307 2214 368 373 0,621048 APETPGGRR SWISS;P02687;MBP_BOVIN 14702 2659 670 1665 1407 412 258 0,741049 DEEMSETAD S-174 CKI SWISS;P11845;IPP2_RABIT 14298 5296 724 3010 3233 468 256 0,871050 FFSSSESGA S-11 CKII SWISS;P04975;CLCB_BOVIN 16057 1730 514 1122 860 606 -92 0,331051 RRRRAASVA SWISS;P12928;P04763;Q64618;KPYR_RAT14267 762 728 745 24 508 220 0,711052 SIRDTPAKN T-199 SWISS;P06748;NPM_HUMAN 11536 961 991 976 21 405 586 0,291053 GGYSLG SWISS;P00698;Q90884;LYC_CHICK 13823 1117 772 944 244 253 519 0,091054 AAASFKAKK S-4 PKC peptide AAASFKAKK 17875 3934 321 2128 2555 87 234 0,471055 ARDIYKNDY Y-323 SWISS;P08941;KROS_CHICK 14557 1537 689 1113 599 257 432 0,391056 DKEVSDDEA S-230 CKII SWISS;P07900;HS9A_HUMAN 12518 3398 896 2147 1769 746 150 0,871057 FRKFTKSER T-84 PKG SWISS;P00516;KGPA_BOVIN 19599 4400 206 2303 2966 55 151 0,361058 RSSMSGLHL S-79 AMP-PK SWISS;P11497;P97902;COAC_RAT 16034 3967 516 2241 2440 576 -60 0,731059 SLVNSRAQE S-136 SWISS;P01036;CYTS_HUMAN 17858 6773 323 3548 4561 510 -187 0,63

1060 RRATVA SWISS;P12928;P04763;Q64618;KPYR_RAT15527 4349 568 2459 2674 654 -86 0,661061 ADSFSLHDA S-5 CKI SWISS;P17931;LEG3_HUMAN 17888 3951 320 2135 2567 109 211 0,511062 ATRRSYVSS S-13 PKA SWISS;Q28115;GFAP_BOVIN 20880 6336 138 3237 4383 184 -46 0,771063 DNIDSQGRN S-401 GRK2 SWISS;P07550;B2AR_HUMAN 24488 279 55 167 159 138 -83 0,131064 GDSESGEEE S-85 SWISS;P11831;SRF_HUMAN 24333 2664 59 1361 1842 256 -197 0,171065 YSLGSALRP S-41 PKC SWISS;P20152;VIME_MOUSE 20157 3924 173 2048 2652 1050 -877 0,171066 SPHQSEDEE S-75 CKII SWISS;P13834;UGS1_RABIT 16635 6166 446 3306 4045 1087 -641 0,321067 RKRSRAE SWISS;P02278;H2B_HUMAN 17242 7413 387 3900 4968 450 -63 0,771068 RNASTNDSP T-413 beta-ARK SWISS;P04274;B2AR_MESAU 17438 11985 362 6174 8219 249 113 0,991069 SAELYSNAL Y-716 SWISS;P09619;PGDR_HUMAN 16500 10725 468 5597 7253 515 -47 0,851070 NRQLSSGVS S-86 PKA SWISS;P14602;HS27_MOUSE 9242 2575 1110 1842 1036 628 482 0,241071 VGPDSD S-389 SWISS;P02340;P53_MOUSE 8293 1125 1139 1132 10 571 568 0,121072 RDDTYTAHA Y-539 SWISS;P00522;ABL_DROME 11881 132 958 545 584 432 526 0,471073 APTPGGRR SWISS;P02687;MBP_BOVIN 15641 1635 558 1096 761 400 158 1,001074 STNDSPL S-416 beta-ARK SWISS;P04274;B2AR_MESAU 14682 3275 673 1974 1840 436 237 0,911075 RRKASGPPV S-35 PKA SWISS;P02253;H11_BOVIN 15456 687 583 635 74 517 66 0,391076 SEENSKKTV S-150 CKI SWISS;P02663;CAS2_BOVIN 16509 1447 467 957 693 546 -79 0,261077 PASPSPQRQ S-553 Cdk5-p23 SWISS;P17599;SYN1_BOVIN 14161 4588 737 2662 2723 375 362 0,691078 RRASR SWISS;P12928;P04763;Q64618;KPYR_RAT16434 5565 478 3021 3597 455 23 0,801079 RFFGSDRGA S-45 PKC SWISS;P02687;MBP_BOVIN 14587 1251 686 968 399 335 351 0,481080 PRTPGGRR SWISS;P02687;MBP_BOVIN 12222 1207 929 1068 