Microtubule affinity-regulating kinase 4: structure, function, and regulation

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Cell Biochemistry and Biophysics ISSN 1085-9195Volume 67Number 2 Cell Biochem Biophys (2013) 67:485-499DOI 10.1007/s12013-013-9550-7

Microtubule Affinity-Regulating Kinase 4:Structure, Function, and Regulation

Farha Naz, Farah Anjum, Asimul Islam,Faizan Ahmad & Md. Imtaiyaz Hassan

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REVIEW PAPER

Microtubule Affinity-Regulating Kinase 4: Structure, Function,and Regulation

Farha Naz • Farah Anjum • Asimul Islam •

Faizan Ahmad • Md. Imtaiyaz Hassan

Published online: 8 March 2013

� Springer Science+Business Media New York 2013

Abstract MAP/Microtubule affinity-regulating kinase 4

(MARK4) belongs to the family of serine/threonine kinases

that phosphorylate the microtubule-associated proteins

(MAP) causing their detachment from the microtubules

thereby increasing microtubule dynamics and facilitating

cell division, cell cycle control, cell polarity determination,

cell shape alterations, etc. The MARK4 gene encodes two

alternatively spliced isoforms, L and S that differ in their

C-terminal region. These isoforms are differentially regu-

lated in human tissues including central nervous system.

MARK4L is a 752-residue-long polypeptide that is divided

into three distinct domains: (1) protein kinase domain

(59–314), (2) ubiquitin-associated domain (322–369), and

(3) kinase-associated domain (703–752) plus 54 residues

(649–703) involved in the proper folding and function of

the enzyme. In addition, residues 65–73 are considered to

be the ATP-binding domain and Lys88 is considered as

ATP-binding site. Asp181 has been proposed to be the

active site of MARK4 that is activated by phosphorylation

of Thr214 side chain. The isoform MARK4S is highly

expressed in the normal brain and is presumably involved

in neuronal differentiation. On the other hand, the isoform

MARK4L is upregulated in hepatocarcinoma cells and

gliomas suggesting its involvement in cell cycle. Several

biological functions are also associated with MARK4

including microtubule bundle formation, nervous system

development, and positive regulation of programmed cell

death. Therefore, MARK4 is considered as the most suit-

able target for structure-based rational drug design. Our

sequence, structure- and function-based analysis should be

helpful for better understanding of mechanisms of regula-

tion of microtubule dynamics and MARK4 associated

diseases.

Keywords Microtubule affinity-regulating kinase �Microtubule dynamics � Tumor proliferation �Microtubule-associated protein � Cell differentiation �Cell polarity � Alzheimer’s disease

Introduction

The protein homologs PAR-1 (partition-defective), KIN1,

and MARK (MAP/Microtubule affinity-regulating kinase)

belonging to the Ser/Thr protein kinase family are basically

involved in cell polarity, microtubules stability, protein

stability, intracellular signaling, cell cycle control, cell

division, and Alzheimer disease (AD), etc. [1]. These

proteins share conserved primary structural organization

consisting of four distinct structural domains, namely cat-

alytic kinase domain, ubiquitin-associated domain, kinase-

associated domain, and ATP-binding domain (Fig. 1).

Interestingly, the N-terminal catalytic domain is highly

conserved, whereas the remaining domains are consider-

ably less conserved [2]. The PAR-1 plays major role in the

anteroposterior (A/P) axis formation and germ line deter-

minants polarization [3]. KIN1 is directly associated in

maintaining cell morphology, cell shape, cell growth rate,

cell wall composition, cell separation, cell polarization, and

fission of yeast cell [4]. Mutations in gene products lead to

defect in the partitioning of the Caenorhabditis elegans

zygote [5]. Microtubule affinity-regulating kinase (MARK)

F. Naz � A. Islam � F. Ahmad � Md. I. Hassan (&)

Centre for Interdisciplinary Research in Basic Sciences,

Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India

e-mail: [email protected]

F. Anjum

Female College of Applied Medical Science, Taif University,

Al-Taif, Kingdom of Saudi Arabia

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Cell Biochem Biophys (2013) 67:485–499

DOI 10.1007/s12013-013-9550-7

Author's personal copy

phosphorylates microtubule-associated proteins (MAPs) at

the KXGS motif, a part of the microtubule-binding domain

[1]. After phosphorylating MAPs, MARK disrupts the

binding of MAPs to the microtubule and thereby changing

its dynamics.

The MARK family contains four related proteins,

MARK1, MARK2 (EMK1), MARK3 (C-TAK1), and

MARK4 (MARKL-1), and their genes are, respectively,

present on chromosome 1, 11, 14, and 19 in the human

genome [6]. The MARK gene family contains 28 pseudo-

genes, and each pseudogene exists in several alternatively

spliced forms. Interestingly, 45 % homology exists among

MARKs family at sequence level. Due to high sequence

similarity, MARKs are considered as a member of the

subfamily adenosine-monophosphate activated protein

kinase (AMPK) of calcium/calmodulin-dependent protein

kinase (CAMPK) family [7]. AMPKs are heterotrimeric

proteins composed of one catalytic subunit (a) and 2 reg-

ulatory subunits (b, c) and are considered as a key regulator

for energy homeostasis. Activation of AMPK has blood

glucose lowering effect capacity. It is, therefore, recom-

mended as a promising anti-diabetic drug target [8, 9].

MARK, AMPK (a-subunit), and other AMPK-like kinases,

MELK, BRSK, NUAK, QSK, SIK, and QIK are known as

AMPK subgroup of CAMK protein kinases and, as shown

in Fig. 2, share a similar architecture [10].

Like AMPK kinases, MARK proteins too have six

conserved segments [11]: (1) N-terminal header segment

with unknown function, (2) catalytic domain that com-

prises activation loop divided into two loops named as

catalytic loop and the P-loop (phosphate-binding loop),

(3) a negatively charged motif linker that resembles to the

common docking site in MAP kinases, (4) a UBA domain

that is presumably an auto-regulator, (5) the most variable

region known as spacer that is essential for the regulation

of MARK activity, and (6) the C-terminal tail containing

kinase-associated domain and possibly having auto-inhib-

itory function. MARKs are phosphorylated on serine,

threonine, and tyrosine residues, and their dephosphoryl-

ated form is catalytically inactive [1]. Nevertheless, phos-

phorylation at Ser218 inactivates MARK4 [12].

Kato et al. [13] had cloned the MARK4 gene located on

the chromosome 19q13.2. The MARK4 exists in two iso-

forms named as MARK4S and MARK4L that are origi-

nated by alternative splicing [13]. The MARK4S, which is

a short protein consisting of 18 exons, encodes a mature

polypeptide of 688 amino acid residues (*75.3 kDa). On

the other hand, MARK4L lacks exon 16, resulting a

shifting of downstream stop codon, which leads to the

formation of longer polypeptide of 752 amino acid residues

long (*82.5 kDa). However, both proteins are identical in

the kinase domain, but they differ in C-terminal tail.

