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• W2023662810 abstract "Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCKα and -β, have been previously identified and characterized. We now describe a novel isoform, MRCKγ, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCKα and -β, which are expressed ubiquitously, MRCKγ mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCKγ showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCKγ promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCKγ. Analysis of the MRCKγ promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCKγ expression. Myotonic dystrophy kinase-related Cdc42 binding kinases (MRCKs) are family members most related to the myotonic dystrophy kinase (DMPK), RhoA-binding kinase (ROK), and citron kinase. Two highly conserved members, MRCKα and -β, have been previously identified and characterized. We now describe a novel isoform, MRCKγ, which is functionally and structurally related to members of this kinase family. We show these kinases to have marked similarities in their genomic organization, substrate phosphorylation, and catalytic autoinhibition. Unlike MRCKα and -β, which are expressed ubiquitously, MRCKγ mRNA was only expressed in heart and skeletal muscle. In cultured cells, MRCKγ showed differential expression with high levels of expression only in certain cell lines. DNA analysis showed that lack of expression is correlated with promoter DNA methylation. We have mapped the methylation sites in the MRCKγ promoter. Significantly, agents that suppressed DNA methylation caused increases in the expression of the kinase in low-expressing cells, further supporting the notion that promoter DNA methylation plays an important role in the expression of MRCKγ. Analysis of the MRCKγ promoter has also revealed two proximal Sp1 sites that are essential for transcriptional activity. We conclude that both promoter DNA methylation and Sp1 binding are important regulators for MRCKγ expression. The Rho GTPases regulate cell morphology, cell growth, and cell polarity (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3875) Google Scholar, 2Burridge K. Wennerberg K. Cell. 2004; 116: 167-179Abstract Full Text Full Text PDF PubMed Scopus (1519) Google Scholar). Numerous downstream effectors have been reported in the last decade; they comprise both kinases and non-kinases. Rho kinase ROK 1The abbreviations used are: ROK, RhoA-binding kinase; MRCK, myotonic dystrophy kinase Cdc42-binding kinase; GST, glutathione S-transferase; KIM, kinase inhibitory motif; MLC-2, myosin light chain-2; MBS, myosin binding subunit; PIM, phosphorylation inhibitory motif; CAT, kinase catalytic domain; CRIB, Cdc42/Rac1-interactive binding; EMSA, electromobility shift assays; HA, hemagglutinin; RT, reverse transcriptase; 5-Aza-CdR, 5-aza-2′-deoxycytidine. interacts specifically with the GTP-bound form of RhoA and organize actin bundling in cultured cells, whereas p21-activated kinases (PAKs) are Rac1 and Cdc42 binders signaling for actin disassembly and focal adhesion dissolution, through their complexes with PAK-interacting exchange factor PIX, G-protein receptor kinase-interacting protein GIT, and paxillin (3Zhao Z.S. Manser E. Loo T.H. Lim L. Mol. Cell. Biol. 2000; 20: 6354-6363Crossref PubMed Scopus (315) Google Scholar, 4Turner C.E. J. Cell Sci. 2000; 113: 4139-4140Crossref PubMed Google Scholar). The non-kinase Rac1/Cdc42-binding Wiskott Aldrich's syndrome protein WASP and related n-WASP are also downstream targets and are essential for actin polymerization, through their interaction with Arp2/3 (5Suetsugu S. Takenawa T. Int. Rev. Cytol. 