197 311 618 0,131081 KRRVSEV S-66 SWISS;P00972;ERTS_RAT 10259 72 1065 569 702 243 822 0,111082 RRRGSSIPQ S-32 PKA SWISS;P07953;F261_RAT 12421 3659 902 2280 1949 289 613 0,311083 SGDTSPRHL S-395 SWISS;P19332;TAU_RAT 13326 1644 820 1232 583 323 497 0,211084 KQITVR SWISS;P11217;PHS2_HUMAN 16777 4958 430 2694 3201 86 344 0,401085 SSKRA S-1 PKC SWISS;P02612;MLRM_CHICK 14924 3246 646 1946 1839 297 349 0,621086 RKRKSSQAL S-28 PKA SWISS;P04625;THA_CHICK 16210 5039 496 2767 3212 265 231 0,821087 AKRSRKE SWISS;P02278;H2B_HUMAN 15992 5026 520 2773 3186 173 347 0,561088 LRRATLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)17222 939 390 665 388 461 -71 0,611089 RRSSSVGYI S-383 PKA SWISS;P02718;ACHD_TORCA 22196 7737 100 3919 5400 69 31 0,881090 SKVTSKAGS S-320 PIR;2144820 22795 9011 88 4549 6309 198 -110 0,641091 RGYSLG S-42 SWISS;P00698;Q90884;LYC_CHICK 23188 2807 82 1445 1927 233 -151 0,211092 AARTPGGRR SWISS;P02687;MBP_BOVIN 23683 802 68 435 519 224 -156 0,311093 SSSESGAPE S-13 CKII SWISS;P04975;CLCB_BOVIN 23874 9733 66 4900 6836 813 -747 0,341094 TKHIYSNLA Y-934 SWISS;P05532;KKIT_MOUSE 16546 7236 460 3848 4791 423 37 0,871095 VDEMYREAP Y-142 EGFR SWISS;P24844;MLRN_HUMAN 17472 7342 358 3850 4938 441 -83 0,751096 YRKGSLKSR S-36 SWISS;P17306;STP1_PIG 20486 10896 151 5523 7598 65 86 0,771097 SNQEYLDLS Y-766 SWISS;P11362;FGR1_HUMAN 20311 13087 162 6625 9139 448 -286 0,681098 RKISASE S-92 GENBANK;L16545 13584 6490 790 3640 4030 636 154 0,971099 VGFMTEYVA T-183 SWISS;P14681;KSS1_YEAST 10099 1723 1075 1399 458 625 450 0,281100 STNEYMDMK Y-721 SWISS;P10721;KKIT_HUMAN 14528 3652 698 2175 2089 144 554 0,54

1101 TPPLSPIDM S-243 ERT SWISS;P05412;AP1_HUMAN 15709 2012 548 1280 1035 327 221 0,881102 VKGATSDEE T-1086 CKII SWISS;P06786;TOP2_YEAST 13561 470 793 631 229 342 451 0,761103 QRATSNVFA S-19 SWISS;P24844;MLRN_HUMAN 19574 3663 207 1935 2444 374 -167 0,381104 SPRKSPRKS S-14 sperm-specificSWISS;P02256;H1_PARAN 32631 12792 6 6399 9041 74 -68 0,471105 RRRASVA SWISS;P12928;P04763;Q64618;KPYR_RAT14653 4460 678 2569 2674 630 48 0,841106 VPTPSPLGP S-362 cdc2 SWISS;P19138;P20426;KC21_HUMAN 13202 4103 833 2468 2312 518 315 0,711107 SVSSSSYRR S-8 PKC SWISS;P20152;VIME_MOUSE 16284 2846 489 1668 1667 211 278 0,481108 TRQASISGP S-566 CaM-II SWISS;P09951;SYN1_RAT 13296 1295 825 1060 332 225 600 0,061109 VRTYTHEVV T-160 SWISS;P24941;CDK2_HUMAN 8708 2300 1129 1714 828 319 810 0,121110 REILSRRPS S-129 GSK3 SWISS;P16220;P21934;CREB_HUMAN 11735 33 973 503 665 295 678 0,04 �1111 GASGSFKL S-103 PKC SWISS;P02253;H11_BOVIN 15397 2145 591 1368 1099 155 436 0,261112 VRRSDRA S-20 SWISS;P02646;TRIC_RABIT 12252 2150 924 1537 867 559 365 0,381113 VTRRTLSMD T-773 PKA SWISS;P17858;K6PL_HUMAN 12128 2256 931 1594 937 502 429 