Abstract
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.
Highlights
Pim-1 [1] is a member of the small family of known protooncogene-encoded serine/threonine protein kinases
Extensive transgenic studies have confirmed that pim-1 is an oncogene in mice (4 – 6), but the only evidence that Pim-1 contributes to human cancers is that the protein and mRNA levels for this kinase are elevated in some human tumors and
We have cloned Pim-1 from Xenopus laevis and characterized a purified glutathione S-transferase (GST) fusion form of this kinase that was expressed in Escherichia coli
Summary
Pim-1 [1] is a member of the small family of known protooncogene-encoded serine/threonine protein kinases. The normal functions of Pim-1 have been obscure, its mRNA and protein expression patterns indicate that it is involved in hematopoietic signal transduction and development of male germ cells [1, 3, 7, 10, 11]. Expression and stability of pim-1 mRNA and protein is tightly regulated at different levels, implying that the kinase is functionally potent [7, 9, 12,13,14]. Up-regulation of Pim-1 by signaling through the GM-CSF receptor family requires the presence of the GM-CSF receptor membrane proximal domain and may involve the JAK2 tyrosine kinase (19 –24). As Pim-1 phosphotransferase activity has not yet been shown to be regulated by upstream kinases, it is possible that an autophosphorylation event may modulate the activity of the kinase. Immobilized metal affinity chromatography (IMAC) and high pressure liquid chromatography (HPLC)-elec-
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