Identification of Yin-Yang Regulators and a Phosphorylation Consensus for Male Germ Cell-Associated Kinase (MAK)-Related Kinase

• DOI: 10.1128/mcb.00816-06
• Publication Date: 2006-11-01T11:27:13Z
• Authors: Zheng Fu; Katherine A. Larson; Raghu K. Chitta; Sirlester A. Parker; Benjamin E. Turk; Matthew W. Lawrence; Philipp Kaldis; Konstantin Galakt...; Steven M. Cohn; Jeffrey Shabanowitz; Donald F. Hunt; Thomas W. Sturgill
• Keywords: 0303 health sciences; Amino Acid Motifs; Molecular Sequence Data; Down-Regulation; Nuclear Proteins; Proteins; Hydrogen Peroxide; Protein Serine-Threonine Kinases; Transfection; Cyclin-Dependent Kinases; Cell Line; Protein Structure, Tertiary; 03 medical and health sciences; Gene Expression Regulation; Consensus Sequence; Phosphoprotein Phosphatases; Humans; Amino Acid Sequence; Phosphorylation; Cyclin-Dependent Kinase-Activating Kinase; Molecular Chaperones

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.