Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase

Mol Cell Biol. 2006 Nov;26(22):8639-54. doi: 10.1128/MCB.00816-06. Epub 2006 Sep 5.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Cell Line
  • Consensus Sequence*
  • Cyclin-Dependent Kinases / metabolism*
  • Down-Regulation
  • Gene Expression Regulation*
  • Humans
  • Hydrogen Peroxide / pharmacology
  • Molecular Chaperones
  • Molecular Sequence Data
  • Nuclear Proteins / metabolism
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / genetics*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteins / metabolism
  • Transfection

Substances

  • BAG6 protein, human
  • Molecular Chaperones
  • Nuclear Proteins
  • Proteins
  • Hydrogen Peroxide
  • CILK1 protein, human
  • Protein-Serine-Threonine Kinases
  • Cyclin-Dependent Kinases
  • cyclin-dependent kinase-activating kinase
  • Phosphoprotein Phosphatases
  • protein phosphatase 5