Bruton��s tyrosine kinase (Btk): function, regulation, and transformation with special...

58 � 2009 The Authors • Journal compilation � 2009 Blackwell Munksgaard • Immunological Reviews 228/2009

Abdalla J. Mohamed

Liang Yu

Carl-Magnus Backesjo

Leonardo Vargas

Rani Faryal

Alar Aints

Birger Christensson

Anna Berglof

Mauno Vihinen

Beston F. Nore

C. I. Edvard Smith

Bruton’s tyrosine kinase (Btk):function, regulation, andtransformation with specialemphasis on the PH domain

Authors’ addresses

Abdalla J. Mohamed1, Liang Yu1, Carl-Magnus Backesjo1, Leonardo

Vargas1, Rani Faryal1,2, Alar Aints1,3,4, Birger Christensson5, AnnaBerglof1, Mauno Vihinen6,7, Beston F. Nore1, C. I. Edvard Smith1

1Clinical Research Center, Department of Laboratory Medicine,

Karolinska Institutet, Huddinge University Hospital, Huddinge,

Sweden.2Department of Biosciences, Comsats Institute of Information

Technology, Chak Shazad Campus, Islamabad, Pakistan.3Department of Hematology and Oncology, Tartu University,

Tartu, Estonia.4Competence Centre for Cancer Research, Tallinn, Estonia.5Division of Pathology, Department of Laboratory Medicine, Karo-

linska Institutet, Huddinge University Hospital, Huddinge, Sweden.6Institute of Medical Technology, University of Tampere, Finland.7Tempere University Hospital, Tampere, Finland.

Correspondence to:

C. I. Edvard SmithClinical Research Center at NovumKarolinska InstitutetHuddinge University HospitalSE-141 86 Huddinge, SwedenTel.: +46 8 58583651Fax: +46 8 58583650e-mail: [email protected]

Acknowledgements

We thank Prof. Kiyoshi Takatsu for the Y16 cell line, Dr

Harry Mellor for the Rif siRNA, Dr Julie G. Donaldson for the

Arf6 construct, Dr Anne Ridley for valuable discussions, and

Marcelo Toro for the FACS sorting. This work was supported

by the Swedish Science Council, the Stockholm County

Council (research grant ALF-projektmedel medicin), the

Cancer Foundation, the European Union FP7 grant EURO-

PADnet, The Magn. Bergvall Foundation and the Ake Wiberg

Foundation. Mauno Vihinen was supported by the Academy

of Finland and the Medical Research Fund of Tampere

University Hospital. Rani Faryal was a recipient of a

Postdoctoral Fellowship from the Higher Education

Commission (HEC), Pakistan.

Immunological Reviews 2009

Vol. 228: 58–73

Printed in Singapore. All rights reserved

� 2009 The Authors

Journal compilation � 2009 Blackwell Munksgaard

Immunological Reviews

0105-2896

Summary: Bruton’s agammaglobulinemia tyrosine kinase (Btk) is a cyto-plasmic tyrosine kinase important in B-lymphocyte development, differ-entiation, and signaling. Btk is a member of the Tec family of kinases.Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) inhumans and X-linked immunodeficiency (Xid) in mice. Activation of Btktriggers a cascade of signaling events that culminates in the generation ofcalcium mobilization and fluxes, cytoskeletal rearrangements, and tran-scriptional regulation involving nuclear factor-jB (NF-jB) and nuclearfactor of activated T cells (NFAT). In B cells, NF-jB was shown to bind tothe Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-jB signaling pathway requires functional Btk. Moreover,Btk activation is tightly regulated by a plethora of other signaling proteinsincluding protein kinase C (PKC), Sab ⁄ SH3BP5, and caveolin-1. Forexample, the prolyl isomerase Pin1 negatively regulates Btk by decreasingtyrosine phosphorylation and steady state levels of Btk. It is intriguingthat PKC and Pin1, both of which are negative regulators, bind to thepleckstrin homology domain of Btk. To this end, we describe here novelmutations in the pleckstrin homology domain investigated for their trans-forming capacity. In particular, we show that the mutant D43R behavessimilar to E41K, already known to possess such activity.

Keywords: IL-2 inducible tyrosine kinase (Itk), Tec protein tyrosine kinase (Tec), bonemarrow tyrosine kinase gene on chromosome X, X-linked agammaglobulinemia, peptidyl-prolyl cis–trans isomerase (Pin 1), nuclear factor jB

Introduction

Bruton’s agammaglobulinemia tyrosine kinase (Btk) is a non-

receptor tyrosine kinase belonging to the Tec family of kinases

(1, 2). Tec family kinases (TFKs) form the second largest fam-

ily of cytoplasmic tyrosine kinases in mammalian cells and

include, in addition to Btk, Tec, Itk, Txk (also known as Rlk),

and bone marrow tyrosine kinase gene on chromosome X

(Bmx). Most of the TFKs (Btk, Itk, Tec, Txk) are predomi-

nantly expressed in hematopoietic cells, whereas Bmx is

essentially restricted to endothelial cells (1, 3–5). In addition

to hematopoietic cells, Tec expression has been detected in

other tissues (6). In mammalian cells, more than one member

of these kinases has been demonstrated to be expressed in the

same tissue or cell suggesting the possibility of redundancy.

For example, T cells express Tec, Itk, and Txk, while B cells

use Btk and Tec. In addition, expression of Itk and Btk is much

higher than Tec in T and B lymphocytes, respectively. The

biological function and intracellular signaling of Tec family

kinases have been described in several reports (3, 7–11).

Gene-targeted animals deficient for Btk or Itk have defined

phenotypes, while single knockouts of Tec or Txk do not

show overt changes (12–18). By contrast, certain knockouts

involving combinations of two members (Btk ⁄Tec and

Itk ⁄Txk) have been shown to display profound phenotypic

changes (16, 19). TFKs have been reported to signal through

several cell surface receptors. A common cellular response

triggered by TFK signaling is a dramatic cytoskeletal remodel-

ing involving actin polymerization (2, 11, 20).

Btk is critical for B-cell development, differentiation,

and signaling (21–23). Moreover, Btk expression is

assumed to be a prerequisite for B-cell proliferation and

survival. Consequently, Btk-deficient B lymphocytes fail to

reach the mature state and are presumably doomed to

premature death. Btk is the only member of the TFKs

reported to be associated with human disease. Thus, indi-

viduals harboring loss-of-function mutations in the gene

encoding Btk virtually lack circulating B lymphocytes, are

unable to generate immunoglobulins of all classes, and

therefore cannot mount humoral immune responses. This

primary immunodeficiency is named X-linked agamma-

globulinemia (XLA). Similarly, a spontaneous mutation in

the mouse-Btk gene (23) leads to X-linked immunodefi-

ciency (Xid), a considerably milder condition than XLA,

while the very same mutation in humans causes classical

XLA, suggesting species differences (23, 24).