MARK4L has kinase-associated 1 domain, which is pres-

ent in all MARK proteins and are structurally identical, but

the corresponding domain in MARK4S is not similar to

any known structures in sequence [11, 13, 14]. Phosphor-

ylation of Thr214 in the activation loop (T-loop) activates

MARK4, whereas phosphorylation of Ser218 inactivates it

[15]. Modification sites of MARK4 in human, mouse, and

rat are shown in Table 1. Moreover, polyubiquitination

inhibits the kinase activation process [16]. Protein kinase C

lamda (PKCk) and cell division control protein 42 (Cdc42)

catalytic domain UBANH T Spacer TailFig. 1 Structural topology of

Ser/Thr kinase family

HUNK

SNRK NIM1

MELK

AMPKα2 AMPKα2 BRSK2 BRSK1

NUAK2 NUAK1 QSK

SIK

QIK

MARK1

MARK2 MARK3

MARK4

Fig. 2 Members of AMPK

subfamily

486 Cell Biochem Biophys (2013) 67:485–499

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interact with MARK4 that is required for the cell polarity

control. Interaction of MARK4 with transforming growth

factor b-inducing anti-apoptotic factor (TGFbIAF) is

involved in asymmetric division of neuroblasts in Dro-

sophila [17].

All these studies suggest that MARK4 is a versatile

protein involved in large number of metabolic processes. It

will, therefore, be worthwhile to review all essentially

important features of MARK4 protein. Here, we have

attempted to gather all necessary information including

gene structure, regulation, protein structure, function, and

interaction of MARK4.

Biosynthesis and Secretion

Northern blot experiments [13, 18, 19] and semi-quantita-

tive PCR-based analysis [14] on different organisms

(human, rat, and mouse tissues) have shown that MARK4

is predominantly expressed in brain followed by testis and

lungs [18]. Generally, MARK4L is highly expressed in

testis, brain, kidney, liver and lungs [14, 18, 19], while

MARK4S levels are elevated in testis, heart, and brain [13,

14]. Northern blot analysis on rat tissues has showed that

MARK4L and S expression levels are equal in the testis

[14], whereas semi-quantitative PCR on mouse samples

showed an elevated level of MARK4L in the testis and

MARK4S in the brain [14]. Immunohistochemistry studies

on rat cerebral cortex and hippocampus brain showed

MARK4L and S expressions in neurons of the gray matter

[14]. Moreover, MARK4 (presumably the L isoforms) was

observed on the tips of neurite-like processes [18].

MARK4 is located near nucleus of the cell and is

associated with the endoplasmic reticulum (ER). It

co-localizes with microtubules in the centrosome when

activated through phosphorylation in order to perform its

function [18]. The exogenous MARK4 co-localizes with

microtubules, centrosomes, and neurite-like processes of

neuroblastoma cells in contrast to MARK1, MARK2, and

MARK3 that exhibit uniform cytoplasmic localization.

However, endogenous MARK4L protein co-localizes with

centrosomes, nucleolus, and midbody [20]. MARK4L

protein is also present in glioma cells, and it is highly

expressed in hepatocarcinoma cell lines, and is involved in

neoplastic transformation, because its expression is

restricted to neural progenitors in brain [21]. Both

MARK4L and S isoforms are localized in the centrosome

and midbody in tumors other than glioma and normal cell

lines [20, 21].

Colony-forming assay suggests that dysregulation of

MARK4 causes cell death [13]. The MARK4 gene is

duplicated and upregulated in glioblastomas [22]. Over-

expression of MARK4S in the early stages of an ischemic

event increases the neuronal cell death and reduces cell

viability. This action can be compared with MARK1 that

causes cell death after overexpression by interfering with

microtubule stability [1].

Gene Structure and Regulation

MARK4 gene, previously known as ‘‘MAP/microtubule

affinity-regulating kinase like 1’’, (MARKL1) is 53,992 bp

long, present at q13.2 position of chromosome 19 that starts

from 45,754,550 bp and ends at 45,808,541 bp. The cDNA of

MARK4S is 3,609 bp long, and it contains an open reading

frame of 2,069 bp with 18 exons that encodes 688 amino

acids, while MARK4L cDNA is 3,529 bp long that encodes

752 amino acids that lacks exon 16. Interestingly, the MARK4

gene promoter has ARP-1, LUN-1, GATA-1, GATA-3, GATA-

2, Elk-1, Pax-5, MIF-1, MZF-1, STAT1-b transcription factor

binding sites (http://www.sabiosciences.com). Surprisingly,

771 SNPs are reported in the MARK4 gene. Pseudogenes of

MARK4 gene are located on both the short and long arms of

chromosome 3 (http://www.genecards.org/cgi-bin/carddisp.

pl?gene=MARK4).

MARK kinases contain several domains, which appear

to be regulated by multiple mechanisms. In general,

MARK activation enhances microtubule dynamics, while

Table 1 Modification sites of MARK4 in human, mouse, and rat

Site Human Site Mouse Site Rat

S26-p GTLGSGRsSDKGPSW S26 GTLGSGRSSDKGPSW S26 GTLGSGRSSDKGPSW

K183-u NIVHRDLkAENLLLD K183 NIVHRDLKAENLLLD K183 NIVHRDLKAENLLLD

T214-p TLGSKLDtFCGsPPY T214-p TLGSKLDtFCGsPPY T214 TLGSKLDTFCGSPPY

S218-p KLDtFCGsPPYAAPE S218-p KLDtFCGsPPYAAPE S218 KLDTFCGSPPYAAPE

T300 LNPAKRCTLEQIMKD T300-p LNPAKRCtLEQIMKD T300 LNPAKRCTLEQIMKD

S423-p YHRQRRHsDFCGPSP S423-p YHRQRRHsDFCGPSP S423 YHRQRRHSDFCGPSP

S438-p APLHPKRsPtSTGEA S438-p APLHPKRsPtSTGDT S438 APLHPKRSPTSTGDT

T440-p LHPKRsPtSTGEAEL T440-p LHPKRsPtSTGDTEL S440 LHPKRSPTSTGDTEL

* Modification sites were given in small letter alphabets

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http://www.sabiosciences.com

http://www.genecards.org/cgi-bin/carddisp.pl?gene=MARK4

http://www.genecards.org/cgi-bin/carddisp.pl?gene=MARK4

its inhibition stabilizes microtubules. MARK is generally

activated through phosphorylation by kinases such as liver

kinase B1 (LKB1) and MARK Kinase (MARKK) at the

threonine residue in the T-loop (MARK1, T215; MARK2,

T208; MARK3, T211; MARK4, T214). Moreover, the

calcium/calmodulin-dependent protein kinase I (CaMKI)

phosphorylates MARK2 at a different site in its kinase

domain for its activation [23]. The glycogen synthase

kinase 3b (GSK3b) inhibits MARK proteins by phos-

phorylating the serine residue, near the threonine activation

site in the T-loop in all the MARKs (MARK1, S219;

MARK2, S212; MARK3, S215; MARK4, S218). This

inhibition occurs because phosphorylated serine is no

longer able to form interactions with other amino acids

present in the activating loop [12]. Par-5 binds MARK

kinases in their catalytic domain [24] or in the spacer

region, and it alters MARKs localization and reduces their

activity, probably by stabilizing the inhibitory interaction

between the KA1 domain and the catalytic domain.