2003; 229: 245-286Crossref PubMed Scopus (57) Google Scholar). A well conserved motif for the interaction of Rac1/Cdc42 (Cdc/Rac1-interactive binding, CRIB motif) has been identified when comparisons were made with a number of the Rac1/Cdc42-binding proteins such as PAK, ACK, and WASP (6Hoffman G.R. Cerione R.A. Cell. 2000; 102: 403-406Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 7Burbelo P.D. Drechsel D. Hall A. J. Biol. Chem. 1995; 270: 29071-29074Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). Members of the myotonic dystrophy kinase-related Cdc42-binding kinase family also possess the CRIB motif, which has preferential binding to Cdc42 (8Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (221) Google Scholar). However, their catalytic domain is closely related to that of Rho kinase, which specifically binds GTP-RhoA. Although the exact function of these kinases remains to be characterized, the catalytic domain homology suggests a similar function to Rho kinases ROKs, which are known to enhance the phosphorylation status of myosin light chain-2 (MLC-2), directly by phosphorylation or indirectly by phosphorylation and inactivation of myosin-targeting subunits of myosin phosphatase (9Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. Iwamatsu A. Kaibuchi K. Science. 1996; 273: 245-248Crossref PubMed Scopus (2444) Google Scholar, 10Muranyi A. Zhang R. Liu F. Hirano K. Ito M. Epstein H.F. Hartshorne D.J. FEBS Lett. 2001; 493: 80-84Crossref PubMed Scopus (85) Google Scholar, 11Tan I. Ng C.H. Lim L. Leung T. J. Biol. Chem. 2001; 276: 21209-21216Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). MRCKs, resembling ROKs, can phosphorylate and activate LIM kinases, which phosphorylate and inactivate cofilin, thereby facilitating actin polymerization events (12Sumi T. Matsumoto K. Shibuya A. Nakamura T. J. Biol. Chem. 2001; 276: 23092-23096Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). At the cellular level, MRCK has been shown to be involved in regulating cell morphology by enhancing Cdc42-induced membrane extensions (8Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (221) Google Scholar), and the dominant negative form of MRCKα can block nerve growth factor-induced neurite outgrowth in PC12 cells (13Chen X.-Q. Tan I. Leung T. Lim L. J. Biol. Chem. 1999; 274Google Scholar). The Drosophila autologue Genghis Khan (GEK) has also been implicated in actin polymerization events during development (14Luo L. Lee T. Tsai L. Tang G. Jan L.Y. Jan Y.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12963-12968Crossref PubMed Scopus (60) Google Scholar). Two genes encoding different isoforms of MRCK have been reported in mammals (8Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (221) Google Scholar). Both isoforms can exist as tetrameric forms through intermolecular interaction of their extended coiled-coil domains (15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar). A region in the distal coiled-coil region (CC2/3) was found to be essential for kinase inhibition, but the exact location has not been mapped previously. Activation of the kinase was observed upon binding of phorbol ester to the neighboring cysteine-rich domain, presumably by releasing the constraint of the inhibitory effect on the catalytic activity (15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar). Similar oligomeric structures with distinctive features have also been reported for the related Rho kinases ROKs (16Amano M. Chihara K. Nakamura N. Kaneko T. Matsuura Y. Kaibuchi K. J. Biol. Chem. 1999; 274: 32418-32424Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 17Chen X.-Q. Tan I. Ng C.H. Hall C. Lim L. Leung T. J. Biol. Chem. 2002; 277: 12680-12688Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and the myotonic dystrophy kinase (DMPK) (18Bush E.W. Helmke S.M. Birnbaum R.A. Perryman M.B. Biochemistry. 2000; 39: 8480-8490Crossref PubMed Scopus (39) Google Scholar). Both MRCKα and MRCKβ are ubiquitously expressed in various mammalian tissues and are abundantly expressed in all cell lines studied (18Bush E.W. Helmke S.M. Birnbaum R.A. Perryman M.B. Biochemistry. 2000; 39: 8480-8490Crossref PubMed Scopus (39) Google Scholar, 19Tan I. Cheong A. Lim L. Leung T. Gene (Amst.). 2003; 304: 107-115Crossref PubMed Scopus (7) Google Scholar). Interestingly, MRCKα is present as multiple species through differential splicing (20Moncrieff C.L. Bailey M.E. Morrison N. Johnson K.J. Genomics. 