In this review, we focus on the function and regulation of

Btk, and in particular, discuss the achievements made over the

past decade with respect to the biochemistry of this remark-

able protein. We also include primary data regarding several

aspects of Btk biology, including the existence of a truncated

form of Btk, as well as novel, site-directed mutants with trans-

forming capability.

Genetics and expression of Btk

The BTK gene consists of 19 exons and spans approximately

37.5 kb on the human X chromosome (25). Scientists on both

sides of the Atlantic, including our laboratory, independently

cloned the gene in 1992 (21, 22). Over 800 mutations affect-

ing Btk have been reported to the international mutation data-

base designated BTKbase (http://bioinf.uta.fi/BTKbase),

including deletions encompassing also three additional genes

(26). Similar to other genetic diseases, the disease-causing

mutations that occur in Btk include missense, nonsense, splice

site, deletions ⁄ insertions and are literally spread over the

entire gene (27–34). However, no disease-related missense

mutation in the Src homology 3 (SH3) domain has been

reported.

Btk is predominantly expressed in B lymphocytes but not in

plasma cells (35). Btk expression in the B-cell lineage is also

developmentally regulated, with marrow-derived hematopoi-

etic stem cells, common lymphoid progenitor cells, develop-

ing B and myeloid lineages showing the highest levels,

whereas resting mature cells prior to activation have reduced

cellular Btk (36). Moreover, with the exception of T lympho-

cytes, all other hematopoietic lineages have been shown to

express Btk. As B lymphocytes are the only cells known to be

affected in XLA, the physiological significance of Btk expres-

sion in other cell types remains to be established. Nonetheless,

there are multiple reports describing defects in other cell types

such as platelets and macrophages (37–39). Recent studies

show that osteoclast development is impaired in Btk-deficient

mice (40, 41). Although changes have been reported to occur

in different cell types of the hematopoietic system, as men-

tioned, the severity in B lymphocytes is far more pronounced.

Thus, in humans Btk is indispensable for B-cell development.

Second, the molecular mechanism(s) underlying the differ-

ence in the degree of severity in phenotype between XLA and

Xid is not known. It is known, however, that Tec partially can

replace Btk in mice (16), while in humans Btk deficiency

alone causes severe disease in spite of the presence of Tec in

human B lymphocytes. In transfection experiments involving

a Btk-deficient chicken B-cell line (DT40-derived), Tec was

demonstrated to be capable of restoring activity and trigger

downstream signaling (42). This cell line is deficient in

calcium signaling (43, 44) and also displays cytoskeletal

features characterized by formation of shorter membrane

protrusions and the degree of aggregation is much less than in

wildtype cells (20).

Btk is the only member in the Tec family reported to be

associated with pathogenesis in humans. In the absence of

Btk, B-cell development is impaired due to a block

between the pro- and pre-B-cell stages leading to severe

reduction of mature B lymphocytes. Accordingly, individu-

als who have loss-of-function mutations in the Btk gene

develop XLA, the hallmark of which is an essentially

complete absence of circulating B cells. These patients are

incapable of producing antibodies and are subject to

recurrent bacterial and enteroviral infections (45). There

are also mouse models of this disease, one of which is Xid.

Mohamed et al Æ Bruton’s tyrosine kinase (Btk)

� 2009 The Authors • Journal compilation � 2009 Blackwell Munksgaard • Immunological Reviews 228/2009 59

Xid manifests as a result of a spontaneous mutation of a

CpG site in the Btk gene changing residue 28 from an argi-

nine to a cysteine (R28C) in the pleckstrin homology (PH)

domain (23). While the identical mutation leads to classi-

cal XLA in humans (24), Xid mice maintain about half the

number of splenic B lymphocytes. The second model,

genetically modified Btk knockout mice, has a similar

phenotype as Xid mice (17, 18). When female mice

heterozygous for the R28C missense mutation and the null

allele were analyzed, we found that none of the alleles

conferred any B-cell survival advantage over the other,

suggesting that this missense mutation functions as a bona

fide null allele (46).

Biochemistry of Btk

It is generally considered that mammals and birds have all five

TFK family members, although only a single avian genome

has been sequenced. Using bioinformatics tools, we recently

demonstrated that a TFK ancestor exists in the unicellular

choanoflagellate Monosiga brevicollis, which is the closest known

relative to metazoans with a sequenced genome (47). The

analysis of the genomes of sponges, insects, hagfish, and frogs

suggests that these species encode a single TFK. TFKs are

non-receptor kinases containing several unique domains char-

acteristic for signaling proteins (1). Btk comprises of several

domains from the N-terminus: the PH, Tec homology (TH),

SH2, SH3, and kinase (SH1) domains (Fig. 1). Each of these

domains has the potential to interact with a plethora of

proteins critical for intracellular signaling. Moreover,

functional association of Btk with many of its partners is

crucial for its activation and regulation. Here we want to

highlight some of the key proteins that execute this.

Btk is a metalloprotein enzyme requiring Zn2+ for optimal

activity and stability. Binding and coordination of Btk to the

Zn2+ion is mediated by a highly conserved zinc finger motif,

also called the Btk motif, located in the TH domain (89, 90).

Mutations affecting Zn2+binding lead to the generation of

extremely unstable protein (31, 61).

Fig. 1. Schematic representation of the Bruton’s tyrosine kinase (Btk) promoter and Btk domains with all known interacting/regulatingpartners. Domains shown: pleckstrin homology (PH); Tec homology (TH), [Btk homology region (BH) and polyproline region (PPR)]; Src-homology3 (SH3); Src-homology 2 (SH2); Kinase (SH1), tyrosine kinase domain. Regulatory phosphorylation sites are indicated by arrows, phosphotyrosinespY223 and pY551 are in red color, and phosphoserines pS21, pS115, and pS180 in blue. The black box contains putative partners for which the siteof interaction has not been identified (20, 31, 48–60, 62–88).



Btk is transcribed from a single gene, and there is no

evidence of alternative splicing. Yet, a shorter, presumably

truncated isoform of the protein is often observed in sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

when an SH3 or C-terminus specific antibody is utilized (Abdalla

J. Mohamed, Liang Yu, C. I. Edvard Smith, unpublished data).

This shorter isoform of Btk is expressed both in endogenous

and in transfected cell lines and seems to be highly unstable.

