Identification of the autophosphorylation sites of the Xenopus laevis Pim-1 proto-oncogene-encoded protein kinase.
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.
Palaty
C K
CK
Department of Medicine, University of British Columbia, Vancouver, British Columbia V5Z 1A1, Canada.
Kalmar
G
G
Tai
G
G
Oh
S
S
Amankawa
L
L
Affolter
M
M
Aebersold
R
R
Pelech
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D017931:DNA Primers; D005260:Female; D005982:Glutathione Transferase; D006801:Humans; D007700:Kinetics; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D051573:Proto-Oncogene Proteins c-pim-1; D011519:Proto-Oncogenes; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D017931:DNA Primers; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010770:Phosphotransferases; C496634:PIM1 protein, human; C496636:Pim1 protein, mouse; C496637:Pim1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051573:Proto-Oncogene Proteins c-pim-1