1999; 57: 297-300Crossref PubMed Scopus (16) Google Scholar), mainly at an internal variable splice site, which is located between the inhibitory region and the phorbol-binding cysteine-rich domain. Whether or not these sequence diversities play any roles in MRCK function remains to be determined. The characterization of yet another member, MRCKγ, has not been reported. We now describe the biochemical and functional characterization of MRCKγ. The expression of this kinase show marked variation in some cell lines, and this is dependent on the methylation status of its promoter DNA as well as on Sp1 binding. Cell Culture, Transfection, and Cell Staining—HeLa, Hct116, and MRC5 cells were cultured in minimum Eagle's medium, MCF7 and MKN28 cells were grown in RPMI medium, and COS-7 cells were maintained in Dulbecco's modified Eagle's medium. All media were supplemented with 10% fetal bovine serum, and cell cultures were maintained in humidified 5% CO2. Subconfluent cells plated on culture dishes for 24 h were transfected with respective DNA constructs using LipofectAMINE (Invitrogen) according to the recommended protocol. For immunostaining experiments, transfected cells were fixed with 4% paraformaldehyde and stained with anti-FLAG antibody (M2; Sigma). Construction of Expression Vectors—A 997-bp DNA fragment covering the CpG islands and the putative Sp1 binding sites of the putative MRCKγ promoter was amplified by PCR from genomic DNA isolated from Hct116 cells using the primer pair 5′-GTC TGA AGC CAC CAA TAT GTC-3′ (forward)/5′-CTG CTC GGC TAC AGT CTG G-3′ (reverse) and Hotstar Taq (Qiagen). The PCR products were cloned into pDrive vector (Qiagen) and sequenced. The HindIII/KpnI DNA fragment was subcloned into pGL3 basic vector to generate pGL3-1 (–844 to +152). To generate 5′ deletion constructs, forward primers (5′-GCT CCT ACT GAA GCT TAA AAC G-3′; 5′-CAG GTA CCA TCT GCC AAA TGG GCG-3′; and 5′-CAG GTA CCT TCA CCT CTA GCC GCG-3′) were paired up with the reverse primer to generate pGL3-2 (–622 to +152), pGL3-3 (–522 to +152), and pGL3-5 (–73 to +152), respectively. pGL3-4 (–357 to +152) was generated by digesting pGL3-1 with NcoI (blunted) and HindIII and subcloned into pGL3 basic vector cut with SmaI and HindIII. ΔBSSHII (deleted from –84 to –36) and Δ SacII (deleted from –307 to –61) constructs were generated by digestion with BSSHII and SacII, respectively, and religated. The primer pair 5′-GCT CCT ACT GAA GCT TAA AAC G-3′ (forward) and 5′-GGA CGG GCA GGT GCG CGC-3′ (reverse) was used to generate pGL3-2Δ3′ (–622 to –23). Full-length MRCKγ was obtained by PCR of the Hct116 cDNA using the primer pair 5′-CAG GAT CCA TGG AGC GGC GGC TGC GC-3′ (forward) and 5′-CTG CGG CCG CCC TAA CAG AGG GCA TCA A-3′ (reverse). The nucleotide sequences encoding the 103 amino acids (residues 670–772) of MRCKγ kinase inhibitory motif (KIM) were obtained by PCR using the primer pair 5′-CAG GAT CCG GTG AGC GGC GGG AGA CG-3′ and 5′-CTG CGG CCG CTG TCA GCC GCT CCT GCA GCT GCA GG-3′. The 309-bp BamHI-digested PCR fragment was subcloned into BamHI/SmaI-digested pXJ40-GST vector for mammalian cell transfection. All sequences were confirmed by DNA sequencing. pXJ40-HA-MRCKγ-CAT and pGEX-MRCKγ-CAT were generated by the digestion of full-length MRCKγ with BamHI/StuI and subcloned into their respective vectors digested with BamHI/SmaI. GST·MRCKγ· CRIB domain was obtained from a pGEX-vector containing the PCR product from primers 5′-CAG GAT CCT TTG TGC GCT CCA AGC TCA T-3′ and 5′-CTG CGG CCG CCC TAA CAG AGG GCA TCA A-3′. Other constructs used have been described previously (8Leung T. Chen X.