0,321114 TEPQYQPGE Y-530 SWISS;P06241;FYN_HUMAN 12228 1822 928 1375 632 451 477 0,261115 TTRRSASKT S-460 PKA SWISS;P02671;FIBA_HUMAN 21503 10685 119 5402 7471 229 -110 0,731116 WLTKTPEGN T-711 p34cdc2 SWISS;P12957;CALD_CHICK 14519 707 701 704 4 307 394 0,251117 RGRSSVYSA S-311 myosin SWISS;P10569;MYSC_ACACA 16720 1523 438 981 767 180 258 0,531118 RKRTLRRL T-678 SWISS;P00533;P06268;EGFR_HUMAN 21000 4957 134 2545 3410 194 -60 0,671119 LRHASLG BOS TAURUS (BOVINE) LIVER PYRUVATE KINASE (FRAGMENT)21264 209 127 168 58 555 -428 0,131120 YGNGYSSNS Y-354 INSR SWISS;P04274;B2AR_MESAU 26349 3075 29 1552 2154 260 -231 0,091121 QTASSPLSP S-42 cdc2 SWISS;P08928;LAM0_DROME 23813 473 67 270 287 867 -800 0,00 �1122 LRKVSKQEE S-239 PKA SWISS;P50552;VASP_HUMAN 19944 9269 189 4729 6420 129 60 0,981123 PQRATSNVF T-18 SWISS;P24844;MLRN_HUMAN 19049 10333 242 5287 7135 454 -212 0,691124 SSNDSTSVS S-286 GRK SWISS;P08172;ACM2_HUMAN 20064 14107 181 7144 9847 675 -494 0,631125 THVASVSDV S-1215 AMP-PK SWISS;P11497;P97902;COAC_RAT 17969 8941 311 4626 6103 761 -450 0,571126 LRRLSTKYR S-39 PKA SWISS;Q05209;PTNC_HUMAN 20182 6990 169 3580 4823 360 -191 0,551127 PRRDSTEGF S-11 PKA SWISS;P09201;F16P_YEAST 19513 109 209 159 71 324 -115 0,491128 SRKDSLDDS S-404 GRK SWISS;P15823;A1AB_RAT 18173 10013 300 5157 6868 491 -191 0,721129 LSEHSSPEE S-45 CKII SWISS;P07516;IPPD_BOVIN 17099 6067 399 3233 4008 641 -242 0,581130 PSPKYPGPQ Y-191 SWISS;P05059;CMGA_BOVIN 13767 1045 777 911 190 384 393 0,271131 SSVLYTAVQ Y-1009 SWISS;P09619;PGDR_HUMAN 11773 475 968 722 348 478 490 0,181132 TLSDSDDED S-263 CKII SWISS;P04198;MYCN_HUMAN 14520 304 700 502 280 433 267 0,411133 LSLDSQGRN S-406 beta-ARK SWISS;P04274;B2AR_MESAU 12954 2049 862 1456 839 598 264 0,581134 PTKRSPQKG S-15 sperm-specificSWISS;P06145;H2B1_STRPU 11512 783 994 889 149 445 549 0,391135 SRRGSESSE S-16 PKA PIR;S04004 13949 2014 757 1385 889 472 285 0,651136 PASAYGSVK Y-23 p60-Src SWISS;P19620;ANX2_PIG 14614 6460 682 3571 4086 321 361 0,741137 PWQVSLRTR S-597 SWISS;P00747;PLMN_HUMAN 11896 2678 956 1817 1218 404 552 0,271138 STSRSLYSS S-50 PKA SWISS;P20152;VIME_MOUSE 14288 895 725 810 120 216 509 0,091139 TRGGSLERS S-394 PKC SWISS;P15823;A1AB_RAT 13294 3453 826 2139 1857 501 325 0,651140 PFKLSGLSF S-100 PKC SWISS;P28667;MRP_MOUSE 12848 764 867 815 73 246 621 0,201141 QEQEYVQAV Y-20 SWISS;P07150;ANX1_RAT 12681 1429 881 1155 388 263 618 0,22