In fact, in some cell lines including primary B lymphocytes, it

is virtually undetectable. Although the nature and origin of

this faster migrating band is not known, we think that it is

generated as a result of alternative translation initiation or pro-

teolysis. In addition to the first methionine (ATG), which as

customary is often utilized, Btk contains several additional

in-frame translation initiation codons that may be responsi-

ble for producing the truncated form. Second, missense muta-

tions that modify the first ATG codon have been reported.

Although patients harboring these types of mutations present

classical XLA, full-length Btk protein, albeit at much lower

levels, could readily be detected in peripheral blood mono-

nuclear cells (PBMCs) (91). This result is consistent with our

own findings, whereby mutating the first ATG to TTG leads to

the production of not only the shorter truncated protein

but also full-length Btk (Abdalla J. Mohamed, Liang Yu,

C. I. Edvard Smith, unpublished data). It has been reported

that the translation machinery of eukaryotic genes can some

times utilize a non-ATG codon but with much less efficiency

(92–94).

Btk activation

The molecular mechanisms that underlie activation of Btk are

currently not fully understood. However, it is generally

believed that post-translational modifications as well as sub-

cellular localization could play key roles. In steady state, Btk is

predominantly cytosolic. Following B-cell receptor (BCR)

engagement, Btk translocates to the plasma membrane where

it gets phosphorylated at Y551 by a Src family kinase (SFK),

presumably Lyn. Phosphorylation of Btk at this particular tyro-

sine, which is in the catalytic domain, is followed by an auto-

phosphorylation event involving another tyrosine in the SH3

domain, Y223. Notably, the initial phosphorylation at Y551

primes the protein for activation, while the role of phosphory-

lation at Y223 is less clear (95). In close cooperation with the

adapter protein B-cell linker protein (BLNK), also known as

SH2 domain-containing leukocyte protein of 65 kDa (SLP-

65), Btk subsequently phosphorylates phospholipase Cc2

(PLCc2), igniting the downstream signaling pathway. Since

Btk is a multidomain protein, it has also the ability to bring

together a diverse array of signaling proteins into a single

platform. In primary B lymphocytes, Btk is almost always

non-phosphorylated and is thus catalytically inactive. How-

ever, many of the experiments that address phosphorylation

of Btk and other tyrosine kinases have been conducted on

heterologous cell types, such as COS-7 and HEK 293T, as well

as transformed B cells. Although some important information

relevant to these questions can be obtained using these cells,

there are two fundamental problems associated with both of

these systems. First, heterologous cell lines, though easily

tranfectable, do not endogenously express Btk or other B-cell-

specific proteins required for activation and signaling. Second,

as most utilized B-cell lines are transformed, they may not

represent the genuine milieu that prevails in primary cells.

Nonetheless, COS-7 and HEK 293T have been invaluable in

the biochemical and functional characterization of cloned

genes.

In spite of the accumulation of a great amount of data in

recent years, the precise mechanism(s) underlying phosphor-

ylation and activation of Btk has not been established. How

does Btk get phosphorylated in the first place? Why does it get

phosphorylated in some cells but not in others? We need to

understand these and similar questions to get deeper insight

into the dynamics of Btk activation. For instance, whereas Btk

and Tec are robustly phosphorylated in the COS-7 cell line, Itk

is not (Abdalla J. Mohamed, Liang Yu, C. I. Edvard Smith,

unpublished data). This situation is reminiscent to that of Syk

and f-associated protein of 70 kDa (ZAP-70) (96). It is

intriguing that these structurally similar kinases (Btk ⁄ Itk,Syk ⁄ZAP-70) behave so differently in the same cellular

environment. It is conceivable that a specific kinase or a phos-

phatase can differentiate between Btk and Itk in the COS-7 cell

line. Second, it is also possible that the two proteins localize

differently within the cell, leading to phosphorylation of one

but not the other. Third, as Btk and Itk differ in their primary

structure (approximately 50% sequence identity), post-

translational modification may be an important process in reg-

ulating their activity. Last but not least, although similar in

structure, the two proteins may still adopt different conforma-

tions in the same milieu.

Btk and Toll-like receptor signaling

The ability of Btk to interact with members of Toll-like recep-

tor (TLR) family (namely TLR4, TLR6, TLR8, and TLR9) and

key proteins from TLR signaling pathways, such as myeloid

differentiation protein 88 (Myd88), MyD88 adapter-like

protein (Mal), and interleukin-1 receptor (IL-1R)-associated

kinase-1 (IRAK-1), suggest the importance of Btk as a compo-



nent of TLR pathways and that this kinase therefore plays an

important role in the function of immune cells of innate as

well as adaptive immunity (97–99).

We have investigated the potential role of Btk in the TLR9

activation and the subsequent production of pro-inflamma-

tory cytokines such as IL-6, tumor necrosis factor-a (TNF-a)

and IL-12p40 (13). Our data show that Btk-deficient B cells

respond more efficiently to CpG-DNA stimulation, producing

significantly higher levels of pro-inflammatory cytokines

when compared to wildtype cells but less of the inhibitory

cytokine IL-10. Quantitative reverse transcriptase polymerase

chain reaction (RT-PCR) analysis presented in our previous

work shows that IL-27 mRNA was significantly increased in

Btk-deficient B cells after CpG-DNA stimulation. In this study,

we also demonstrated important differences in CpG respon-

siveness between transitional 1 (T1) and T2 B-cell survival

and maturation. Interestingly, CpG-DNA stimulation induced

a time-dependent accumulation of transitional T2 B cells in

both Btk-deficient and normal B cells.

Furthermore, stimulation of B cells with TLR9 ligand

CpG leads to transient phosphorylation of Btk, and in the

absence of Btk, TLR9-induced proliferation of B cells is

impaired (100). Collectively, these data suggest that Btk is

a critical molecule in regulating TLR9 activation in splenic

B cells. Finally, elucidation of the physiological role of

TLRs in B cells may provide important clues for under-

standing the molecular mechanisms behind inflammation

and immunity.

Subcellular localization of Btk

Plasma membrane localization of Btk is a critical step in the

phosphorylation and activation of Btk. Following BCR engage-

ment by antigen, phosphatidylinositol-3-kinase (PI3K) is

activated, leading to the generation of the phosphoinositide

phosphatidylinositol-3,4,5-trisphosphate (PIP3). Accumula-

tion of PIP3 in the inner surface of the plasma membrane

results in the recruitment of various signaling proteins includ-

ing Btk. Subsequent binding of the PH domain of Btk to PIP3

is a prerequisite for its activation. Although the exact molecu-

lar mechanisms responsible for targeting Btk to the cell surface

are not well known, the PH domain is thought to play a key

role (20, 101). Deletion of the PH domain renders Btk mainly

cytoplasmic, although a substantial amount translocates to the

nucleus (102). Also, for reasons that are not clear to us, steady

state levels of this form of the protein are higher than wildtype

Btk. Further, though such a PH domain-less version of Btk is

robustly phosphorylated, it cannot properly signal. Even a

subtle alteration in the PH domain could have disastrous

effects on the overall function of the Btk protein. This is sub-

stantiated by the fact that the R28C mutation in the PH

domain (Fig. 2) leads to classical XLA, and, moreover, there

are additional missense mutations in this domain that cause

disease (28, 103).

