title: Substrate specificity and activity regulation of protein kinase MELK.
abstract: 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.
authors.affiliation: Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium. Monique.Beullens@med.kuleuven.be
authors.forename: Monique
authors.initials: M
authors.lastname: Beullens
authors.forename: Sadia
authors.initials: S
authors.lastname: Vancauwenbergh
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Rita
authors.initials: R
authors.lastname: Derua
authors.forename: Hugo
authors.initials: H
authors.lastname: Ceulemans
authors.forename: Etienne
authors.initials: E
authors.lastname: Waelkens
authors.forename: Mathieu
authors.initials: M
authors.lastname: Bollen
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002021:Buffers; D019556:COS Cells; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002453:Cell Cycle; D002460:Cell Line; D002522:Chlorocebus aethiops; D004229:Dithiothreitol / Q000737:chemistry; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D007477:Ions; D007700:Kinetics; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D040081:Protein Array Analysis; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000502:physiology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D013234:Stem Cells / Q000166:cytology; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D002021:Buffers; D007477:Ions; D008024:Ligands; D010455:Peptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; C484610:MELK protein, human; D017346:Protein Serine-Threonine Kinases; D005978:Glutathione; D002118:Calcium; D004229:Dithiothreitol