Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function. Maternal embryonic leucine zipper kinase (MELK) is a protein Ser/Thr kinase that has been implicated in stem cell renewal, cell cycle progression, and pre-mRNA splicing, but its substrates and regulation are not yet known. We show here that MELK has a rather broad substrate specificity and does not appear to require a specific sequence surrounding its (auto)phosphorylation sites. We have mapped no less than 16 autophosphorylation sites including serines, threonines, and a tyrosine residue and show that the phosphorylation of Thr167 and Ser171 is required for the activation of MELK. The expression of MELK activity also requires reducing agents such as dithiothreitol or reduced glutathione. Furthermore, we show that MELK is a Ca2+-binding protein and is inhibited by physiological Ca2+ concentrations. The smallest MELK fragment that was still catalytically active comprises the N-terminal catalytic domain and the flanking ubiquitin-associated domain. A C-terminal fragment of MELK functions as an autoinhibitory domain. Our data show that the activity of MELK is regulated in a complex manner and offer new perspectives for the further elucidation of its biological function. The AMP-activated protein kinase (AMPK) 5The abbreviations used are: AMPKAMP-activated protein kinaseKA1kinase-associated 1 domainMBPmyelin basic proteinMELKmaternal embryonic leucine zipper kinaseNIPP1nuclear inhibitor of protein phosphatase-1UBAubiquitin-associated domainDTTdithiothreitol. is the best characterized member of the subfamily of AMPK-related protein Ser/Thr kinases (1Hardie D.G. Carling D. Carlson M. Annu. Rev. Biochem. 1998; 67: 821-855Crossref PubMed Scopus (1288) Google Scholar, 2Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6458) Google Scholar). It is allosterically activated by AMP and controls processes that restore the energy charge in the cell. The other members of the subfamily of AMPK-related kinases include the protein kinases ARK5, BRSK, MARK, MELK, QIK, NUAK, and SNARK, which fulfill functions in processes as diverse as cell cycle progression, cell survival, cell differentiation, and microtubule stability (3Levebvre D.L. Bai Y. Shahmolky N. Sharma M. Poon R. Drucker D.J. Rosen C.F. Biochem. J. 2001; 355: 297-305Crossref PubMed Google Scholar, 4Suzuki A. Kusakai G. Kishimoto A. Lu J. Ogura T. Lavin M.F. Esumi H. J. Biol. Chem. 2003; 278: 48-53Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar, 6Tassan J.-P. Le Goff X. Biol. Cell. 2004; 96: 193-199Crossref PubMed Scopus (86) Google Scholar). With the exception of maternal embryonic leucine zipper kinase (MELK), which is activated by autophosphorylation, the AMPK-related kinases are activated through phosphorylation of their T-loop by protein kinase LKB1 (5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar, 7Woods A. Johnstone S.R. Dickerson K. Leiper F.C. Fryer L.G.D. Neumann D. Schlattner U. Wallimann T. Carlson M. Carling D. Curr. Biol. 2003; 13: 2004-2008Abstract Full Text Full Text PDF PubMed Scopus (1371) Google Scholar) and the calcium- and calmodulin-dependent protein kinase II (8Hurley R.L. Anderson K.A. Franzone J.M. Kemp B.E. Means A.R. Witters L.A. J. Biol. Chem. 2005; 280: 29060-29066Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). AMP-activated protein kinase kinase-associated 1 domain myelin basic protein maternal embryonic leucine zipper kinase nuclear inhibitor of protein phosphatase-1 ubiquitin-associated domain dithiothreitol. The catalytic domain of the AMPK-related protein kinases is located in the N terminus of their catalytic subunit. Some of the AMPK-related protein kinases, including MELK, have an ubiquitin-associated (UBA) domain adjacent to the catalytic domain and a C-terminal kinase-associated 1 (KA1) domain. The function of the latter domains are poorly understood. UBA domains are known to bind (poly)ubiquitin and have been suggested to thereby prevent additional ubiquitination and proteosomal degradation of the target protein (9Buchberger A. Trends Cell Biol. 2002; 12: 216-221Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Hartmann-Petersen R. Semple C.A.M. Ponting C.P. Hendil K.B. Gordon C. Int. J. Biochem. Cell Biol. 2003; 35: 629-636Crossref PubMed Scopus (28) Google Scholar, 11Mueller T.D. Kamionka M. Feigon J. J. Biol. Chem. 2004; 279: 11926-11936Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Davies G.C. Ettenberg S.A. Coats A.O. Mussante M. Ravichandran S. Collins J. Nau M.M. Lipkowitz S. Oncogene. 