Although predominantly cytoplasmic, a small fraction of

Btk can at any given time be detected in the nucleus, suggest-

ing that it is a nucleocytoplasmic shuttling protein (102).

Thus, it is becoming increasingly clear that Btk not only trans-

locates to the nucleus but also that it seems to be rapidly

exported. In some B cells, the fraction of Btk that is inside the

nucleus can be as high as 50% (Abdalla J. Mohamed, Liang

Yu, C. I. Edvard Smith, unpublished data). The molecular

mechanism(s) responsible for nuclear localization and ⁄or

export of Btk as well as its potential role in the nucleus remain

to be established. Although motifs resembling classical nuclear

localization signals (NLS) appear in the PH domain, they do

not seem to play a role in the nuclear translocation process.

Similarly, nuclear shuttling of Btk is independent of phos-

phorylation, since a kinase-defective mutant can also translo-

cate into the nucleus. Of note, deletion of the PH–TH domain

doublet or a truncation extending into the N-terminal part of

the SH3 domain renders Btk slightly more nuclear (102). In

Fig. 2. The structure of the pleckstrin homology (PH) domain andthe Btk motif corresponding to residues 1–170 of the humanBruton’s tyrosine kinase (Btk). The figure is based on the experimentalstructure in Protein Data Bank (entry 1bwn). Cyan ribbon runs alongbackbone of the molecule, except for orange for the C-terminal a-helix,and blue for the Btk motif of TH domain. Serines 21 (top) and 115(bottom), which can be phosphorylated, are in magenta. The yellow sidechains represent residues glutamate 41 (right), aspartate 43 (middle),and glutamate 45 (left), respectively. The arginine 28 residue is in pink.IP4 molecules are green. The long Btk-specific loop is not visible in thisstructure. The blue sphere denotes the Zn2+ion in the Btk motif.



addition, mutation of Y223, but not Y551, increases the frac-

tion of Btk in the nucleus (Abdalla J. Mohamed, Liang Yu,

C. I. Edvard Smith, unpublished data).

Nuclear export of Btk has been shown to be mediated by

the nuclear export receptor CRM1 (exportin), since treatment

of cells with the antibiotic leptomycin B leads to nuclear arrest

of Btk (102). Thus, these data suggest that Btk is continuously

shuttling between the cytosol and the nucleus. Furthermore,

we have conclusive evidence that Btk interacts with CRM1

both in endogenous and in transfected settings (Abdalla J.

Mohamed, Liang Yu, C. I. Edvard Smith, unpublished data).

At present, we do not know the nuclear export signals that Btk

utilizes to exit from the nucleus. Very little is known about

the function of Btk inside this compartment. However, as

nuclear Btk is less phosphorylated as compared with the form

residing in the cytosol, it is possible that a nuclear phosphatase

is responsible and that this could be of functional importance

for the regulation of this kinase. Alternatively, Btk may have

unknown nuclear substrates. As described in the section ‘Role

of Btk in transcription’, there are reports suggesting a role for

Btk in the activation of the transcription factor Bright. This

activity is independent of Btk’s catalytic function, as Bright

does not seem to be phosphorylated by Btk (75).

For signaling proteins in particular, nucleocytoplasmic shut-

tling could be one way of regulating and ⁄ or fine-tuning their

activity. It creates a forum for compartmentalization. In the

case of kinases like Btk, activation could be elicited following

signals at the plasma membrane, whereas inactivation and

termination of signaling may occur in the nucleus.

Plasma membrane localization of Btk

As discussed in the previous section, plasma membrane associ-

ation of Btk is a prerequisite for its phosphorylation and

subsequent activation. Thus, to properly signal during BCR

engagement, Btk has to traffic first to the cell surface. As the

PH domain is thought to mediate plasma membrane targeting

of Btk, it is not surprising that mutations in this domain have

profound effects on this activity. For instance, a great majority

of the disease-causing mutations in the PH domain of Btk

frequently affect arginine 28 (32). This alters the positive

charge on the surface of the PH domain (Fig. 2), abolishing its

binding to membrane-resident lipid ligands and consequently

the recruitment to the plasma membrane after stimulation of

the cell (61, 104).

By contrast, when the glutamate at position 41 in the PH

domain of Btk is substituted by lysine (E41K), Btk achieves a

gain-of-function state with increased membrane targeting and

tyrosine phosphorylation level (105). In addition, this

gain-of-function mutation has been shown to display robust

transformation potential in the anchorage-dependent soft agar

assay. According to the structure of the PH domain, the E41K

mutant binds to two PtdIns(3, 4, 5)P3 molecules compared

with the wildtype that binds to only one (104). Therefore, it

is possible that this type of mutation enables Btk to spend

more time at the inner surface of the plasma membrane, lead-

ing to sustained activation of the protein. To determine

whether creation of additional positively charged amino acids

in the PH domain leads to increased activity of Btk, we

mutated asparagine 43 (D43) and glutamate 45 (E45) to

either lysine (K) or arginine (R) and tested them, as described

in the next sections. The E41K mutant was also combined

with E45K or E45R. According to the X-ray crystal structure of

Btk, these residues are all located on the surface of the PH

domain, within the region that binds to the cell membrane

(Fig. 2).

Using a Btk–green fluorescence protein (GFP) fusion

construct, subcellular localization of the mutants as well as

wildtype and the kinase inactive form of Btk were character-

ized in HeLa cells. As shown in Fig. 3, wildtype Btk displays

typical cytoplasmic distribution in these cells. However, as

expected, a small fraction of Btk is also detected in the

nucleus. We had shown previously that overexpression of the

Btk mutant E41K in COS-7 cells results in a pronounced

induction of extensive lamellipodia and some filopodia forma-

tions (20). In sharp contrast, HEK 293 cells transfected with

the same construct predominantly display filopodia formation

(Abdalla J. Mohamed, Liang Yu, C. I. Edvard Smith, unpub-

lished data). In HeLa cells, formation of ‘hairy’ actin filament

bundles or microspike structures were the most pronounced

form of cytoskeletal alterations found, following expression of

the E41K mutant in these cells. The nature of the cytoskeletal

structures formed in the different cell lines depends largely on

the endogenous expression of the small GTPases Rac, Rho,

and Cdc 42. For example, Rac is probably the major small

GTPase expressed in COS-7, which could explain the forma-

tion of lamellipodia in this cell line. In fact, lamellipodia for-

mations in COS-7 were successfully blocked with dominant

negative Rac1 (20).

