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.

T Y Shih,P E Stokes,G W Smythers,R Dhar,S Oroszlan

doi:10.1016/s0021-9258(18)33830-4

Abstract

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.

Highlights

(v-rasHno)f Harvey murine sarcoma virus (Ha-MuSV) product directly responsible for the malignant transformation is the 21,000-dalton protein (p21) whichis theimmedi- induced by these viruses [2,3,4,5]
In the par- An important question is what is the molecular mechanism tially purified Ha-MuSV p21, the protein shows a guanine nucleotide-bindingactivity 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 by which the p21 protein transforms the normal cells into the malignant state
The results show that themajor phosphoryla- membrane [9]

Summary

RESULTS

Used as thesource of Ki-MuSV p21: Ki-NIHDT,NIH 3T3 cells transformed by theKi-MuSV [2] and Ki-CI'LS/Mo-Mul,V, a virus producing C127 cells transformed by Ki-MuSV anda helper Moloney murineleukemiavirus(Mo-MuLV). At the highest level of 3 X 20pg (Fig. 3, P3), intermediate [y-,"'P]GTP The latter phosphorylates the a single major phosphopeptide (spot 1)was obtained. Preincubation of the immunoprecipitates with GDP re- protease digestion experiments suggest that under optimal generation systems to convert the bound GDP to the GTP conditions, there is only one major phosphopeptide derived form should prevent the p21 phosphorylation by [y-""P]ATP. PAGE, the gel pieces containing approximately the same radioactivity as judgedby autoradiography of the p21 proteins were digested with three portions of trypsin at 3 X 5 pg/ml.

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DISCUSSION