2004; 23: 7104-7115Crossref PubMed Scopus (73) Google Scholar). In some proteins, UBA domains appear to function as dimerization domains (13Bertolaet B.L. Clarke D.J. Wolff M. Watson M.H. Henze M. Divita G. Reed S.I. J. Mol. Biol. 2001; 313: 955-963Crossref PubMed Scopus (102) Google Scholar). Between the UBA and the KA1 domains, MELK contains a TP dipeptide-rich domain that is phosphorylated in mitotically arrested cells and mediates binding to the transcription and splicing factor NIPP1 (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Preliminary evidence implicates MELK in various cellular processes. MELK binds tightly to the zinc finger-like protein ZPR9 and causes its nuclear accumulation (15Seong H.-A. Gil M. Kim K.-T. Ha H. Biochem. J. 2002; 361: 597-604Crossref PubMed Google Scholar, 16Seong H.-A. Kim K.-T. Ha H. J. Biol. Chem. 2003; 278: 9655-9662Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In the nucleus, ZPR9 itself interacts with the transcription factor B-Myb, a regulator of cell proliferation and differentiation, and enhances its transcriptional activity. Another interactor of MELK is the transcription and splicing factor NIPP1, but the binding of NIPP1 requires the phosphorylation of MELK on a specific threonine in its TP dipeptide-rich domain (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Because wild-type MELK, but not a NIPP1-binding mutant, is a potent inhibitor of pre-mRNA splicing in nuclear extracts and because the MELK-NIPP1 interaction is increased during mitosis, it has been proposed that MELK contributes to the ending of pre-mRNA splicing just before mitosis (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). A third protein ligand of MELK is Cdc25B, a protein-tyrosine phosphatase that triggers mitosis by the activation of protein kinase Cdk1. Davezac et al. (17Davezac N. Baldin V. Blot J. Ducommun B. Tassan J.-P. Oncogene. 2002; 21: 7630-7641Crossref PubMed Scopus (88) Google Scholar) reported that the ectopic expression of MELK induces an accumulation of cells in G2 and that this effect was counteracted by the overexpression of Cdc25B. Intriguingly, MELK is expressed at high levels in both embryonic (18Heyer B.S. Warsowe J. Solter D. Knowles B.B. Ackerman S.L. Mol. Reprod. Dev. 1997; 47: 148-156Crossref PubMed Google Scholar, 19Heyer B.S. Kochanowski H. Solter D. Dev. Dyn. 1999; 215: 344-351Crossref PubMed Scopus (48) Google Scholar) and neural stem cells (20Geschwind D.H. Ou J. Easterday M.C. Dougherty J.D. Jackson R.L. Chen Z. Antoine H. Terskikh A. Weissman I.L. Nelson S.F. Kornblum H.I. Neuron. 2001; 29: 325-339Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 21Easterday M.C. Dougherty J.D. Jackson R.L. Ou J. Nakano I. Paucar A.A. Roobini B. Dianati M. Irvin D.K. Weissman I.L. Terskikh A.V. Geschwind D.H. Kornblum H.I. Dev. Biol. 2003; 264: 309-322Crossref PubMed Scopus (56) Google Scholar), indicating that it may also play a role in stem cell functions of multipotency and self-renewal. Finally, MELK possibly contributes to oncogenesis because its expression is increased in tumor-derived progenitor cells (22Hemmati H.D. Nakano I. Lazareff J.A. Masterman-Smith M. Geschwind D.H. Bronner-Fraser M. Kornblum H.I. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15178-15183Crossref PubMed Scopus (1555) Google Scholar) and in cancers of nondifferentiated cells (23Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barette T. Pandey A. Chinnaiyan A.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9309-9314Crossref PubMed Scopus (819) Google Scholar). The MELK ligands ZPR9 (15Seong H.-A. Gil M. Kim K.-T. Ha H. Biochem. J. 2002; 361: 597-604Crossref PubMed Google Scholar), NIPP1, 6M. Beullens, V. Vulsteke, S. Vancauwenbergh, and M. Bollen, unpublished data. and Cdc25B (17Davezac N. Baldin V. Blot J. Ducommun B. Tassan J.-P. Oncogene. 