Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase.
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.
Fu
Zheng
Z
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, VA 22908-0735, USA.
Larson
Katherine A
KA
Chitta
Raghu K
RK
Parker
Sirlester A
SA
Turk
Benjamin E
BE
Lawrence
Matthew W
MW
Kaldis
Philipp
P
Galaktionov
Konstantin
K
Cohn
Steven M
SM
Shabanowitz
Jeffrey
J
Hunt
Donald F
DF
Sturgill
Thomas W
TW
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
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