-Q. Tan I. Manser E. Lim L. Mol. Cell. Biol. 1998; 18: 130-140Crossref PubMed Scopus (221) Google Scholar, 15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar). Immunoprecipitation and Kinase Assays—COS-7 cells expressing HA·MRCKα·CAT or HA·MRCKγ·CAT alone or co-expressed with GST· MRCKγ·KIM were lysed in lysis buffer containing 25 mm HEPES, pH 7.3, 150 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 20 mm β-glycerol phosphate, 1mm sodium vanadate, 5% glycerol, 0.5% Triton X-100, and 1× complete protease inhibitor mixture (Roche Applied Science), and immunoprecipitations were performed essentially as described (15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar) using either gluthathione-Sepharose (Amersham Biosciences) or anti-HA antibody (12CA5; Roche Applied Science). Kinase assays with 10 μg of substrates ((histone H1, myelin basic protein, GST fusion of myosin light chain-2 (MLC-2), or GST fusion of the phosphorylation inhibitory motif of myosin phosphatase MBS85 (GST·PIM)) and 0.1 μg of the immunoprecipitated kinase were carried out as described previously (15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar). The reactions were stopped by the addition of sample buffer, and the proteins were resolved by 11% SDS-PAGE, dried, and autoradiographed. Western Blot Analysis—Protein expression was detected by Western immnunoblotting. Cells were harvested in lysis buffer, and 100 μg of proteins were separated by 7.5% SDS-PAGE. MRCKα, -β, and -γ proteins were detected by specific antibodies raised against MRCKα, -β, and -γ, respectively. In Vitro GTPase Binding Assay—GST fusion proteins (50 ng) of MRCKα and MRCKγ containing the CRIB domain were resolved by SDS-PAGE and transblotted onto a polyvinylidene difluoride membrane. Immobilized proteins were first renatured for 3–4 h at 4 °C in renaturing buffer (phosphate-buffered saline containing 1% bovine serum albumin, 0.1% Triton X-100, 0.5 mm MgCl2 and 5 mm dithiothreitol) and subjected to probing with solution containing either [γ-32P]GTP-labeled GST·Cdc42 or TC10 as described previously (21Manser E. Leung T. Salihiddin H. Tan L. Lim L. Nature. 1993; 363: 364-367Crossref PubMed Scopus (262) Google Scholar). Probed filters were washed three times and autoradiographed. Northern Blot Analysis—Blots containing mRNA from various human tissues were obtained from Clontech and were hybridized with a C-terminal [32P]-labeled 3-kb NheI/NotI fragment from full-length MRCKγ (nucleotides 1610–4661). Cell Treatment with 5-Aza-CdR and Trichostatin A and Reverse Transcriptase-PCR—HeLa cells were plated onto 90-mm2 dishes 18–24 h prior to experiments. 5-Aza-2′-deoxycytidine (5-Aza-CdR; Sigma) was freshly added onto cells every 24 h at a final concentration of 1 μm for 48 h. 100 ng/ml trichostatin A (Sigma) was added at the last 12 h of the cell treatment with 5-Aza-CdR. Total RNA from treated or untreated cells with drugs was obtained using the Qiagen RNeasy® RNA isolation kit. cDNA was reverse-transcribed from the total RNA using M-MuLV reverse transcriptase (New England Biolabs). PCR was then performed using Hotstar Taq and the CRIB domain primers. The PCR conditions were as follows: 95 °C for 15 min; 40 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s; and a final extension of 72 °C for 5 min. Primers for glyceraldehyde-3-phosphate dehydrogenase (5′-TCC ATC ACC ATC TTC CAG-3′ (forward) and 5′-ATG AGT CCT TCC ACG ATA CC-3′ (reverse)) were used for similar PCR as control. For amplification of the internal splice site, primers 5′-TAG CAG GAA GAC CTT CTG TAG-3′/5′-TGG AGC TGC AGT CAG CGC T-3′ were used. The PCR products were subcloned into pDrive vector and sequenced. Determination of Transcriptional Initiation Site—RT-PCR was performed using Hct116 poly(A)+RNA to determine the transcriptional initiation site. Several primers close to the putative initiation site of MRCKγ (including primer 1, 5′-GAA GGT GAC AGC GGG GAGG-3′; primer 2, CCA GGT GAG GGC GCG CTG-3′; primer 3, 5′-CGG CGC CCA GGT GAG GGC-3′; and primer 4, 5′-GGC TCC CAT TGG CCG GCG-3′) were synthesized to pair up with the reverse primer 5′-CTG CTG AGC TCG TGG TGC A-3′. Various annealing temperatures for each primer pair were used for optimizing reaction conditions using genomic DNA as template, and the right-sized products from RT-PCR were subcloned into pDrive vector for DNA sequencing. Luciferase Reporter Assay—Hct116 cells were transfected with various MRCKγ luciferase reporter gene constructs, and cells were harvested for luciferase assays using the single luciferase assay system (Promega). pXJ40-HA-Sp1 was also co-transfected with pGL3-2 to determine the effect of Sp1 on the promoter activity. A cytomegalovirus promoter-β-galactosidase reporter (pCMV-β-galactosidase) construct was also included to monitor transfection efficiency. Preparation of Whole Cell Extracts and Electromobility Shift Assays (EMSA)—Preparation of whole cell extracts from HeLa, MKN28, and COS-7 transfected cells was performed according to Manley et al. (22Manley J. Fire A. Cano A. Sharp P.A. Gefter M. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3855-3859Crossref PubMed Scopus (735) Google Scholar), and EMSA was performed essentially as described previously (23Garg L.C. Dixit A. Webb M.L. Jacob S.T. J. Biol. Chem. 1989; 264: 2134-2138Abstract Full Text PDF PubMed Google Scholar). The oligonucleotide used for EMSA was the consensus Sp1 binding site (underlined) in MRCKγ promoter (5′-CCC CGG CCC GCC CCC GCA CTT-3′; see Fig. 5B). The oligonucleotide was 5′-end-labeled with [γ-32P]ATP and annealed with its complementary strand (5′-AAG TGC GGG GGC GGG CCG GGG-3′). A DNA fragment of 205 bp (–263 to –59) with sequences spanning the tandem Sp1 binding sites was also used for EMSA. The 32P-radiolabeled primer pair (5′-CAG GTA CCG CGC CTC CTG GGT CA-3′/5′-GCT AGA GGT GAA GGT GGG-3′) was used for PCR of the 205-bp DNA fragment. Unlabeled competitors were added in 100-fold excess to the reaction mix prior to the addition of radiolabeled probe. For supershift assays, 2 μg of the polyclonal Sp1 antibody (Santa Cruz Biotechnology) were added to the binding reaction prior to the addition of radiolabeled probe. The DNA-protein complex was then size-fractionated by using 5% PAGE in 0.5× Tris-borate EDTA buffer at 150 V at 4 °C. The gel was dried and autoradiographed. Preparation of Genomic DNA and McrBC Digestion—The genomic DNA from various cell lines was prepared using a DNeasy® tissue kit (Qiagen) according to the manufacturer's instructions. 250 ng of genomic DNA were cleaved with McrBC (New England Biolabs) in a final reaction volume of 25 μl at 37 °C for 1 h. The heat-inactivated cleavage mixture (2.5 μl) was used for PCR using primers 5′-GTC TGA AGC CAC CAA TAT GTC-3′ and 5′-CTG CTC GGC TAC AGT CTG G-3′. Bisulphite Modification and Sequencing—For mapping of methylated cytosine residues, genomic DNA samples from Hct116 and HeLa cells were modified by bisulphite reaction using the CpGenome™ DNA modification kit (Intergen Inc.), and the final products were amplified with primers 5′-GGG TTT AAT TTA AGA GTT ATT TTG-3′ (forward)/5′-CTA TCA CCT TCC TCC-3′ (reverse), which were specific for the modified MRCKγ sequence. PCR was performed with the BD Advantage™ 2 system (BD Biosciences) and started at 95 °C for 1 min followed by 5 cycles of 95 °C for 30 s, 65 °C for 30 s, and 68 °C for 1 min, with a decrease of 2 °C in annealing temperature per cycle. After another 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min and a final extension of 68 °C for 3 min, the PCR products were cloned into pDrive vector, and 20 separate clones for each cell lines were sequenced for analysis. MRCKγ Is a Novel Member of the MRCK Serine/Threonine Kinase Family—We have previously reported two members, MRCKα and MRCKβ, in this class of Rho GTPase-binding serine/threonine kinases. Sequences of a related kinase have been reported in the databases (e.g. accession number XM_290516), but hitherto, no reports of its biochemical and functional characterization have been reported. We have now isolated and characterized the human cDNA of this novel kinase. The sequence codes for a new member of the MRCK family, which we have termed MRCKγ (Fig. 1A). The organization of domains in MRCKγ is similar to the other MRCKs, and comparisons with MRCKα revealed the following sequence identity (in percent) in the various domains: kinase (73%), cysteine-rich (54%), pleckstrin homology (47%), Citron homology (42%), and CRIB (53%). The coiled-coil domain in MRCKγ is much shorter (mainly in the proximal coiled-coil 1 (CC1) region) and more diverse (29%). Within the coiled-coil domain, there is a highly conserved motif (66% identical to