IDENTIFICATION OF THE AUTOPHOSPHORYLATION SITES OF THE XENOPUS-LAEVISPIM-1 PROTO-ONCOGENE-ENCODED PROTEIN-KINASE

Pim-1 is an oncogene-encoded serine/threonine kinase expressed primari ly in cells of the hematopoietic and germ line lineages. Previously id entified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim- 1 encoded a protein of 324 residues, which exhibited 64% amino acid id entity with the full-length human cognate, Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and i n COS cells. Phosphoamino acid analysis revealed that recombinant Pim- 1 autophosphorylated on serine and threonine and to a more limited ext ent on tyrosine. Electrospray ionization mass spectroscopy was underta ken to locate these phosphorylation sites, and the primary autophospho rylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high c onserved Asp-Phe-Gly motif in catalytic subdomain VII, is also feature d in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs we re expressed in bacteria as GST fusion proteins and in COS cells, Thes e mutants confirmed that Ser-190 is a major autophosphorylation site o f Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 res idue may serve to activate this kinase.