Moreover, MARK2 is also inhibited by interaction with

PAK5 (p21-activated kinase) at the catalytic domain [25].

Another novel proposed mechanism for MARK autoinhi-

bition is dimerization, which commonly occurs in many

kinases and MARK proteins [11].

The structure of MARK family proteins is highly con-

served. Hence, regulation of MARK4 is seemingly identical

to that of the other MARK members. MARK4 is acti-

vated by LKB1 that phosphorylates Thr214 in the T-loop

[17, 26]. Recently, it was demonstrated that polyubiquiti-

nated MARK4 interacts with the deubiquitinating enzyme,

ubiquitin-specific protease (USP9X). Non-USP9X-binding

mutants of MARK4 are hyperubiquitinated and are not

phosphorylated at Thr214. So polyubiquitination may

inhibit LKB1 activation of MARK4. The proposed model

suggests that in the steady-state MARK4 UBA domain is

bound by ubiquitin and cannot interact with the catalytic

domain, making the T-loop less accessible to LKB1.

Alternatively, ubiquitin may cover and hide the Thr214 site

or induce conformational modifications favoring the activ-

ity of phosphatases [16]. It has also been demonstrated that

MARK4 interacts with aPKC [17] and could, therefore, be

phosphorylated and inactivated by this kinase, as reported

for MARK2 and MARK3 [27].

Structure of MARK4

MARK4 sequences have been taken from different mam-

mals from Swiss-Prot-TrEMBL protein sequence database

(www.expasy.org), and these are analyzed. Results of

multiple sequence alignment suggested that these proteins

exhibit high degree of sequence similarities throughout the

course of evolution. The details are shown in Fig. 3.

MARK4S and L isoforms have 688 and 752 amino acid

residues, respectively. The MARK4L has Ser/Thr protein

kinase catalytic domain [S_TKc (59–310)], ubiquitin-

associated domain [UBA (331–368)], kinase-associated

domain 1[KA1 (649–752)], and protein kinases catalytic

domain [PKc_like (58–306)]. However, the MARK4S

contains UBA (331–368), STKc_AGC catalytic domain of

AGC family (65–98), and S_TKc (59–310). The structural

topology of MARK4 starts from residue 1 to 59 N-terminal

header, followed by Ser/Thr kinase catalytic domain

starting from 59 to 314 residues. This region is followed by

membrane-targeting motif (T-region) which starts from

314 to 322 residues, followed by ubiquitin-associated

domain (322–369 residues), followed by least-conserved

spacer region (369–649 residues) and KA1 domain tail

(residue 649–752). The tail domain of MARK4 is least

conserved in MARK1, 2, 3, MARK4S, and MARK4L.

MARK4L has kinase-associated 1 domain, which is pres-

ent in all MARK proteins and is structurally identical, but

the corresponding domain in MARK4S has no homology to

any known structures [11, 13, 14]. MARK isoforms 1, 2,

and 3 terminate with the 4-residues motif ELKL, but this is

replaced by DLEL in MARK4.

The crystal structure of MARK4 is not yet available in

protein data bank (PDB). We have predicted the structure

of MARK4 using homology modeling tools like I- TAS-

SER [28], SWISS MODEL and validated by SAVES

(http://nihserver.mbi.ucla.edu/SAVES/), which includes

PROCHECK that examines various structural properties

such as the bond length, bond angles, steric clashes, and

stereochemical parameters. The final atomic coordinates of

MARK4 model are taken for structure analysis (Fig. 4).

Crystal structures of three MARK isoforms (MARK1, 2

and 3) have already been determined [29–31]. Interest-

ingly, we have noticed that all these isoforms have a

similar domain organization including that of MARK4

[11]. Structure comparison with other known proteins is

done by using DALI server (Table 2). The structural fea-

tures of the three major domains of MARK4 are described

here in detail.

The Catalytic Domain

The kinase domain of MARK4 has a bilobed structure like

many other kinases [32]. The catalytic domain along with

other domains attains a bilobal flexible structure and sub-

sequently forms a cleft for the substrate and ATP. Such

Fig. 3 Multiple sequence alignment of MARK4 from mammalian

sources. The phosphorylated residues are highlighted in gray; binding

site is highlighted in green, and active site is highlighted in sky blue.

The amino acid sequences were taken from Swiss-Prot-TrEMBL

protein sequence database (www.expasy.org) (Color figure online)

c


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http://www.expasy.org

http://nihserver.mbi.ucla.edu/SAVES/

http://www.expasy.org

MARK4_Homo -----MSSRTVLAPGNDR----NSDTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 51MARK4_Cavia -----MSSRTALAPGNDR----NSDTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 51MARK4_Orangutan -----MSSRTVLAPGNDR----NSDTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 51MARK4_Mus -----MSSRTALAPGNDR----NSDTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 51MARK4_Rattus -----MSSRTALAPGNDR----NSDTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 51MARK4_Canis ----------------------MLEAHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 38MARK4_Equus PP---LLHRVAGQVEEAT----MFPTHGTLGSGRSSDKGPSWSSRSLGARCRNSIASCPE 61

**********************************

MARK4_Homo EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 111MARK4_Cavia EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 111MARK4_Orangutan EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 111MARK4_Mus EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 111MARK4_Rattus EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 111MARK4_Canis EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKTQLNPSSLQKLFREVRIMK 98MARK4_Equus EQPHVGNYRLLRTIGKGNFAKVKLARHILTGREVAIKIIDKMQLNPSSLQKLFREVRIMK 121

***************************************** ******************

MARK4_Homo GLNHPNIVKLFEVIETEKTLYLVMEYASAGEVFDYLVSHGRMKEK-----EAR------A 160MARK4_Cavia GLNHPNIVKLFEVIETEKTLYLVMEYASAGEVFDYLVSHGRMKEK-----EAR------A 160MARK4_Orangutan GLNHPNIVKLFEVIETEKTLYLVMEYASAGEVFDYLVSHGRMKEK-----EAR------A 160MARK4_Mus GLNHPNIGKISLFRSVWTTALMVISRG---EVFDYLVSHGRMKEK-----EAR------A 157MARK4_Rattus GLNHPNIG----------------------EVFDYLVSHGRMKEK-----EAR------A 138MARK4_Canis GLNHPNIVKLFEVIETEKTLYLVMEYASAGEVFDYLVSHGRMKEK-----EAR------A 147MARK4_Equus GLNHPNIVKLFEVIETEKTLYLVMEYASAGECLDYLVSHGRMKEKRPLPSSARPLVGQTG 181