With the exception of E45K and E45R, all other PH domain

mutants displayed a pattern similar to E41K, and this pattern

was more pronounced after serum stimulation (Fig. 3, data

not shown). The cytoskeletal changes in the form of protru-

sions induced by expressing E45K and E45R were much less

pronounced. The most striking difference, however, was seen

after introducing the D43R mutant. The cells get rounder and

smaller following serum stimulation but to some degree keep



their microspike-like structures. As mentioned earlier, in

steady state, Btk is predominantly cytoplasmic, although

sometimes it can also show a punctuate staining all over the

cell. In serum-stimulated cells, Btk translocates mainly to a

perinuclear compartment, seemingly residing on filaments

reaching to the plasma membrane. The kinase-inactive form

of Btk failed to induce the microspike formations, indicating

that both membrane association and kinase activity are impor-

tant for this phenotype (Abdalla J. Mohamed, Liang Yu, C. I.

Edvard Smith, unpublished data). In addition, membrane tar-

geting was not clearly altered for the PH domain mutants

D43K and D43R compared with E41K, whereas membrane

tethering of the E45 mutants was reduced (Abdalla J.


Table 1 summarizes the cell biological and functional charac-

terization of the different PH domain mutants.

The structures formed by the expression of E41K in COS-7

cells show similarities to the cytoskeletal structures appearing

when a constitutively active form of Rif or Arf6 is expressed

in cells (106, 107). Co-transfection of siRNA targeting Rif or

dominant negative form of Arf6 with Btk E41K did not block,

however, the microspike formation (Abdalla J. Mohamed,

Liang Yu, C. I. Edvard Smith, unpublished data).

Transforming potential of Btk

An important question is whether Btk is oncogenic or not.

This issue has been debated since the cloning of the BTK gene

and has not been conclusively resolved. There are numerous

reports implicating a number of tyrosine kinases in cancer

(108–110). Most of these concern receptor tyrosine kinases

belonging to the growth factor receptor family. However,

there are also non-receptor tyrosine kinases that are critical for

cancer development, the most well-known being Abl. As

many of the tyrosine kinases are important in cell proliferation

and survival, they are also prone to support tumor growth. In

addition, some tyrosine kinases are important in cell migra-

tion, a property of cancer cells. For example, SFKs are believed

to play a key role in growth and metastasis of many types of

human malignancies. Although Btk is known to be crucial for

important biological process in B-cell development, such as

proliferation and survival, its relation to cancer is complex.

Thus, there are at least five rather different scenarios where

Btk has been implicated in tumor formation: (i) inactivating

mutations of Btk found in lymphoid tumors, (ii) inactivating

mutations of Btk as a cause of colorectal cancer, (iii) a role for

splice variants of Btk in leukemia, especially chronic myeloge-

nous leukemia, (iv) Btk-defective mice are resistant to certain

experimentally induced tumors, such as the pristine-induced

plasmacytomagenesis (111), suggesting that Btk-deficient B

cells are less likely to get transformed, and (v) activating

mutations causing a transforming phenotype in experimental

Steady state

Btk

D43R

E41K

Stimulated

Fig. 3. Confocal images of HeLa cells transfected with expressionconstructs for Bruton’s tyrosine kinase (Btk)–green fluorescenceprotein (GFP). Wildtype Btk (upper panel); D43R (middle panel); E41K (lowerpanel). The left panels show serum-starved cells and the panels to the rightafter serum stimulation. Red indicates F-actin stained with phalloidinconjugated to rhodamine, and blue indicates nucleic acid, stained withDAPI.

Table 1. Characteristics of transforming Btk mutants

Btk mutant Btk localization* Microspikes*Transformingcapacity�

WT Cytoplasmic ) )E41K Cytoplasmic ⁄ membrane ++ +E41R Cytoplasmic ⁄ membrane ++ )D43K Cytoplasmic ⁄ membrane ++ )D43R Cytoplasmic + ⁄ ) +E45K Cytoplasmic ⁄ membrane + )E45R Cytoplasmic ) )E41K + E45K Cytoplasmic ⁄ membrane ++ +E41K + E45R Cytoplasmic + +

*Data are from HeLa cells transfected with the various Bruton’s tyrosinekinase (Btk)–green fluorescence protein (GFP) constructs. Forty hourslater, subcellular localization and microspike formation was determinedusing confocal microscopy. Perinuclear localization was normally seen.�The transforming potential of the mutants was investigated in the IL-5-dependent Y16 mouse B-cell line. Transformation of these cells rendersthem IL-5 independent.



settings, as already discussed, where Btk achieves a

gain-of-function state with increased membrane targeting and

tyrosine phosphorylation (105).

Concerning (i) the inactivating mutations of Btk found in

lymphoid tumors, there are both reports claiming such an

association in human leukemia (112) and others that did not

find evidence for this association (113). The strongest argu-

ment in support comes from knockout mice with inactivated

BLNK. Here, it is very clear that combining this null mutation

with Btk deficiency potentiates tumor development (114,

115). Regarding (ii) inactivating mutations of Btk as a cause

of colorectal cancer, there are several such reports (reviewed

in 45, 116), and very recently two additional patients were

described (117). Owing to that Btk is normally not expressed

in colon-derived cells, it is likely that any association is indi-

rect and caused for example by an altered bacterial flora,

which subsequently predisposes for cancer.

With regard to (ii) a role for splice variants of Btk in leuke-

mia, especially chronic myelogenous leukemia, Btk has been

implicated as tumor-inducing through an effect dependent on

different splice variants induced by the fusion protein Bcr–Abl

(118, 119). The interpretation of these findings was that lack

of Btk expression, or expression of dominant-negative splice

variants, in B-cell precursor leukemia cells can inhibit differ-

entiation beyond the pre-B-cell stage and protect from radia-

tion-induced apoptosis. However, while the SH3 domain of

Btk may serve as a substrate for the Abl cytoplasmic tyrosine

kinase (120, 121), a recent study found no evidence for a

connection (121). These authors reported that inhibition of

Btk did not affect the ability of Bcr–Abl to transform primary

murine hematopoietic cells in colony-forming and B-cell

transformation assays.

