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Characterization of the pim-1 oncogene-encoded protein kinase

University of British Columbia

Pim-1 is an oncogene-encoded serine-threonine kinase, expressed primarily in hematopoietic and germ cells. Previously identified only in mammalian systems, pim-1 was cloned and sequenced from Xenopus laevis, the African clawed frog. The coding region of X. laevis Pim-1 encoded a protein of 324 amino acids, which exhibited 64% amino acid sequence identity with the full-length human protein. PCR was also used to demonstrate the existence of Pim-1 in Pisaster ochraceus, the purple sea star. The high sequence conservation observed in the catalytic domain of Pim-1 between divergent species support a conserved and important function for this kinase. The full-length coding regions of both human and X. laevis Pim-1 were expressed as recombinant bacterial fusion proteins which exhibited phosphotransferase activity towards exogenous substrates as well as serine, threonine and tyrosine autophosphorylation activity. A kinase-inactive mutant was engineered to serve as a negative control. The phosphorylation site consensus sequence for recognition by Pim-1 was defined by stepwise alterations in the amino acid sequences of peptide substrate analogues to determine which of the amino acid residues surrounding the substrate phosphorylation site were critical for kinase recognition. The optimal substrate peptide for Pim-1 was determined to be K/R - K/R - R - K/R - L – S/T - X, where X is an amino acid residue with a small side chain. Studies were undertaken to determine the autophosphorylation sites of the GST-Pim-1 kinase, using electron spray ionization mass spectroscopy (ESI-MS). The autophosphorylation sites of the GST-Pim-1 were identified as Ser-4, Ser-190 and Thr- 205. These sites were conserved amongst all Pim-1 homologues. An additional site was identified on the GST protein, Thr-17. To assess the importance of the Ser-190 site on phosphotransferase activity, the Ser-190 residue was changed to alanine and to glutamic acid using PCR site-directed mutagenesis. These mutants were expressed in bacteria as GST-fusion proteins, and their activites were compared to the wild-type. Together with Pim-1-specific antibodies, the optimal Pim-1 peptide substrate was used to study endogenous Pim-1 during X. laevis and P. ochracheus oocyte maturation. Pim-1 did not exhibit maturation-induced activation in sea star oocytes and the quantity of Pim-1 remained constant during the oocyte maturation process. This is the first study carried out to investigate this kinase in the oocyte system.

Thesis/Dissertation

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2009-04-24

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http://hdl.handle.net/2429/7540

Experimental Medicine

University of British Columbia

UBCV

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