******* * :************ .** .

MARK4_Homo KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 220MARK4_Cavia KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 220MARK4_Orangutan KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 220MARK4_Mus KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 217MARK4_Rattus KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 198MARK4_Canis KFRQIVSAVHYCHQKNIVHRDLKAENLLLDAKANIKIADFGFSNEFTLGSKLDTFCGSPP 207MARK4_Equus RVPPIVSAVHYCHQKNIVHRDLKAENLLLDAEANIKIADFGFSNEFTLGSKLDTFCGSPP 241

:. ***************************:****************************

MARK4_Homo YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 278MARK4_Cavia YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 278MARK4_Orangutan YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPXXXXTPSLQELRERVLRGKYRVPFY 280MARK4_Mus YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 275MARK4_Rattus YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 256MARK4_Canis YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 265MARK4_Equus YAAPELFQGKKYDGPEVDIWSLGVILYTLVSGSLPFDG--HNLKELRERVLRGKYRVPFY 299

*********************************** * *************************************************** .*:****************

MARK4_Homo MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 338MARK4_Cavia MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 338MARK4_Orangutan MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 340MARK4_Mus MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 335MARK4 Rattus MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 316MARK4_Rattus MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 31MARK4_Canis MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 325MARK4_Equus MSTDCESILRRFLVLNPAKRCTLEQIMKDKWINIGYEGEELKPYTEPEEDFGDTKRIEVM 359 ************************************************************

MARK4_Homo VGMGYTREEIKESLTSQKYNEVTATYLLLGRKTEEGGDRGAPGLALARVRAPSDTTNGTS 398MARK4 Cavia VGMGYTREEIKEALTSQKYNEVTATYLLLGRKTEEGGDRGTPGLALARVRAPSDTTNGTS 398MARK4_Cavia VGMGYTREEIKEALTSQKYNEVTATYLLLGRKTEEGGDRGTPGLALARVRAPSDTTNGTS 39MARK4_Orangutan VGMGYTREEIKEALTSQKYNEVTATYLLLGRKTEEGGDRGAPGLALARVRAPSDTTNGTS 400MARK4_Mus VGMGYTREEIKEALTNQKYNEVTATYLLLGRKTEEGGDRGAPGLALARVRAPSDTTNGTS 395MARK4_Rattus VGMGYTREEIKEALTNQKYNEVTATYLLLGRKTEEGGDRGAPGLALARVRAPSDTTNGTS 376MARK4_Canis VGMGYTREEIKEALTSQKYNEVTATYLLLGRKTEEGGDRGTPGLALARVRAPSDTTNGTG 385MARK4_Equus VGMGYTREEIKEALTSQKYNEVTATYLLLGRKTEEGGDRGTPGLALARVRAPSDTTNGTS 419 ************:**.************************:******************.

MARK4_Homo SSKGTSHSKGQRSSSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGEAELKEERLPGRKAS 458MARK4_Cavia SSKGTSHSKGQRSSSSTYQRQRRHSDFCGPSPAPLHPKRSPTSTGDAELKEERLPGRKAS 458MARK4_Orangutan SSKGTSHSKGQRSSSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGEAELKEERLPGRKAS 460MARK4_Mus SSKGSSHNKGQRASSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGDTELKEERMPGRKAS 455MARK4_Rattus SSKGSSHNKGQRTSSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGDTELKEERLPGRKAS 436_MARK4_Canis SSKGTSHSKGQRSSSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGDAELKEERLPGRKAS 445MARK4_Equus SSKGTSHSKGQRSSSSTYHRQRRHSDFCGPSPAPLHPKRSPTSTGETELKEERLPGRKAS 479 ****:**.****:*****:**************************::******:******


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structural features provide different conformations in the

activation/inactivation state. The smaller N-terminal lobe

consists of five b-strands and single a-helix, whereas

the larger C-terminal lobe might be composed mainly of

a-helices, with a cleft between them, which have nucleotide

binding and active site. The C-terminal lobe comprises the

activation segment, which is essential for the coordination

of nucleotide and substrate in the catalytically active state.

The N- and C-lobes are connected with a flexible hinge

region that plays a role in opening and closing of the cleft.

This region consists of 5 residues of alanine and one resi-

due of glycine in MARK4, whereas two residues of glycine

are present in MARK1, 2, 3 [11]. The regulatory loop is the

most variable part in all structures solved so far. We can

say that MARK4 regulatory loop might be different from

other MARKs. This linker is connected with the UBA

domain through hydrophobic contacts. MARK kinases are

activated by phosphorylation in the T-loop that contains a

conserved sequence, LDTFCGSPP. Subsequently, these

phosphorylated MARKs phosphorylate other substrate

proteins. In the inactive state, the T-loop is disordered and

folded into the cleft blocking the access of substrate peptide

and ATP. The phosphorylation sites (threonine and serine)

are nevertheless accessible for kinases. Once they are

phosphorylated (activation), the T-loop is reoriented and

folds out of the cleft, which becomes open enabling ATP

MARK4_Homo CSTAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 518MARK4_Cavia CSAAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 518MARK4_Orangutan CSAAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSMSTPNNLPPSMMTRRNTYVCTERP 520MARK4_Mus CSAVGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 515MARK4_Rattus CSAAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 496MARK4_Canis CSAAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 505MARK4_Equus CSAAGSGSRGLPPSSPMVSSAHNPNKAEIPERRKDSTSTPNNLPPSMMTRRNTYVCTERP 539

** ******************************** *************************:.******************************** ***********************

MARK4_Homo GAERPSLLPNGKENSSGTPRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGGQVRDRR 578MARK4_Cavia GAERPSLLANGKENSSGTPRVPPASPSSHSLAPPSGERSRLARGSTVRSTFHGGQVRDRR 578MARK4_Orangutan GTERPSLLPNGKENSSGTPRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGGQVRDRR 580MARK4_Mus GSERPSLLPNGKENSSGTSRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGGQVRDRR 575MARK4 Rattus GSERQSLLPNGKENSSGTSRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGGQVRDRR 556MARK4_Rattus GSERQSLLPNGKENSSGTSRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGGQVRDRR 55MARK4_Canis GAERPSLLPNGKENRCASGRNGPWTSPRMAHEATPQPTGRPRPTTNLFTKLTS------- 558MARK4_Equus GAERPSLLPNGKENSSGTPRVPPASPSSHSLAPPSGERSRLARGSTIRSTFHGG------ 593 *:** ***.***** ..: * * :.. : ... .* .