MRCKα), termed the KIM because of its unique property of interacting with, and inhibiting activity of, the kinase (Figs. 1, A and B, and 2A) (see also Ref. 15Tan I. Seow K.T. Lim L. Leung T. Mol. Cell. Biol. 2001; 21: 2767-2778Crossref PubMed Scopus (69) Google Scholar). MRCKγ has a molecular mass of about 160 kDa, when compared with 180 kDa for the other known family members, the main difference being due to the shorter CC1 domain. Phylogenetic analysis of the MRCK family showing its relationship to the mammalian and invertebrate counterparts is presented in Fig. 1C. The gene is located at chromosome 11q13. Analysis of the genomic sequence reveals the coding sequence to reside within 36 exons (Fig. 1, A and B) with an organization that is markedly similar to that of MRCKα (19Tan I. Cheong A. Lim L. Leung T. Gene (Amst.). 2003; 304: 107-115Crossref PubMed Scopus (7) Google Scholar) and MRCKβ (Ref. 20Moncrieff C.L. Bailey M.E. Morrison N. Johnson K.J. Genomics. 1999; 57: 297-300Crossref PubMed Scopus (16) Google Scholar and data not shown). However, the overall size of the gene is only about 30 kb, which is far more compact than the 250 kb reported for MRCKα and the 130 kb reported for MRCKβ.Fig. 1Genomic and sequence organization of human MRCKγ.A, DNA and deduced amino acid sequences of human MRCKγ. The bolded letters represent the exon-intron boundaries. The amino acid sequence is represented in order from the N terminus to the C terminus, and the kinase domain, kinase inhibitory motif, cysteine-rich domain, pleckstrin homology domain, Citron homology domain, and Cdc42/Rac-interactive binding motif are highlighted. B, genomic structure of the human MRCKγ gene. In the top panel, the exons are boxed and numbered, and introns known to be above 1 kb are shown. Lower panel, protein domain arrangement of MRCKγ organized from deduced amino acid sequence. Corresponding exons and amino acid sequences in the subdomains are linked by dotted lines. Subdomain arrangements for MRCKα and -β were also shown for comparison. N, N-terminal region; C, CRIB motif; CC, coiled-coil domain; V, internal variable region; CR, cysteine-rich C1 domain; PH, pleckstrin homology domain; CNH, Citron homology domain. C, a phylogenetic tree of members of the MRCK family. Amino acid sequences of the various MRCKs from NCBI and Fugu databases were analyzed using the Clustal phylogenetic analysis from DNASTAR. The numerals denote the number of residues that are varied from each other.View Large Image Figure ViewerDownload (PPT)Fig. 2Biochemical characterization and cellular localization of MRCKγ.A, a, substrate specificity of MRCKα·CAT and MRCKγ·CAT. Kinase assays with histone H1 (H1), myelin basic protein (MBP), GST·myosin light chain (GST·MLC-2), and GST·PIM were used as substrates, which were marked with asterisks. b, the inhibitory effects of MRCKγ·KIM on the catalytic activities of MRCKα and MRCKγ. HA·MRCKα·CAT and HA·MRCKγ·CAT constructs were expressed alone (lanes 1 and 3) or co-expressed with GST·MRCKγ·KIM construct (lanes 2 and 4). Immunoprecipitations were carried out using anti-HA antibody (lanes 1 and 3) or glutathione-Sepharose beads (lanes 2 and 4), and the immunoprecipitates recovered were assayed for kinase activity using GST·MLC-2 as substrate. Blotted filters were also immunostained with anti-HA and anti-GST antibodies after autoradiography. c, KIM motif alignment. KIM motifs of various MRCKs from human and Drosophila were aligned with the Clustal method (DNASTAR). Conserved residues are boxed in black, and the numbers indicate the positions of residues. B, sequence alignment of the CRIB sequences of human hMRCKα, hMRCKβ, and hMRCKγ, Fugu fMRCKα, Drosophila GEK, and Caenorhabditis elegans ceMRCK was performed as described in the legend for Fig. 1. The bottom panels show the in vitro GTPase binding assay of MRCKα and MRCKβ. Purified GST fusion proteins containing the CRIB domain of MRCKα and MRCKγ immobilized on polyvinylidene difluoride filters were assayed for binding with [γ-32P]GTP-labeled Cdc42 (left panel) or [γ-32P]GTP-labeled TC10 (right