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Volume 272, Number 16, Issue of April 18, 1997 pp. 10514-10521
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of the Autophosphorylation Sites of the Xenopus laevis Pim-1 Proto-oncogene-encoded Protein Kinase

(Received for publication, December 17, 1996, and in revised form, February 14, 1997)

Chrystal K. Palaty , Gabriel Kalmar ^¶ , Georgia Tai , Stella Oh ^¶ , Lawrence Amankawa , Michael Affolter , Ruedi Aebersold and Steven L. Pelech ^**

From the  Department of Medicine, University of British Columbia, and ^** Kinetek Pharmaceuticals, Inc., Vancouver, British Columbia V5Z 1A1, the ^¶ Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 156, Canada, and the  Department of Biotechnology, University of Washington, Seattle, Washington 98105

Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified 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 identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation 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 conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured 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 were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.

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