MARK4_Homo AGGGGGGGVQ-NGPPASPTLAHEAAPLPAGRPRPTTNLFTKLTS-KLTRRVADEPERIGG 636MARK4 Cavia AGAGGGGCVQ-NGPPASPTLAHEAAPLPTGXPRTTTNLFTKLTS-KLTRRVTDEPERIGG 636MARK4_Cavia AGAGGGGCVQ NGPPASPTLAHEAAPLPTGXPRTTTNLFTKLTS KLTRRVTDEPERIGG 63MARK4_Orangutan AGGGGGGGVCRHGPPASHPLAYEVAPXPAGRPRTITNLFTKLTS-KLTRRVADEPERIGG 639MARK4_Mus AGSGSGGGVQ-NGPPASPTLAHEAAPLPSGRPRPTTNLFTKLTS-KLTRRVTDEPERIGG 633MARK4_Rattus AGGGSGGGVQ-NGPPASPTLAHEAAPLPSGRPRPTTNLFTKLTS-KLTRRVTDEPERIGG 614MARK4_Canis ----------------------------------------KLTR-----RVTDEPERIGG 573MARK4_Equus -------------------------------------------------QVRDR--RAGG 602

:* *. * **

MARK4_Homo PEVTSCHLPWDQTETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 696MARK4_Cavia PEVTSGHLPWDQAETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 696MARK4_Orangutan PEVTSCHLPWDQTETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 699MARK4_Mus PEVTSCHLPWDKTETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 693MARK4_Rattus PEVTSCHLPWDKAETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 674_MARK4_Canis PEVTSCHLPWDQTETAPRLLRFPWSVKLTSSRPPEALMAALRQATAAARCRCRQPQPFLL 633MARK4_Equus GGGGGVQ---NGPPASPTLAHEATPLPTGRPRPTTNLFTKLTSKLTRRVTLDPSKRQNSN 659 . : : . ::* * : . .: .**. *:: * . : . :

MARk4_Homo ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 752

MARK4_Cavia ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 752

MARK4_Orangutan ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 755

MARK4_Mus ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 749

MARK4_Rattus ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 730

MARK4_Canis ACLHGGAGGPEPLSHFEVEVCQLPRPGLRGVLFRRVAGTALAFRTLVTRISNDLEL 689

MARK4_Equus RCVSG-ASLPQ-----GSKINSRTNLRESGDLRSQVAIYLGIKRKPPPGCSDSPGV 709

*: * *. *: :: . .. * * :** *. . *:. :

Fig. 3 continued


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and substrate access [12]. The catalytic domain of MARK3

and MARK4 is highly similar showing root mean square

deviation value of 1.702 (226–226 atoms).

UBA Domain

The UBA domain of MARK4 is small and globular, and it

consists of three short helices (a1, a2, and a3). However,

this domain is not structurally similar to other ubiquitin-

associated proteins, and it does not bind ubiquitin. The

structural topology and fold of UBA domains of MARK3

and MARK4 are conserved [31]. The crystal structure

analysis of human MARK1 [30], rat MARK2 [30], and

human MARK3 [31] revealed the importance of a con-

served tyrosine residue (Tyr361) within a3 helix of the

UBA domain. This Tyrosine residue (Tyr364 in MARK4)

is presumably responsible for the stability of MARKs

because it contributes to the hydrophobic core and a p–pinteraction with the side chain of Arg331 (Arg334 in

MARK4). It has been observed that UBA domains of

MARK1 and MARK2 have inhibitory effect on the kinase

activity even after phosphorylation by the activator kinase

MARKK [30].

UBA domain interacts with the N-terminal lobe of cat-

alytic domain of MARKs locking it in an open (inactive)

conformation. Based on these observations, two different

roles were proposed for MARK UBA domain including

auto-inhibitory and positive regulatory. Since UBA domain

prevents substrate and ATP binding by locking the kinase

in an open conformation [30], this open conformation

increases the accessibility of the activation loop for acti-

vating or deactivating kinases [31]. Finally, it was sug-

gested that the UBA domain plays several significant roles

as inhibitory, activating, and stabilizing, depending on the

phosphorylation state of the kinase domain or cofactor

interactions [11].

The spacer domain is the most variable region in MARK

isoforms, which varies in size between 369 and 649 resi-

dues in the MARK4. Structure analysis suggests that this

spacer domain has the characteristic features of natively

unfolded protein that appears to be essential for the regu-

lation of MARK activity. Sequence analysis suggests that

this spacer region contains three phosphorylation sites at

Ser423, Ser438, and Thr440, which are conserved in

MARK2 [33]. Phosphorylation at these sites stimulates

relocation of MARK from cell membranes to the

cytoplasm.

The C-terminal tail (KA1 domain)

The C-terminal tail is 50 residues long as seen through

protein family sequence homologies (pfam: PF02149) [34]

and Prosite: PS50032 [35], but experimental studies sug-

gested that the C-terminal folded domain of MARK is

approximately double to the original KA1 motif (*100

residues) [36]. The C-terminus ELKL motif of MARK1, 2,

and 3 is important for the proper folding of the KA1

domain, but this conserved motif is replaced by DLEL in

MARK4. A comparison of MARK/PAR-1/KIN1/MELK

with KA1 structure suggests that conserved residues are

clustered at a specific site, formed by the N-terminal region

(b4 and b5) and the N-terminal half of a2, which give

hydrophobic concave surface that is surrounded by con-

served positively charged residues. This concave surface is

the binding site for negatively charged regions of cyto-

skeletal proteins, as this domain plays essential role in

subcellular localization. This replacement may form highly

negative domain so that positive charge residues may be

easily attached.

Recently, it is considered that kinase-associated-1

domains drive MARK/PAR1 kinases to membrane targets

by binding acidic phospholipids [37]. This domain pri-

marily contains positively charged residues that interact

with negatively charged regions of (1) cytoskeletal pro-

teins, (2) MARK catalytic domain, and (3) MARK CD

domain [36]. In the yeast, C-terminal tail physically

interacts with the N-terminal kinase domain (presumably

when it is in an open conformation) with an auto-inhibitory

effect [38]. Therefore, this domain is also considered

Fig. 4 Overall structure of MARK4 generated by I-TASSER is shown

in cartoon model using PyMOL. The protein kinase catalytic domain,

ubiquitin-associated domain, and kinase-associated domain are illus-

trated in red, sky blue, and pink respectively (Color figure online)


123


as another auto-inhibitory domain. The KA1 (Kinase

Associated) domain was also reported to be involved in

protein localization [36].

