Phosphorylation site substrate specificity determinants for the Pim-1 protooncogene-encoded protein kinase.
Pim-1 is an oncogene-encoded serine-threonine kinase that is expressed primarily in cells of the hematopoietic system and germ line. The full-length coding regions of both human and Xenopus laevis Pim-1 were expressed as recombinant bacterial fusion proteins that autophosphorylated in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pim-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ribosomal protein S6 (residues 229-249). The optimal substrate peptide for Pim-1 was determined to be Lys/Arg-Lys/Arg-Arg-Lys/Arg-Leu-Ser/Thr-X, where X is an amino acid residue with a small side chain. These results were confirmed using X. laevis Pim-1 expressed in COS cells. These findings could permit the identification of physiological substrates of Pim-1 and predict the location of phosphorylation sites within these proteins.
Palaty
C K
CK
Department of Medicine, University of British Columbia, Vancouver, Canada.
Clark-Lewis
I
I
Leung
D
D
Pelech
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D016384:Consensus Sequence; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D051573:Proto-Oncogene Proteins c-pim-1; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry*; D013379:Substrate Specificity; D014981:Xenopus
D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; C496634:PIM1 protein, human; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1