title: Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase.
abstract: MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.
authors.affiliation: Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908-0735, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Fu
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Larson
authors.forename: Raghu K
authors.initials: RK
authors.lastname: Chitta
authors.forename: Sirlester A
authors.initials: SA
authors.lastname: Parker
authors.forename: Benjamin E
authors.initials: BE
authors.lastname: Turk
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Lawrence
authors.forename: Philipp
authors.initials: P
authors.lastname: Kaldis
authors.forename: Konstantin
authors.initials: K
authors.lastname: Galaktionov
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cohn
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Sturgill
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D016384:Consensus Sequence; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D015536:Down-Regulation; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism; D014162:Transfection; D000095043:Cyclin-Dependent Kinase-Activating Kinase
chemical_list: C092009:BAG6 protein, human; D018832:Molecular Chaperones; D009687:Nuclear Proteins; D011506:Proteins; D006861:Hydrogen Peroxide; C097276:CILK1 protein, human; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722413:CCRK protein, human; C000722416:CDK7 protein, human