Phylogenetic Analysis

The origin and evolutional study of MARK4 was analyzed

by generating a phylogenic tree by using variety of methods

including neighbor joining, boot strapping with sequence of

MARK4, MARK3, MARK2, MARK1 of human, equus,

heterocephalus, canis, bovin, xenopus, cricetulus, rat,

mouse, danio, cavia, bos, and the related proteins (KIN1,

PAR1, PAR6). This phylogenetic analysis has a simple user

interface and is integrated with the multiple sequence

alignment editors for maximum flexibility. The amino acid

sequences were aligned using the ClustalW program

(GCG). The resultant alignments were used to construct

the phylogenetic tree by using Mac Vector software system

(http://www.macvector.com). We constructed the phylo-

genetic tree and found out that PAR1, KIN1, and MARKs

are originated from the common parent gene (Fig. 5), and

Table 2 Structural comparison of MARK4 with other known protein by using DALI

Name of protein PDB ID Identity (%) Nt Na Z score RMSD

50-Amp-activated protein kinase catalytic subunit 2y94-A 32 432 427 41.1 0.9

Serine/threonine-protein kinase MARK2 3iec-C 83 311 276 32.2 2.5

Serine/threonine kinase 6 1ol5-A 33 265 258 32.0 2.1

3-phosphoinositide-dependent protein kinase 1 3orx-B 32 281 263 31.6 1.7

Polo-like kinase 1 3d5w-A 32 285 265 31.3 2.5

G Protein-coupled receptor kinase 6 2acx-A 25 495 265 31.2 2.2

Loc398457 protein 2bfy-A 32 271 262 31.1 2.4

Pkb-like 3nuu-A 32 277 257 31.1 1.6

Serine/threonine-protein kinase Plk1 3kb7-A 29 290 262 31.0 2.0

Serine/threonine-protein kinase 12-A 3ztx-A 32 270 262 30.8 2.4

G Protein-coupled receptor kinase 6 3nyn-B 23 553 291 30.7 14.6

Rac-beta serine/threonine-protein kinase 1o6 l-A 34 317 266 30.7 2.2

Aurora kinase A 3uod-A 33 266 256 30.7 2.2

Rac-alpha serine/threonine-protein kinase 4ekk-A 35 319 266 30.7 2.1

Calcium/calmodulin-dependent protein kinase type 2wel-A 34 304 287 30.6 24.4

V-Akt murine thymoma viral oncogene homolog 1 3mvh-A 35 312 266 30.6 2.2

Camp-dependent protein kinase, alpha-catalytic 1rej-A 34 335 264 30.6 2.0

Phosphorylase B kinase gamma catalytic chain 2y7j-B 35 281 262 30.4 2.1

Death-associated protein kinase 3 1yrp-A 35 276 255 30.3 1.8

Rhodopsin kinase 3c4w-B 30 519 269 30.3 2.6

V-Akt murine thymoma viral oncogene homolog 1 3ocb-A 35 316 265 30.3 2.1

Protein kinase C theta 2jed-A 31 326 265 30.2 1.9

Protein kinase C alpha type 3iw4-A 31 332 266 30.2 1.9

Peripheral plasma membrane protein cask; 3mfu-A 35 302 260 29.6 2.0

Beta-adrenergic receptor kinase 1 3pvu-A 22 609 294 29.6 12.7

Calcium/calmodulin-dependent protein kinase Ii 3kk8-A 37 284 263 29.5 3.9

Proto-oncogene serine/threonine-protein kinase Pi 1ywv-A 29 274 250 28.2 2.1

Pimtide 2c3i-B 29 266 248 28.2 2.1

P38a 3oht-A 21 331 247 18.5 4.0

Serine/threonine-protein kinase/endoribonuclease 3p23-C 24 390 253 21.5 8.1

Map kinase-activated protein kinase 3; 3r1n-A 35 274 215 21.0 2.0

Cell division protein kinase 7 1ua2-A 30 287 230 21.5 2.6

Mitogen-activated protein kinase 13 3coi-A 25 344 255 21.9 3.4

Nt Total number of residues

Na Total number of residues aligned


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http://www.macvector.com

the MARK, PAR1, and PAR6A have evolved from KIN1

family. PAR6A and PAR6G are more closely related to

KIN1 when compared to PAR1. PAR1 lineage divides into

two lineages, one leads to invertebrate MARK family and

the other to vertebrate MARK family. MARK of mamma-

lian has evolved from invertebrates MARKs. Invertebrates

MARKs give MARK4 and other MARKs too. MARK3 and

MARK1 are evolved from the same ancestral gene and they

are closely related to each other.

Functions of MARK4

Regulation of Programmed Cell Death

Programmed cell death, a major component of the ischemic

cascade, depends on the changes of gene expression during

cerebral ischemia [39, 40]. MARK4 is upregulated in the

early stages of ischemic events [19]; hence, it was sug-

gested that MARK4 may regulate programmed cell death

Fig. 5 Phylogenetic tree of MARKs, KIN1 and PAR generated by MAC VECTOR software


123


and may promote cell death cascade [19]. However, the

different experimental results also suggest that the upreg-

ulation of MARK4 increases the likelihood of neuron to

die, and its overexpression in heterologous cells lead to

decreased cell viability [19].

ATP Binding

MARK4 catalyzes phosphorylation of proteins by using

ATP. ATP binds on ATP-binding domain of MARK4 at

Lys88 and phosphorylates the substrate protein [19]. The

catalytic domain of MARK4 along with CD domain linker

and the UBA domain form a bilobal structure which creates

a cleft for substrate and ATP.

Gamma-Tubulin Binding

It is seen that tandem affinity-purified MARK4 protein is

complexed with a, b, and c tubulin [18]. Moreover,

MARKs phosphorylate tau and related MAPs on their

tubulin-binding domains and subsequently catalyze their

detachment from microtubules in vitro and in the cultured

cells [1, 41, 42].

Microtubule-Dependent Transport

Like other MARK members, MARK4 phosphorylates

MAP proteins leading to an increase in microtubule

dynamics and cell shape alterations [17]. It has been

reported that MARK proteins, especially MARK2, control

microtubule-dependent transport [43]. In addition, MARK

co-localization with clathrin-coated vesicles (CCV) was

recently demonstrated, confirming a function of MARK4 in

the regulation of microtubule-dependent transport of CCV

during endocytosis [44].

Protein Serine/Threonine Kinase Activity

MARK4 itself gets phosphorylated at Thr214 and Ser218

to perform its function. When it is phosphorylated at

Thr214, it becomes functionally active, and when it gets

phosphorylated at Ser218, its activity gets inhibited [19]. It

phosphorylates serine motif in the microtubule-binding

domain of microtubule-associated protein, which is critical

for microtubule binding.

Tau Protein Kinase Activity

The binding of tau is controlled by phosphorylation in

spatial and temporal fashions [45]. MARKs phosphorylate

tau causing its detachment from microtubules [1, 41, 42].

Tau in the growth cone usually loses its stabilizing effect

after the phosphorylation by MARK4 [18]. Overexpression

of MARKs causes the disruption of the cellular microtu-

bule network in the cell [1, 46]. The Ser262 of tau is

phosphorylated by MARK, from the neurofibrillary

deposits as observed in the AD brains [47]. This finding is

in close agreement with the fact that tau purified from

neurofibrillary deposits is unable to bind to microtubules,

which can be restored by dephosphorylation [48]. Other

experiments also suggest that MARK4 phosphorylates the

microtubule-binding domains of tau at Ser262 [18].

Ubiquitin Binding

UBA domain of MARK4 is considered to mediate inter-

action with ubiquitin [49]. Binding of a ubiquitin oligomer

with UBA domain may detach this domain from catalytic

domain and thus enabling kinase activity of MARK4 [11].

