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 (RPLT¹⁰⁸⁰S) 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.

We are especially grateful to all the generous colleagues who have made our studies possible by provision of their reagents and advice: Richard Honkanen, Sandra Rossie, Elizabeth Goldsmith, Xiaoshan Min, Andrew Hubberstey, Bert Vogelstein, Robert Fisher, and Sally Kornbluth. We are indebted to Katrina Clines for her support and technical assistance with the yeast two-hybrid screen. We thank members of Sturgill Lab (Carol Chrestensen and Darlene Bruce) for discussions and support and members of John Lawrence Lab (Dale Choi and Thurl Harris) for advice on lentivirus infection.

This work was supported by NIH grants GM62890 (to T.W.S.), GM37537 (to D.F.H.) and DK064751 (to S.M.C.).