2002; 21: 7630-7641Crossref PubMed Scopus (88) Google Scholar) are also MELK substrates, but the significance of their phosphorylation is not clear. A major limitation in studying the role of MELK as a protein kinase is that it is not known what controls its activity and that MELK expressed in mammalian cells seems to be inactive (5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar). This has prompted us to what the activity and substrate specificity of MELK, expressed in or mammalian We show here that MELK has a rather broad substrate specificity and that its activity is regulated by autophosphorylation, Ca2+ and reducing and and and of was of MELK MELK and the MELK and in the with the The on The MELK have been (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). to the of the and The of the by Cell and and cells in with the cells with and in at and the cell by at and the used for The cell with to protein for at with with and with the in at and used for kinase and with kinase of MELK and with the substrates for 1 at in a at and with substrates by of the to and with with myelin basic protein as substrate on and by kinase contains in of The was as in the the kinase was for at in the at and MELK. the the was with with with and with before The kinase was on a and Ca2+ of MELK or NIPP1 on a and the binding was as I. L. H. H. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The Ca2+ in was with the the of and of of by was as M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). was for at in a at and to The autophosphorylation sites of by as as by this was to a for at with and to an for at in the with The was by an with for at in the the with and the was with in The on a in and with a of in The by a in the or by on an The latter was with a of the and of the phosphorylation sites the and the The autophosphorylation sites of also by a MELK was with in as (5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar). to with and with of a in the an for the in an to a The with and with The was and in for by on an in as was on a high to an The on a in and with a at MELK a with a by et al. (5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar), expressed MELK was active and phosphorylated the a substrate of the subfamily of AMPK-related protein kinases Another used substrate for kinases, the was an in substrate of MELK. MELK also phosphorylated a broad of proteins, such as and the splicing NIPP1, and some the substrate of MELK, we a for substrates The used of that in phosphorylation sites. on this phosphorylated by MELK. The best MELK substrates factor and not show and also the and and the surrounding the MELK phosphorylation sites of and NIPP1 we have that of factor is phosphorylated by MELK data that MELK has a rather broad substrate specificity in MELK an catalytic domain of MELK is located in its N The C-terminal of MELK comprises a UBA a TP dipeptide-rich and a KA1 domain the latter domains the catalytic activity of MELK, we various MELK of the KA1 domain or the KA1 domains increased the the or as substrates the additional of the UBA as in in an inactive indicating that the UBA domain is for the expression of catalytic activity. with this we that the of the UBA domain MELK also in an inactive MELK was inactive the of the of the UBA domain. Our that the N-terminal of MELK is active than the protein suggested that the C-terminal of MELK functions as an autoinhibitory domain. we that the catalytic activity of MELK or was inhibited by the of With as a was with MELK we an fragment to which to the domain the KA1 was not The latter was because the of the KA1 domain increased the catalytic of MELK that the of by was for by a with the increased for as with the of wild-type MELK The of by and was also the was used as but the of was less with this substrate and to of with and by we that expressed MELK was phosphorylated on and tyrosine and that the of phosphorylation was further by of the with The phosphorylation on and be by with the catalytic of protein the phosphorylation on tyrosine was by with a protein-tyrosine phosphatase The phosphorylation of MELK is to be an because the inactive (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which is in the of kinase was not phosphorylated at We have used to the autophosphorylation sites of expressed wild-type MELK and This in the of autophosphorylation the with the of the UBA and KA1 domains not which was in with the data of Because MELK contains a tyrosine in its catalytic that is not in the catalytic of the other members of the subfamily of protein kinases and because MELK is also the member of this subfamily that is known to be on we have it is that is by MELK. In with this we that the autophosphorylation on tyrosine was in the that this was still