Owing to the remarkable effects of the Btk PH domain

mutants in HeLa cells, the interest was to further characterize

the mutants and determine how they behave in B cells. We

therefore chose to use the mouse B-cell line Y16. This cell line

strictly requires the cytokine IL-5 for optimal growth. Thus,

in the absence of IL-5, the cells cannot proliferate and eventu-

ally go into apoptosis. Next, we stably expressed the different

mutants in the form of Btk–GFP fusions in Y16 cells. GFP+

cells were then sorted from the GFP) cells by fluorescence

activated cell sorting (FACS) and were studied in a digital con-

focal microscopy. In the Y16 cells stably expressing the Btk

mutant E41K, we could readily detect membrane protrusions

(Abdalla J. Mohamed, Liang Yu, C. I. Edvard Smith, unpub-

lished data). By contrast, there were no visible protrusions in

cells expressing the other mutants. Interestingly, the E41R

mutant showed clear accumulation in one or two cytoplasmic

spots located close to the nucleus, which could indicate co-

localization with centriols in the microtubule organizing cen-

ters (MTOCs) (Abdalla J. Mohamed, Liang Yu, C. I. Edvard

Smith, unpublished data). Similar observations were found in

the rat basophilic cell line RBL 2H3 (Abdalla J. Mohamed,

Liang Yu, C. I. Edvard Smith, unpublished data). In addition,

we noticed an enhanced proliferation of some of the mutants,

as indicated by the rapid acidification of cell culture medium.

Analysis by cell count and the colorimetric cell proliferation

reagent WST-1 showed a higher proliferation rate of E41K,

D43R, and the double mutants E41K + E45K or E45R com-

pared with the wildtype Btk–GFP (Abdalla J. Mohamed, Liang

Yu, C. I. Edvard Smith, unpublished data). This result is con-

sistent with previous findings showing that the E41K mutant

can support growth of Y16 cells in the absence of IL-5 (105).

To further determine whether these mutants could also

support IL-5-independent growth of the cells, we propagated

the cells in the presence or absence of IL-5. The data clearly

show proliferation of cells expressing E41K, D43R, and

E41K +E45R, even though IL-5 was excluded from the

growth medium, indicating that these mutants have a trans-

forming potential (Fig. 4, data not shown). Owing to that

E41K induces an increased proliferation rate, it is likely that

this is the dominating feature also of the double mutants, as

neither of the E45 mutations further alters the effect. By con-

trast, cell proliferation following introduction of the E45

mutants alone was more similar to cells expressing wildtype

Btk. Interestingly, the D43R mutant induced a pronounced

cell-proliferative effect. This finding may suggest that the E45

Fig. 4. Growth curve of Y16 cell lines expressing the different formsof Bruton’s tyrosine kinase (Btk)–green fluorescence protein (GFP).Y16 cells stably expressing the different forms of Btk–GFP were culturedin the absence of IL-5 over 6 days. The OD values obtained using theWST-1 reagent, during 1 h of incubation, are proportional to the num-ber of living cells as detected by a colorimetric cell proliferation assay.



mutants are located too far away from the active membrane-

binding region of the PH domain and therefore do not con-

tribute to enhanced activation, in spite of changing the net

surface charge of Btk similar to the E41 and D43 mutants.

Moreover, as the E41 + E45 double mutants have an

increased effect whereas the E45 single mutants do not, the

overall structural conformation of the PH domain with an E45

mutation is probably not altered. The results are summarized

in Table 1.

Enhanced electrostatic polarization seems to increase the

affinity for PtdIns(4,5)P2 and PtdIns(3, 4, 5)P3 (101, 122). In

the crystal structure of the Btk PH domain with the E41K

mutation, soluble Ins(1, 3, 4, 5)P4 was used to mimic the

PtdIns(3, 4, 5)P3. A second PtdIns(3, 4, 5)P3 bound to the

E41K mutant, indicating a role of positively charged regions

in binding phosphoinositides. E41K has also been shown to

transform fibroblasts in vitro (105, 123). When overexpressed

in B lymphocytes, it disrupts the architecture of lymphatic

tissues (124). Thus, the extension of electrostatic polarization

with positively charged amino acids in transgenic mice over-

expressing the Btk E41K results in a more severe B-cell defect

than the Xid phenotype (18). This mutant not only blocks the

development of follicular recirculating B cells but also causes

an enhanced blast formation of splenic B cells in vitro (124).

Recent findings show an increased pre-B cell tumor incidence

in mice defective in both Btk and BLNK and that Btk E41K

could substitute for SLP-65 as a tumor suppressor (114).

Regulation of Btk function – an overview

Much of the regulation of Btk function remains elusive. More-

over, information about structure–function relationship of

this cytoplasmic tyrosine kinase is scanty. Although a com-

plete and thorough understanding of the three-dimensional

structure of Btk is not available, experiments indicate that the

molecule is extended under certain conditions and in steady

state exists as a monomer (125, Abdalla J. Mohamed, Liang

Yu, C. I. Edvard Smith, unpublished data). However, crystal

structures formed using the PH domain of Btk have suggested

that dimers could be formed in the membrane (61, 126).

Unlike SFKs, Btk does not possess a negative regulatory

C-terminal tyrosine to control its activity. Also there is no

evidence of intra or intermolecular interaction(s) involved

in the regulation of full-length Btk, while there is ample

evidence for such interactions when various forms of TH–SH3

domain doublets are studied (127–129).

Thus, it may well be that Btk predominantly relies on pro-

tein–protein interaction mechanism(s) to regulate its own

kinase activity. Interestingly, most of the identified Btk-inter-

acting proteins seem to be negative regulators. One of the ear-

liest described negative regulators of Btk was protein kinase C

(PKC) (67, 69, 70, 130–132). By phosphorylating a key

serine in the TH domain (S180), PKCb has been shown to

interfere with the plasma membrane targeting and subsequent

activation of Btk (68, 131). In stark contrast, PKCh activates

Btk, while Btk downregulates the activity of PKCh (70, 132).

Remarkably, PKCb-deficient mice display the same phenotype

as Xid mice (133). Recently, another protein termed inhibitor

of Btk (IBtk) has been shown to physically associate with Btk

and downregulate its kinase activity (82). However, little is

known about how this happens, although the same group

recently reported that IBtk specifically binds to the PH domain

of Btk (134). Also, Sab (synonymous SH3BP5) has been

shown to inhibit the autophosphorylation and transphosph-

orylation activity of Btk and thus negatively regulates BCR sig-

naling (62, 63), as also shown in genetic studies in Drosophila

melanogaster (135).

It has been demonstrated that caveolin-1 is expressed in B

lymphocytes and functionally interacts with the kinase

domain of Btk (64, 136). Interaction of Btk with caveolin-1

led to dramatic downregulation of the kinase activity of Btk.

