Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

Abstract

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp6-vsrc present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp6Ovsrc. The detection of these variant forms of pp60v"src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implication is that modified, highly active forms of the pp60ovsrc protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60vsrc, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.The protein responsible for the oncogenic capabilities of Rous sarcoma virus (RSV) is pp6Ov-sr, a phosphoprotein encoded by the viral src gene (2,20,35,46). pp6,v-src appears to possess at least one enzymatic activity, that of a tyrosine-specific protein kinase (6,12,13,18,21,28,29,31,41). pp6Ov-src as isolated from transformed cell lysates itself contains two major phosphorylated residues: a phosphoserine located near the amino-terminal end of the protein (ser-17), which appears to be the result of phosphorylation by a cellular cyclic AMP-dependent protein kinase (7,8), and a phosphotyrosine residue, initially mapped to the carboxyterminal one-third of the polypeptide (7,8) and subsequently identified as the tyrosine residue at amino acid position 416 in the pp60v-src molecule (33, 42). Phosphorylation of tyrosine 416 may be the result of pp6Ov-src autophosphorylation since this same tyrosine residue was also found to be phosphorylated in vitro by purified pp60-src enzyme preparations (9,34,42 ed with these mutants were morphologically transformed and possessed pp6O-src tyrosine kinase activities similar to wild-type levels. These two studies therefore seem to indicate that phosphorylation of pp60-src, at either tyr416 or ser-17, plays little, if any, role in the function of src.We too have been interested in evaluating the functional consequences of phosphorylation, specifically tyrosine phosphorylation, on the pp6ov-sr, protein. However, our approach to this problem differ...

Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.

“…5 lanes 1 and 2), 10 (lanes 3 and 4), or 100 (lanes 5 and 6) FiM ATP were immunoprecipitated and resolved by gel electrophoresis. The bands corresponding to pp6f-vsrc were excised, reelectrophoresed in the presence of 5 (lanes 1, 3, and 5) or 50 (lanes 2, 4, and 6) ng of S. aureus V8 protease, and analyzed by autoradiography. The positions of unproteolyzed pp60v-src and the V8 proteolytic products corresponding to the amino-terminal 34,000-dalton (N34) and carboxy-terminal 26,000-dalton (C26) fragments are denoted in the margin.…”

“…In this paper, we describe kinetic studies of phosphorylation of amino-and carboxy-terminal sites in membrane-bound pp6Ov-src and suggest that both kinetic parameters and the presence of phosphoprotein phosphatases may limit the extent of amino-terminal phosphorylation. While this work was in progress, Collett et al (5) and Brown and Gordon (1) reported the identification of variant forms of pp6ov-src containing amino-terminal phosphotyrosine in transformed cell lysates. Our work with membrane vesicles complements and extends these findings.…”

Alterations in pp60c-src accompany differentiation of neurons from rat embryo striatum.

“…4), indicating that not all the pp60csrc molecules in this band were phosphorylated at these additional sites. Second, although phosphorylation of Ser-12 (32) or of unidentified NH2-terminal tyrosines (10,13,72) can cause small decreases in the mobility of fibroblast pp60csrc, there is no known in vivo phosphorylation event which results in such a dramatic shift in the mobility of pp60c-src. Third, the 61-kDa protein does not appear to be VOL.…”

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

“…While the IgG kinase assay is capable of displaying a 10-fold enhancement of pp60csrc kinase activity in the presence of cooperating factors (D. Shalloway et al, manuscript submitted), it does not accurately display kinase activity differences associated with labile pp60v`src tyrosine phosphorylation (9,41). While the monoclonal antibody exogenous substrate kinase assay measures activitation of pp60csrc kinase activity by polyoma middle T (4), it is possible that antibody binding or the presence of contaminating factors perturbs the results.…”