catalytically active an substrate We have also used N-terminal by degradation to autophosphorylation sites in the catalytic A that was was phosphorylated on both Thr167 and Ser171 The latter was not by possibly because Ser171 was phosphorylated In the of autophosphorylation sites that we mapped to 16 of autophosphorylation sites by in a new MELK not the the residue was by a tyrosine indicating that MELK is not a specificity protein kinase and the autophosphorylation of MELK was of its that the autophosphorylation an rather than an In further with this we that wild-type MELK phosphorylated the catalytic domain of MELK a the catalytic of MELK and including the autophosphorylation sites and Ser171 We have the role of autophosphorylation of the catalytic domain of MELK by was that autophosphorylation on this is for activity. was indicating that this the phosphorylation of both and that phosphorylation of Ser171 is for activity but is not by an of or an or an not the activity of of a or an residue also no effect on the activity of The of MELK of MELK to and to an substrate was on the of reducing agents A of the protein kinase activity was with was a less potent than was and not MELK at The for reducing agents suggested that MELK is by the of or The are at located in the catalytic UBA domains because was also the of a that no required reducing agents to be active MELK the or of of the that are in the catalytic and UBA domains indicating that contains at that be and thereby a of the that MELK is a both in the and of indicating that its activation by reducing agents is not the of the of or MELK by the of the that MELK was active in the of the Ca2+ we have the activity of MELK is by a to various of we that the kinase of and inhibited in a manner by as we a at Ca2+ of 1 This to the Ca2+ that is the of cells with a of a Ca2+ Ca2+ less We data with the as kinase that the at 1 to Ca2+ not with the The data prompted us to MELK is a Ca2+-binding we that both MELK and and bind In a of the protein NIPP1 not bind Ca2+ MELK in data with expressed MELK MELK has expressed in mammalian we cells and cells with an expression for MELK or MELK with an N-terminal The the cell with In it is to what we for expressed MELK, expressed in cells was on for activity. the kinase activity of was inhibited by 1 Ca2+ but not by of Furthermore, the activity of was not by of Thr167 an but was Thr167 was by an data that MELK expressed in mammalian cells has as expressed MELK. of protein kinases have a rather substrate specificity that is by the of the that their phosphorylation sites D.J. 1998; PubMed Scopus Google Scholar). MELK phosphorylated an of the and the flanking the phosphorylation sites not to a a sequence be for the 16 mapped autophosphorylation sites Our data that MELK has a rather broad substrate specificity in This does not that MELK has also substrates in it be that MELK interacts in with that function as substrate to the regulation of protein phosphatase-1 H. Bollen M. Rev. 2005; Scopus Google Scholar). of the MELK fragment that was catalytically active of the catalytic domain and the UBA domain the UBA domain of MELK a inactive because UBA domains have been implicated in (poly)ubiquitin binding (9Buchberger A. Trends Cell Biol. 2002; 12: 216-221Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 10Hartmann-Petersen R. Semple C.A.M. Ponting C.P. Hendil K.B. Gordon C. Int. J. Biochem. Cell Biol. 2003; 35: 629-636Crossref PubMed Scopus (28) Google Scholar, 11Mueller T.D. Kamionka M. Feigon J. J. Biol. Chem. 2004; 279: 11926-11936Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Davies G.C. Ettenberg S.A. Coats A.O. Mussante M. Ravichandran S. Collins J. Nau M.M. Lipkowitz S. Oncogene. 2004; 23: 7104-7115Crossref PubMed Scopus (73) Google Scholar) and in the dimerization of (13Bertolaet B.L. Clarke D.J. Wolff M. Watson M.H. Henze M. Divita G. Reed S.I. J. Mol. Biol. 2001; 313: 955-963Crossref PubMed Scopus (102) Google Scholar). Our data the reported evidence that a UBA domain is required for the expression of catalytic activity by a protein Because UBA domains are also in other members of the AMPK-related kinases, the J.