We envisage that following BCR stimulation, activated Btk

translocates to lipid rafts and caveolae. Lipid rafts and caveolae

are lipid-rich plasma membrane compartments known to

function as signaling platforms for regulating the activity of

intracellular signaling molecules. Thus, caveolae may function

as a temporary station for deactivating Btk and other signaling

proteins. Caveolae-resident Btk may have two fates: (i) it is

either degraded or (ii) it may be reactivated upon BCR signal-

ing. Subsequently, activated Btk exits the caveolae compart-

ment and translocates to the cytosol to be reused.

Pin1 negatively regulates Btk

Btk is a remarkably stable protein with a half-life of more than

20 h in the mouse B-lymphocyte cell line A20 (Abdalla J.


In stark contrast, the half-life is considerably reduced when

heterologous systems for over expressing Btk are used. The

reason for this discrepancy is not clear. Our laboratory

recently reported that Pin1 (protein interacting with NIMA1)

regulates phosphorylation and steady state levels of Btk (65)

(Fig. 5). Pin1 is a peptidyl–prolyl cis–trans isomerase [prolyl

isomerase (PPIase)] involved in controlling a diverse array of

proteins ranging from enzymes to transcription factors. Pin1

is a mitotic regulator containing two functional domains: an

N-terminal, substrate-binding WW domain and a C-terminal

PPase domain responsible for isomerase activity. Pin1 is a



master regulator in key biological processes important in

health and disease, and various studies have shown altered

expression of Pin1 in a number of pathological conditions

including cancer and neurodegenerative diseases (137). By

binding to phosphorylated serines or threonines located N-

terminally of a proline residue, Pin1 changes the conforma-

tion of protein substrates influencing their function or stabil-

ity. Initially, we became interested to find out whether Pin1

could also regulate Btk. Visual inspection of the amino acid

sequence of Btk shows five potential Pin1-binding sites that

are spread over the entire protein.

It is becoming increasingly clear that serines 21 and 115,

both conserved residues, are bona fide Pin1-binding sites that

are differentially regulated during the cell cycle. S21 mediates

binding to Pin1 during mitosis while S115 is used during

interface (Fig. 6). By alternately binding to these phosphory-

lated sites, Pin1 can modulate the kinase activity of Btk in a

cell cycle-dependent manner. Interestingly, Pin1-binding

leads also to destabilization of Btk. Pin1-mediated regulation

of Btk requires an intact PH domain and a catalytically active

protein, as a PH domain-less and a kinase-deficient mutant are

not affected (65). How does interaction with Pin1 lead to

dephosphorylation of Btk? As Pin1 usually changes conforma-

tion of its substrates, it is conceivable that Btk is made

conformationally available to the action of a neighboring

phosphatase. Alternatively, Pin1 may itself recruit a specific

phosphatase and bring it in close proximity to Btk, making

dephosphorylation possible. In fact, we have preliminary

evidence suggesting that Btk can associate with one of the sub-

units of the serine threonine phosphatase PP2A (Abdalla J.


PP2A is a conformation-specific phosphatase (dephosphory-

lates only the trans pSer ⁄Thr–Pro isomer) that cooperates with

Pin1 to effectively dephosphorylate substrates (138). It has

also been reported that the catalytic activity of Itk is inhibited

by cyclophilin A (CypA), another prolyl isomerase (139,

140). Although the molecular mechanism(s) responsible for

the regulation of Btk activity by Pin1 is speculative, those

underlying the destabilization of Btk are even less clear. Our

previous work on this disqualifies both the proteasome and

the lysosome as the end station for Pin1-mediated Btk degra-

dation.

Our working hypothesis is the following (Fig. 6): upon BCR

stimulation, activated PI3K causes the accumulation of

increased levels of PIP3 serving as a tether for Btk, with the net

result that there is an increase of membrane attached Btk. Sub-

sequently, a membrane-bound SFK transphosphorylates Btk at

Y551, a highly conserved site in the activation loop of cyto-

plasmic PTKs. This post-translational modification is followed

by autophosphorylation at Y223 in the SH3 domain, the role

of which remains somewhat elusive (95). Functionally active,

membrane-bound Btk is now ready to phosphorylate down-

stream substrates including PLCc2. Meanwhile, additional

phosphorylation events are known to occur at serine and ⁄ or

threonine sites. Serine ⁄ threonine phosphorylation of Btk

could be critical for Btk regulation, and as mentioned, phos-

phorylation of S21 and S115 in the PH domain leads to the

recruitment of the PPase Pin1. The protein kinase(s) responsi-

ble for phosphorylation at these sites is unknown. Regarding

the outcome of this event, we think that Pin1-binding to Btk

can attenuate the signaling activity of Btk in two ways:

dephosphorylation and destabilization of Btk protein.

Role of Btk in transcription

Although the exact role of Btk in the nucleus is not clear,

increasing evidence suggests that it is involved in transcrip-

tional regulation. Btk interacts with several regulators of tran-

scription and phosphorylates some of these, thereby inducing

the activity of transcription factors. One such factor is nuclear

factor jB (NF-jB), which is involved in regulating the expres-

Fig. 5. Pin1 negatively regulates Bruton’s tyrosine kinase (Btk). In thepresence of Pin1, Btk is dephosphorylated and subsequently degraded.Immunoprecipitation analysis of COS-7 cells transfected with constructsencoding Btk and Pin1. The upper panel represents phosphorylated Btk(decorated with an anti-phosphotyrosine antibody), and the lower panelis stained with an anti-Btk antibody. Open arrowhead represents post-translationally modified Btk with increased molecular weight (M.W).Filled arrowhead represents Btk with normal M.W. *Represents truncatedform of Btk (only shown in the upper panel, see text for details).



sion of hundreds of genes, and in Btk-deficient B cells the

activity of NF-jB is profoundly impaired (141, 142). Another

transcriptional target of Btk is nuclear factor of activated T cells

(NFAT) (143), which is more known for its role in T-lym-

phocyte transcription, with the prototype target being the

gene encoding IL-2. Following BCR ligation, Btk also binds

and phosphorylates the transcription factor BAP-135 ⁄TFII-I

(53). Phosphorylated TFII-I translocates to the nucleus and

binds regulatory enhancer elements to modulate gene expres-

sion (144, 145). Btk has been shown to associate with another

transcription factor, Bright (75). Although Bright is not a

direct substrate for Btk, its activity was shown to be dependent

on functionally active Btk. Further, interaction of Btk and

Bright is thought to take place in the nucleus (in contrast to

most other effects of Btk on transcriptional regulators). More

recently, it has been demonstrated that in addition to Btk,

Bright can associate with TFII-I to induce immunoglobulin

heavy chain transcription (76, 146). Btk induces also the tran-

scriptional co-activation activity of the Btk-associated protein

BAM 11 (147). Finally, Btk has been shown to phosphorylate

signal transducer and activator of transcription 5A (STAT5A)

in the chicken B-cell line DT-40 (148). However, we have

recently found that different sublines of DT-40 are highly

divergent, suggesting that such interactions must be verified

in other cells (149). Although Btk physically interacts with a

number of transcription factors, there is no evidence that it is

capable of binding directly to DNA.