But standard binding assays were not able to detect any

interaction between the presumed UBA domains of MARK

and diversity of ubiquitin and ubiquitin-related species

[50]. NMR techniques have showed the residual interaction

between the isolated MARK3 UBA domain with mono-

ubiquitin at low affinity (Kd [ 2 mM), found in close

agreement with the biochemical assays [31]. Based on

these observations, it was suggested that UBA domains of

AMPK-like kinases have been evolved from canonical

UBA domains. Moreover, kinase domain looses the

potential of ubiquitin binding because it cannot tightly

interact with it [11].

Association with Centrosomes

MARK4 is associated with microtubules, centrosomes, and

neurite-like processes of neuroblastoma cells [18]. Fur-

thermore, it is localized in microtubular structures and

phosphorylates microtubule-associated proteins [18].

Overexpression of MARK4 reorganizes microtubule net-

work and leads to the regulation of centrosomal activities

such as amplification and positioning of centrosomes [18].

Human Glioma

MARK4L expression is increased in glioma and hepato-

carcinoma cell lines, which reveals the functional impor-

tance of MARK4 in cancerous cells. It is involved in

building and maintenance of these cell lines by its trans-

location inside and outside of the nucleolus [20]. There is

also genomic duplication of MARK4L identified by

hybridization techniques in the glioblastoma cell lines [20,

51, 52]. MARK4L colocalizes with centrosomes in all

mitotic stages. The localization of MARK4L in nucleoli

during interphase is also observed in the human glioma

cells and reveals a possible connection between the kinase

and centrosomal aberrations in the glioma cell lines [20].


123


Nervous System Development

MARK4 gene is found rapidly and transiently upregulated

after the ischemic event of brain particularly in the hip-

pocampus. Many genes are also overexpressed in the

injured brain as compared to the healthy brain, including

LKB1 which phosphorylates MARK4. Overexpression of

MARK4S in hepatocytes leads to a reduction in cell

vitality. Thus, it is hypothesized that MARK4S upregula-

tion in the early stages of an ischemic event might increase

the probability of neuron death [19]. MARK4L and S

isoforms have been analyzed by competitive PCR in

human, mouse normal brain and neural progenitors, and it

is observed that MARK4S expression levels are found

increased during neuronal differentiation, whereas

MARK4L expression is maintained at the same levels in

differentiated neurons [14]. Therefore, the S isoform is

suggested to be a neuron-specific marker in the CNS.

Nevertheless, both MARK4L and S proteins are found

expressed in neurons by means of immunohistochemistry,

suggesting that both forms play important role in the

neurons [14]. By analyzing frequent rearrangements

involving chromosome 19q13 in the most frequent tumors

in the CNS, an amplified region encoding MARK4 has

been discovered [53]. The region was never found deleted

in these tumors, suggesting that the gene is probably

important for cancer cells [22].

Other Functions

MARK4L probably participates in cell cycle progression

and helps in coordinating cytokinesis. MARK4 interacts

with microtubules in glial tumors and is found to be

associated with centrosomes and midbody [20]. Since it is

upregulated in some tumors, MARK4 could indeed have a

role in cell proliferation [13, 22]. MARK4 directly interacts

or binds with many proteins to perform respective func-

tions, including cytoskeleton remodeling [17]. Nucleolar

localization of a protein may also influence its stability by

protecting from proteasomal degradation, since protea-

somes are present in the nucleoplasm rather than in

nucleoli [54].

Association with Diseases

MARK4 directly plays role in pathological phosphoryla-

tion of tau protein in the AD [18]. This tau protein belongs

to the MAP family which includes MAP2, MAP2c, and

MAP4 [55]. Tau protein binds and stabilizes MTs, partic-

ularly in neuronal axons and leads to the microtubule-

dependent axonal transport of vesicles and organelles by

motor proteins [56]. MARK4 (or other MARK family

members) phosphorylates at the Lys-Xaa-Gly-Ser (KXGS)

motifs of tau protein, which is responsible for the detach-

ment and destabilization of the MTs. Hyperphosphoryla-

tion of tau is an earlier feature of AD, followed by irregular

aggregation of tau [57–59]. All these observations clearly

indicate the importance of MARK4 in the AD [60, 61]. The

toxic effect of tau on neurons is frequently observed in its

aggregation [62]. However, before the formation of

aggregation, hyperphosphorylation of tau, mis-sorting into

the somatodendritic compartment, the loss of synapses, and

mitochondrial dysfunction has also been observed [63]. It

is not clear yet whether the aggregation of tau is the cause

of its toxic effect or merely a consequence of it.

It has been observed that MARK4L isoform is down-

regulated in normal brain, whereas upregulated in all glio-

mas and overexpressed by intrachromosomal duplication

[14, 21, 52]. RT-PCR data from the glioma cell lines have

shown expression levels of MARK4L isoform increasing

with malignancy [21]. The MARK4L protein is highly

expressed in cancers other than gliomas, including hepa-

tocarcinoma and leukemia cell lines [13]. In addition to its

restricted expression by human neural progenitors in the

CNS [22], the L isoform plays a preferential role in cycling

cells. It has been reported that the MARK4S isoform is

scarcely expressed in gliomas and mainly expressed in adult

brain [14, 21], and it is upregulated in the early stages of an

ischemic event that increases the likelihood of neuronal

death [19]. Experimental overexpression of MARK4S in

hepatocytes causes reduction in cell vitality. The two

complementary datasets support the general view that

MARK4 may be involved in cell cycle control. MAPs can

compete with motor proteins for microtubule binding and

thereby inhibit transport at elevated levels. [19].

MARK4 as a Suitable Target for Drug Design

MARK4 is an important therapeutic target, and its inhibi-

tors can be used as potential drugs [64]. Earlier we have

already discussed that MARK4 is directly involved in the

aggregate formation in AD. Furthermore, MARK4 is

phosphorylated by other kinases such as LKB1 and

MARKK, which is essential for its proper function. We can

design potential inhibitor against these kinases, which

would significantly bind at the active site or binding site of

these kinases, to cure AD [19]. Expression levels of

MARK4L isoform are enhanced with malignancy, and

overexpression of MARK4S leads to reduction in cell

vitality and neuronal cell death. Therefore, it may be

suggested that MARK4 inhibitors can be used to cure

cancer and related ailments. MARK4 is significantly

upregulated in cerebral ischemia and leads to the reduction

in cell viability. Hence, MARK4 may also be an interesting

drug target for stroke [19].


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MARK4 Mutations

Since most kinases are involved in cell proliferation,

mutations in protein kinase genes are often implicated in

cancer initiation and its development. Most of the acti-

vating alterations occur inside the catalytic domain

including the ATP-binding site. There are 518 protein

kinase genes which are involved in 210 diverse human

cancers due to five significant MARK4 mutations [65].

These mutations are missense mutations in exon 12 (spacer

region) that occurred in two colorectal adenocarcinomas

(R377Q and R418C), silent mutations in exon 5 (Y137Y)

and exon 9 (I286I), and intronic mutation (exon 8 ?5

C [ T; kinase domain). Interestingly, in Peutz-Jeghers

syndrome, few SNPs are found without any mutation in

MARK4 gene [66].

