Characterization of the phosphorylation sites and the surrounding amino acid sequences of the p21 transforming proteins coded for by the Harvey and Kirsten strains of murine sarcoma viruses.
The transforming protein coded for by the onc gene (v-rasHa) of Harvey murine sarcoma virus (Ha-MuSV) is the 21,000-dalton protein (p21) which is the immediate agent responsible for the virus-induced malignant transformation of normal cells. The p21 proteins of Ha-MuSV and the closely related Kirsten murine sarcoma virus are heavily phosphorylated in vivo. In the partially purified Ha-MuSV p21, the protein shows a guanine nucleotide-binding activity and, in addition, a very unique autophosphorylating activity at a threonine residue using as phosphoryl donor GTP but not ATP. In the present study, we compared the tryptic peptide maps of the Ha-MuSV p21 phosphorylated in vivo and in vitro. The results show that the major phosphorylation site is identical. Since the GTP-specific phosphorylation is very unique and distinct from all other known protein kinases, the present observation suggests that the in vitro enzymatic activity is responsible for the p21 phosphorylation in vivo. We have analyzed the amino acid sequence surrounding the major phosphorylation site of the Ha-MuSV p21 by automated Edman degradations of the tryptic phosphopeptides. Threonine residue 59 from the initiator methionine residue 1 of the p21 protein is the phosphorylated amino acid residue, and the surrounding amino acid sequence is NH2...-Thr-Cys-Leu-Leu-Asp-Ile-Leu-Asp-Thr-Thr(P)-Gly-Gln-Glu-Glu-Tyr-...COOH. The p21 proteins of both the Ha-MuSV and the closely related Kirsten murine sarcoma virus share the same phosphopeptide. The amino acid sequence of the phosphorylation site is distinct from all other known protein kinases.
D000595:Amino Acid Sequence; D000818:Animals; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D007708:Kirsten murine sarcoma virus / Q000235:genetics*; D051379:Mice; D015689:Oncogene Protein p21(ras); D010446:Peptide Fragments / Q000032:analysis; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D009053:Sarcoma Viruses, Murine / Q000235:genetics*; D014357:Trypsin; D014764:Viral Proteins / Q000235:genetics*
D010446:Peptide Fragments; D010748:Phosphopeptides; D014764:Viral Proteins; D014357:Trypsin; D015689:Oncogene Protein p21(ras)