-P. Le Goff X. Biol. Cell. 2004; 96: 193-199Crossref PubMed Scopus (86) Google Scholar), it be to fulfill the function in kinases as The UBA domain is a domain of that a T.D. Kamionka M. Feigon J. J. Biol. Chem. 2004; 279: 11926-11936Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). It is to this domain contributes to the expression of kinase activity. The UBA domain does not appear to be in the dimerization of MELK because that MELK is a It is that the UBA domain has a function and is for the of the catalytic domain. The C-terminal of MELK, the TP dipeptide-rich and KA1 domains, functions as an autoinhibitory domain and we the major to the TP dipeptide-rich domain. The KA1 domain was not but the TP dipeptide-rich domain was also a less potent inhibitor than the C-terminal of the that the KA1 domain also contributes to in MELK. This is in with a on the AMPK-related members and which also have a C-terminal KA1 domain that was to be autoinhibitory and to with the N terminus of the kinases M. G. Mol. Biol. Cell. 2005; PubMed Scopus Google Scholar). by is a member of the AMPK-related protein kinases in that it does not require an protein kinase for activation and is activated by autophosphorylation 5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google and this various we have no less than 16 autophosphorylation in the catalytic domain and in the domain and its flanking of phosphorylation sites and not by possibly because MELK was phosphorylated on sites. have also that the of phosphorylation sites in a protein a of are Curr. Chem. Biol. 2001; PubMed Scopus Google Scholar, M. S. Chem. 2004; PubMed Scopus Google Scholar). In the of for the of phosphorylation sites. that Thr167 and located the and in the activation or to be for MELK to be active as a protein kinase sites are in other AMPK-related protein kinases and the to Thr167 has been to be phosphorylated by protein kinase LKB1 (5Lizcano J.M. Goransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Makela T.P. Hardie D.G. Alessi D.R. EMBO J. 2004; 23: 833-843Crossref PubMed Scopus (1088) Google Scholar). It is not yet known the to Ser171 is also phosphorylated in other AMPK-related protein Intriguingly, the phosphorylation of Ser171 not be by an MELK also its T-loop on a that is not in other AMPK-related protein kinases In this MELK is to the specificity tyrosine protein kinases because also their activation on a tyrosine but their substrates on and threonine G. Morrice N. V. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). It has been that the tyrosine autophosphorylation by is by a and an in the of the kinase G. Morrice N. V. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). are and the the tyrosine kinase activity is and function as protein Ser/Thr This does not to MELK, because autophosphorylation on is not for its activation and expressed MELK also on We have not yet the role of autophosphorylation of in the autoinhibitory domain is that the of this domain and thereby to the activation of the It be that the autoinhibitory domain also contains phosphorylation sites for other protein kinases (14Vulsteke V. Beullens M. Boudrez A. Keppens S. Van Eynde A. Rider M.H. Stalmans W. Bollen M. J. Biol. Chem. 2004; 279: 8642-8647Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). the domain phosphorylation sites for protein kinases and the phosphorylation of of in mitotically arrested cells mediates the of the transcription and splicing factor It has not yet been the binding of NIPP1 the activity of MELK. by to its regulation by and the N-terminal of MELK is by of at the on reducing agents also to MELK that is expressed in mammalian cells and is the major for to kinase with MELK in mammalian cell are of D.J. Biochem. J. 2004; PubMed Scopus Google Scholar, D. Curr. Biol. 2004; PubMed Scopus Google Scholar, C. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and we not which to MELK. by MELK is inhibited by Ca2+ that are the of of a Ca2+ Ca2+ less which is possibly for by the of a binding for Ca2+ that the effect of the binding of Ca2+ to the high binding MELK is the member of the AMPK-related kinases that is known to be regulated by suggested that Ca2+ binds to the catalytic domain of MELK but its does not an Ca2+-binding additional is to the Ca2+-binding of MELK in In to a kinase activity has us to in some the for the activity of MELK as a protein a of we have here that MELK is regulated by reducing and data be in studying the physiological regulation and functions of MELK. We J. of for on the Ca2+ binding We for MELK used for
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