Work from our laboratory demonstrated recently that

transcription of Btk is suppressed following inhibition of the

NF-jB signaling pathway (85). By contrast, overexpression of

the NF-jB subunit p65 led to a dramatic increase of the pro-

moter activity of Btk. Notably, visual inspection of the Btk

promoter region was sufficient to identify two tandem

repeats, which turned out to be bona fide NF-jB-binding ele-

ments. Both of these putative NF-jB-binding elements were

shown to be functionally active both in vitro and in vivo. More-

over, both of the NF-jB subunits p50 and p65 were found to

bind to the Btk promoter (85). In Xid mice as well as in Btk-

deficient cells, the NF-jB signaling pathway is known to be

out of order. As expected, we also found that Btk is required

for the activation of the NF-jB signaling pathway. Collec-

tively, these findings show that Btk can positively regulate its

Fig. 6. Schematic model showing Bruton’s tyrosine kinase (Btk) regulation by the prolyl isomerase Pin1. B-cell receptor (BCR) ligation activatesphosphatidylinositol-3-kinase (PI3K) resulting in the production of the phosphoinositide, phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 gen-eration leads to recruitment of several intracellular signaling proteins including Btk. Once present at the cytoplasmic surface of the plasma membrane,Btk gets phosphorylated and becomes fully active. Functionally competent Btk executes downstream signaling duties, such as phosphorylation of phos-pholipase Cc2 (PLCc2). At the same time, the enzymatic activity of Btk has to be controlled. Thus, active Btk readily interacts with Pin1 using thePin1-binding motifs S21 ⁄ Pro and S115 ⁄ Pro in the pleckstrin homology (PH) domain. Pin1 hypothetically cooperates with the serine threonine phos-phatase PP2A to facilitate dephosphorylation of Btk. Finally, dephosphorylated Btk is degraded.



own promoter via NF-jB signaling (Fig. 7). Moreover, Btk

and NF-jB form an autoregulatory network, where Btk not

only regulates its own transcription but also that of NF-jB

(Fig. 8).

We have conducted microarray analysis over the last few

years to study the effect of Btk deficiency (46, 150–153).

Expression profiling provides a complete picture of the tran-

scriptome and, among other things, can identify crucial regu-

latory factors. These studies revealed that Id2, a negative

regulator of B-lymphocyte development (154), is upregulated

in Btk deficiency. Conversely, a positive regulator of B1a cell

development (155), NFATc1, was found to be 3.8-fold

downregulated in primary, unstimulated Btk-deficient splenic

B cells as well as in transitional type 1 (T1) B cells (151, 152).

Interestingly, NFATc1 is also known to be a downstream tar-

get of the Itk kinase, suggesting that this transcription factor

can be a common denominator for the signaling of at least

certain TFKs (156, 157). To this end, we have recently com-

pared expression profiles in Itk-deficient primary cells with

normal and cyclosporin A-treated cells, and several interesting

observations have been made (158). Importantly, regarding

NFATc1, Btk may thus have a dual role, both as an upstream

activator of the protein and additionally in positively regulat-

ing transcript levels of NFATc1 (Fig. 8). Thus, the effects

caused by NFATc1 alone could be key for the phenotype

observed in Btk deficiency.

Role of Btk in survival ⁄ apoptosis

As discussed in the previous section, Btk is critical for B-cell

proliferation and differentiation. While normal primary B

lymphocytes start cycling following BCR cross-linking, those

that are deficient in Btk stay quiescent and fail to expand.

Whether this is merely due to aberrant cell cycle, an increase

in cell death, and ⁄or senescence is a subject of intense debate.

However, an interesting point regarding this issue is the pre-

sumed role of Btk in apoptosis. Numerous reports have

described possible involvement of Btk in programmed cell

death. Although there are conflicting reports in the literature

that address this particular question, it is becoming increas-

ingly clear that Btk could be at the crossroads of cell survival,

proliferation, and apoptosis (159, 160). Under normal physi-

ological conditions, Btk is required for cell proliferation, dif-

ferentiation, and signaling. However, it is conceivable that Btk

could be assigned for a special mission related to apoptosis

when cells are subjected to extreme growth conditions or an

exogenous insult. The fact that heterologous cell lines stably

overexpressing Btk, or B lymphocytes expressing an extra

copy of Btk, could readily be derived is a clear indication that

in steady state, Btk does not induce apoptosis. However,

expression of Btk in these cell lines is not stable, instead Btk is

robustly downregulated. In conclusion, over the years, XLA

and Btk have been instrumental for the analysis of many

aspects of B-cell biology. As indicated in this article, while

much knowledge has been gained, many mysteries remain

unresolved, making Btk a highly interesting and challenging

molecule for future studies.

Fig. 7. Schematic model showing reciprocal regulation of Bruton’styrosine kinase (Btk) and nuclear factor jB (NF-jB) signaling. B-cellreceptor (BCR)-dependent activation of Btk induces signaling pathwaysthat converge on the transcription factors NF-jB and nuclear factor ofactivated T cells (NFAT). In both cases it is mediated by protein kinase C(PKC). NF-jB subsequently induces transcription of the Btk gene(depicted) as well as of other NF-jB-dependent genes, including IjB(not shown).

Gene Protein

NF-κBNF-κB

NFAT NFAT

Btk Btk

Fig. 8. Schematic representation of a transcriptional regulatorynetwork involving Bruton’s tyrosine kinase (Btk). Nuclear factor jB(NF-jB) positively autoregulates its transcription and also induces tran-scription of Btk. The Btk protein positively regulates both NF-jB- andnuclear factor of activated T cells (NFAT)-mediated signaling. NFATforms an autoregulatory transcriptional unit. Negative feedback loops arealso involved for NF-jB, which induces inhibitor of NF-jB (IjB), butthis is not included in the scheme for simplicity.



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Transcript of Bruton���s tyrosine kinase (Btk): function, regulation, and transformation with special...

Bruton��s tyrosine kinase (Btk): function, regulation, and transformation with special...

Transcript of Bruton��s tyrosine kinase (Btk): function, regulation, and transformation with special...