panel). C, Northern blot analysis of MRCKγ. The mRNA blot was purchased from Clontech and probed with a 3-kb DNA fragment from the 3′ end of human MRCKγ. S. Muscle, smooth muscle. D, analysis of the internal splice site in MRCKγ. A primer pair specific for covering the internal splice site in the human sequence was used to amplify total cDNA prepared from MKN 28 cells. For a control, a primer pair specific for sequence in the C-terminal region was used to amplify the same cDNA. The marker lane (Mr) is shown in bp. The numbered boxes represent exons present in the corresponding bands as revealed by DNA sequencing. The bottom panel represents the deduced amino acid sequence of exons involved in alternate splicing at the internal splice site. E, HeLa cells were stained with anti-FLAG antibody for detecting the expressed MRCKγ. The arrows show MRCKγ localized mainly at the cytoplasm, with higher density at the leading edges.View Large Image Figure ViewerDownload (PPT) Biochemical Characterization, Expression, and Cellular Localization of MRCKγ—The marked homology of the kinase domain of MRCKγ with those of MRCKα/β and Rho kinases ROK inferred similar substrate specificity. A GST kinase domain fusion protein of MRCKγ was tested for this. Like MRCKα, MRCKγ kinase phosphorylated the known substrates, MLC-2 and a fragment from the myosin binding subunit of myosin phosphatase MBS85 (GST·PIM; Fig. 2A) but not histone H1 nor myelin basic protein. The conserved KIM motif of MRCKγ (amino acid residues 677–765 in CC2/3) also bound the kinase domain of MRCKα and MRCKγ and inhibited their catalytic activity (Fig. 2A). These results are consistent with MRCKγ sharing similar substrate specificity and conserved auto-inhibitory mechanism with the other MRCK kinases. The CRIB domain responsible for Cdc42/Rac1 interaction in MRCKγ is far less conserved when compared with other MRCK counterparts (Fig. 2B). We therefore tested the binding of GST·MRCKγ·CRIB to RhoA, Rac1, Cdc42 as well as the related TC10 GTPase. Although this CRIB domain can bind Cdc42, it binds more strongly to TC10 (Fig. 2B). It binds Rac1 weakly but not RhoA (data not shown). It seems that MRCKγ differs from the other MRCK isoforms in that it may interact preferentially with Rho GTPases other than Cdc42 and Rac1. Apart from TC10, the identity of any other GTPase binding and their physiological roles remain to be determined. Northern blot analysis of MRCKγ revealed that a 6-kb message was expressed in human heart and skeletal muscle (Fig. 2C). By immunological analysis, the protein was found to be also highly expressed in a number of cell lines, including MKN28 cells (Fig. 3A). RT-PCR of MKN28 cells showed a differential splicing event at the internal variable region within exons 21–23 (Fig. 1B), with the major product containing exons 21/22/23 and minor products containing alternative exons 21/22a/23 or 22/23 alone (Fig. 2D). Extensive splicing events in this region have been documented for MRCKα (20Moncrieff C.L. Bailey M.E. Morrison N. Johnson K.J. Genomics. 1999; 57: 297-300Crossref PubMed Scopus (16) Google Scholar), and MRCKγ has adopted a similar but simpler processing. However, other splicing events occurring at the CRIB domain and C-terminal of MRCKα were not observed with MRCKγ. When expressed in HeLa cells, FLAG-tagged MRCKγ was mainly cytoplasmic with a higher density at the leading edges (Fig. 2E). Distinctive Expression Pattern of MRCKγ in Various Cell Lines Determined by Promoter Activity—We used specific antibodies to the different isoforms of MRCKs to evaluate their relative expression in the soluble fractions derived from a variety of human and mammalian cell lines. Although the protein expression of MRCKα and MRCKβ was ubiquitous, that of MRCKγ was more restricted. High expression was detected in MKN28, HCT116, and MCF7 cells but not in HeLa, MRC5, and COS-7" @default.
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• W2023662810 date "2004-08-01" @default.
• W2023662810 modified "2023-09-29" @default.
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