Interaction with other Proteins

In order to predict functional interaction networks of

MARK4, we have performed interaction analysis on

STRING server [67]. MARK4 shows close interactions

with many proteins that are essential for maintaining cell

physiology (Table 3). Interaction of calcium binding pro-

tein 39 (CAB39) and STE20-related adaptor alpha

(STRAD alpha) are found to be essential for the activation

of MARK4 [26]. In addition, MARK4 interacts with serine/

threonine kinase 11 (STK11) during cell arrest as evident

from tandem mass spectrometry analysis [17]. MARK4 is

polyubiquitinated in vivo and interacts with the deubiqui-

tinating enzyme, USP9X [16]. Interestingly, Knock out

USP9X allele showed an increased polyubiquitination of

MARK4, whereas overexpression of USP9X inhibits

ubiquitination. Topological analysis has revealed that

MARK4 is a substrate of USP9X and ubiquitin monomers

attached to MARK4 through Lys29 and/or Lys33 instead of

more common Lys48/Lys63. Furthermore, non-USP9X-

binding mutant of MARK4 is hyperubiquitinated and not

phosphorylated at its T-loop residue, suggesting that

polyubiquitination may inhibit MARK4.

Some proteins bind with phosphorylated protein only

and control multiple cellular processes. The 14–3–3g iso-

forms of 14–3–3 proteins interact with MARK4 and

directly regulate it [17, 68]. Furthermore, PAR-6A and the

PAR-1 proteins interact with MARK2 and MARK4,

Table 3 Interaction of MARK4 with other proteins calculated through STRING*

S. No Protein code Full name Sequence length E/T Score* Reference

1 CAB39 Calcium binding protein 39 341 T 0.970 [17]

2 STK11 Serine/threonine kinase 11 433 E/T 0.967 [16]

3 USP9X Ubiquitin-specific peptidase 9 2,570 E/T 0.958 [70]

4 MARK2 MAP/microtubule affinity-regulating kinase 2 745 E/T 0.933 [70]

5 USP16 Ubiquitin-specific peptidase 16 823 E 0.927 [71]

6 USP21 Ubiquitin-specific peptidase 21 565 E 0.927 [71]

7 STRADB STE20-related kinase adaptor beta 418 T 0.921 [17]

8 STRADA STE20-related kinase adaptor alpha 431 T 0.920 [26]

9 MAP4 Microtubule-associated protein 4 1,152 E/T 0.811 [18]

10 TUBG1 Tubulin, gamma 1 451 E 0.799

11 PRKCG Protein kinase C, gamma 697 E 0.754 [17]

12 YWHAZ Tyrosine 3-monooxygenase/ tryptophan

5-monooxygenase activation protein

245 E 0.733

13 MAP2 Microtubule-associated protein 2 1,827 E/T 0.715 [18]

14 PARD6A Par-6 partitioning defective 6 homolog alpha 346 E/T 0.708 [17]

15 MAPT Microtubule-associated protein tau 776 E/T 0.700 [14]

16 MYBBP1A MYB binding protein (P160) 1a 1,332 E 0.667

17 KCTD20 Potassium channel tetramerization domain containing 20 419 E 0.667

18 KIAA0802 KIAA0802 1,586 E 0.667

19 SMARCA4 SWI/SNF related 1,679 E 0.667

20 MYO18A Myosin XVIIIA 2,054 E 0.667

* STRING (http://www.string-db.org/)


123


http://www.string-db.org/

respectively. However, MARK4 also interacts with PAR-

6A. PKCk is also found to be more intimate partner of

MARK4. Experimentally, interaction of USP16 with

MARK4 is observed, which is essential for the deubiqui-

tination of histone H2A. The USP21 regulates centrosome

and microtubule-associated functions via binding through

MARK4 [69]. Since, MARK4 is activated by LKB1

through STRAD, it is suggested that STRAD also indi-

rectly regulates the function of MARK4.

Interaction analysis shows that MARK4 readily phos-

phorylates tau and related MT-associated proteins MAP2

and MAP4 [18]. Gamma tubulin, present at microtubule

organizing centers (MTOC) such as the spindle poles or the

centrosome, indicates its involvement in the minus-end

nucleation of MT assembly [17, 18]. STRING analysis also

indicates that protein kinase C gamma is a calcium-acti-

vated, phospholipid-dependent, serine- and threonine-

specific enzyme and also interacts with MARK4 [17]. In

addition to this tyrosine 3-monooxygenase/tryptophan

5-monooxygenase activation protein, zeta polypeptide

(YWHAZ) is an adapter protein involved in the regulation

of large number signaling pathways. It binds with many

proteins including MARK4 due to the presence of phos-

phoserine or phosphothreonine motif. Such binding mod-

ulates the activity of the binding partner [17]. PAR-6

partitioning defective 6 homolog alpha (PARD6A) is an

adapter protein that actively participates in asymmetrical

cell division and cell polarization processes. Presumably, it

is also involved in the formation of epithelial tight junc-

tions and interacts with MARK4.

Some cytoskeleton binding proteins like Rho guanine

nucleotide exchange factor 2 (ARHGEF2) interact with

MARK4 and phosphatase 2A, which is associated with MT

and regulate interaction of Tau with MARK4 [17].

MARK4 is directly involved in cytoskeleton dynamics as

evident from its precipitation with a, b, and c tubulin,

myosin, and actin [17, 18]. MARK4 is considered to play a

role as a messenger of the Wnt-signaling pathway.

Concluding Remarks

MARK4 is a Ser/Thr kinase, and its structure is divided

into three distinct domains that are associated with distinct

functions. MARK4 is expressed through almost every

organ of human body. However, its highest concentration is

reported to be in testis and brain. Upregulation and over-

expression of MARK4 are observed in glial tumors and

AD. MARK4 is a potential future target for structure-based

rational drug design. MARK4 is evolved from other

MARK genes with several gene mutations. We have pre-

cisely modeled three-dimensional structure of MARK4 and

described it as well. The active site residue (Asp181) and

ATP-binding residue (Lys88) are located in kinase domain

of MARK4. Apart from three domains, most of the parts of

MARK4 are highly unstructured. We have analyzed

interaction of MARK4 with various proteins which are

essential for maintaining pathophysiology of cells. Altered

interaction of MARK4 with its binding proteins may cause

severe ailments or cell death. Our sequence, structure- and

function-based analysis will be helpful in better under-

standing of mechanism of regulation of microtubule

dynamics and MARK4 associated diseases.

Acknowledgments F.N. expressed her thanks to the Council of

Scientific and Industrial Research (CSIR) for fellowship. M.I.H. and

F.A. are thankful to the Department of Science and Technology,

Indian Council of Medical Research, University Grants Commission

and CSIR for financial assistance.

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Microtubule affinity-regulating kinase 4: structure, function, and regulation

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