Half-life of the Rous sarcoma virus transforming protein pp60src and its associated kinase activity.

Abstract

The half-life of metabolically labeled pp6(Yrc of the Prague A strain of Rous sarcoma virus and of several transformation-defective, temperature-sensitive mutants was investigated by pulse-labeling infected cells with [35S]methionine, chasing for different times, and immunoprecipitating pp6(Yrc with tumor-bearing rabbit serum. These experiments showed that pp6(Yrc has a short half-life of approximately 60 min under normal physiological conditions and that the mutant pp6(Src proteins have similar half-lives to the wild type, irrespective of whether the cells are kept at the nonpermissive (42°C) or permissive (35°C) temperature. The half-life of the ppWsr-associated kinase activity was determined by monitoring its decay by the immunoglobulin G heavy chain assay after the cells had been treated with several inhibitors of protein synthesis. In these experiments the kinase half-life was much longer than expected from the half-life of pp6O!rc. The apparent contradiction between the half-lives of the kinase activity and the 135S]methionine-labeled pp6fysc protein could be resolved by the observation that treatment of cells with inhibitors of protein synthesis stabilized pp6(Yrc, resulting in a greatly extended half-life. Inhibitors of protein synthesis also extended the half-life of the gag precursor polypeptide, Pr76, suggesting that a host factor(s) may be required for the efficient intracellular processing of this polypeptide to the gag proteins.The continual expression of the src gene of Rous sarcoma virus (RSV) is required for maintenance of the transformed phenotype in infected cells (19). A 60,000-dalton phosphoprotein, pp61)Src, has been shown to be a product of the src gene (4,5,18), and a protein kinase activity capable of phosphorylating in vitro several substrates at tyrosine has been demonstrated to be intimately associated with pp6&Wrc (6,7,10,13). Despite extensive biochemical investigation of pp60srC, little is known about the in vivo h'alf-life of the molecule itself and its associated kinase activity (25). In this paper we describe experiments aimed at determining the half-lives of metabolically labeled pp6(sYc and the associated protein kinase activity. We show that pp60Yrc metabolically labeled with [35S]methionine has a short half-life of approximately 60 min. In experiments in which the decay of the pp6&frc-associated kinase activity was followed in cells treated with cycloheximide and other inhibitors of protein synthesis, a long half-life of many hours was observed. These results could be reconciled by our observation that the protein synthesis inhibitors used to demonstrate the decay of pp64src-associated kinase activity greatly prolonged the half-life of metabolically labeled pp60sc.

Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

“…The half-life of total cellular protein (22 h) was identical in cultures infected with NYN4 and NY501. The half-life of p60vsrc (8.5 h) (48,58) was significantly shorter than that of p60c-src (25 h). This difference in half-lives, combined with the similar synthetic rate (Fig.…”

Requirement of Phosphatidylinositol 3-Kinase-Dependent Pathway and Src for Gas6-Axl Mitogenic and Survival Activities in NIH 3T3 Fibroblasts

“…After this time, the cells were washed with cold PBS and Src proteins were immunoprecipitated essentially as described previously (37). Equal amounts of total lysates were incubated for 1 h on ice with an anti-Src polyclonal antibody (67). The immunocomplexes were equilibrated in kinase buffer and incubated for 20 min at 30°C in the presence of [␥- 32 P]ATP to monitor kinase activation.…”

Features of the pp60v-src carboxyl terminus that are required for transformation.

“…pp60r-sn' protein synthesis and equilibrium levels were compared (Fig. 5) by immunoprecipitation from cells labeled for periods which were short (1 h) or long (27 h) relative to the apparent pp6Ov-s" half-life (about 8 h for SR strains of v-src [22,47,68] stable than pp6o-src (Table 3). pp60RS l-src and pp60RS8-src are only moderately less stable than pp60vsrc.…”

“…Ci/mmol; New England Nuclear Corp.) per ml for 1 h. Cells 5). This period is long enough for the intracellular amino acid pool to come to equilibrium (45) but short compared with the half-lives of the pp60v-"r' proteins (previously estimated to be 8 h [22,47,68]). Relative pp60(rc equilibrium levels were determined from similar comparisons by using cells labeled for a long period (27 h) compared with the pp6Osr( half-life.…”