PHOSPHORYLATION SITE SUBSTRATE-SPECIFICITY DETERMINANTS FOR THE PIM-1PROTOONCOGENE-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 ful l-length coding regions of both human and Xenopus laevis Pim-1 were ex pressed as recombinant bacterial fusion proteins that autophosphorylat ed in vitro and exhibited phosphotransferase activity towards various exogenous substrates. The consensus sequence for phosphorylation by Pi m-1 was defined by stepwise replacement of the amino acids in peptide substrate analogues based on the carboxyl-terminal segment of human ri bosomal protein S6 (residues 229-249). The optimal substrate peptide f or 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 resu lts 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 thes e proteins.