1142 SRTPSLPTP S-214 PKA PIR;2144820 12745 123 875 499 532 529 346 0,521143 PKKGSKKAV S-14 PKA SWISS;P02278;H2B_HUMAN 15724 3306 547 1926 1951 101 446 0,271144 LELSDDDD S-4 CKII PIR;A34928 13857 646 767 707 85 226 541 0,221145 TDDGYMPMS Y-608 INSR SWISS;P35570;IRS1_RAT 14858 2107 653 1380 1028 262 391 0,461146 TRRLTGFLP T-30 SWISS;P06738;PHSG_YEAST 19312 2401 220 1310 1542 255 -35 0,471147 PPEKTEEEE T-357 SWISS;P30936;SSR3_RAT 22034 3012 106 1559 2055 503 -397 0,131148 RTGRSGSV SWISS;P12798;KPBB_RABIT 23071 2961 83 1522 2035 232 -149 0,471149 KAEEYILKK Y-381 SWISS;P02714;ACHG_TORCA 19008 6141 248 3194 4167 85 163 0,651150 KRPSFRAKA SWISS;P02687;MBP_BOVIN 20070 7669 180 3925 5296 2 178 0,151151 PTPSAPSPQPKG S-8 SWISS;P04177;TY3H_RAT 16723 3903 437 2170 2450 390 47 0,781152 QRYSSDPTG S-1071 SWISS;P00533;P06268;EGFR_HUMAN 17454 13692 360 7026 9427 831 -471 0,691153 KEDTYTAHA Y-313 SWISS;P03949;ABL1_CAEEL 19999 12474 186 6330 8689 186 0 0,931154 KRRLSFSET S-90 SWISS;P01582;IL1A_MOUSE 21375 1493 123 808 969 96 27 0,901155 KRAKAKTTKKR T-9 SWISS;P02612;MLRM_CHICK 42642 6123 1 3062 4329 1 0 0,531156 KIQASFRGH S-34 PKC SWISS;P35722;NEUG_BOVIN 35666 20700 3 10351 14635 122 -119 0,491157 KRSNSVDTS S-129 PKA SWISS;P13834;UGS1_RABIT 17789 9780 332 5056 6681 691 -359 0,641158 KRKQISGRGL SWISS;P11217;PHS2_HUMAN 17217 378 391 384 9 549 -158 0,301159 SQESSEEEQ S-91 SWISS;P17095;HMGY_MOUSE 13218 672 831 751 113 652 179 0,791160 KKDVTPVKA T-53 SWISS;P10156;H1_TETTH 14708 2611 669 1640 1373 271 398 0,531161 KSRWSGSQQ S-499 PKC SWISS;P04049;KRAF_HUMAN 14095 2862 744 1803 1498 473 271 0,741162 LYSGSEGDS S-79 SWISS;P11831;SRF_HUMAN 13829 406 771 588 258 569 202 0,331163 KKRFSFKKS S-157 PKC SWISS;P12624;MACS_BOVIN 25976 1908 30 969 1328 11 19 0,071164 KYLASASTM S-15 CaM-II SWISS;P02687;MBP_BOVIN 14966 2179 641 1410 1088 132 509 0,131165 PRRVSRRRR S-21 PKC SWISS;P02335;PRT1_CLUPA 13057 1069 851 960 154 486 365 0,291166 SRKRSGEAT S-774 PKA SWISS;P00511;K6PF_RABIT 12996 2425 857 1641 1109 381 476 0,431167 KMKDTDSEE T-79 CKII SWISS;P02593;CALM_HUMAN 14091 1638 745 1191 631 367 378 0,421168 LFRLSEHSS S-42 CKII SWISS;P07516;IPPD_BOVIN 12349 1507 912 1209 421 339 573 0,111169 QDENTVSTS T-307 GRK SWISS;P08172;ACM2_HUMAN 11934 190 953 572 539 422 531 0,551170 KQSPSSSPT S-321 SWISS;P19836;CTPT_RAT 12290 5619 918 3268 3324 626 292 0,761171 LLRPSRRVR S-341 SWISS;P30936;SSR3_RAT 16602 359 452 405 66 199 253 0,251172 QMALTPVVV T-172 CAK SWISS;P30285;CDK4_MOUSE 13171 1739 837 1288 638 230 607 0,131173 SRSRSPGRP S-84 RS SWISS;P23913;LBR_CHICK 15663 2880 553 1717 1645 237 316 0,481174 KRKRSRKES S-32 PKA SWISS;P02278;H2B_HUMAN 30897 13260 11 6636 9369 9 2 0,841175 LQAISPKQS S-315 PKC SWISS;P19836;CTPT_RAT 26829 4023 24 2023 2828 204 -180 0,171176 QRRRSLEPP S-16 PKA SWISS;P00523;Q91345;Q92013;SRC_CHICK22052 3139 105 1622 2145 281 -176 0,23

Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling

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Transcript of Tetrahydroxyquinone induces apoptosis of leukemia cells through diminished survival signaling