Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants.

Abstract

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp6Osrc. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp6Osrc domains that are required for transformation but do not grossly affect tyrosine kinase activity.The transforming src gene of Rous sarcoma virus (RSV) encodes a phosphoprotein with a molecular weight of 60,000, pp6Osr , which is required for the transformation of chicken embryo fibroblasts (CEF) in vitro (30,47). pp605'' is a cyclic-AMP-independent tyrosine protein kinase (6,10,12,24,31,36,37), the presence of which leads to an increase in the to, al cellular phosphotyrosine levels in transformed cells (13,14). The transformation mechanism of this protein is still unknown, but the bulk of pp6O0s appears to be localized at the inner surface of the plasma membrane (27,29,45,50), suggesting that it exerts its effects in this cellular compartment. Moreover, several proteins showing increased phosphotyrosine levels have been identified in RSV-transformed cells (4,15,39,49). These include vinculin (53), a 50-kilodalton (Kd) protein (28), a 36-Kd protein (23, 48), and three non-rate-determining glycolytic enzymes (17). Although the presence of several of these phosphoproteins may not be a prerequisite for morphological transformation or other transformation parameters, individually they could be involved in the appearance of specific features of transformation, as certain parameters may be divorced from each other and thus can be controlled independently (1-3, 16, 40, 51, 56, 61).The mechanism by which pp6os5' transforms cells has been studied with mutants of src. Work with temperaturesensitive (ts) mutants helped to establish the importance of the src gene and its kinase activity in maintenance of the transformed state (27,33,55). Mutants generated by in vitro mutagenesis of the cloned src gene are being used to define important regions of the molecule with respect to kinase activity and transforming ability (7,20,58). Such studies, taken together, suggest that pp605r( has perhaps more than one domain (of which the kinase site is one) that are implicated in full expression of the transformed phenotype (26,34,38,44,59).We have previously isolated and mapped a number of temperature-sensitive src mutations of the Prague strain of RSV, finding that the lesions were distributed along the length of the gene (25,62 Immunoprecipitation and gel electrophoresis. Cells were washed three times in phosphate-buffered saline and lysed in buffer A (0.1 M NaCl, 1 mM EDTA; 10 mM Tris-hydrochloride [pH 7], 1% Nonidet P-40, 1 mg of bovine serum albumin per ml, 40 mM NaF, 50 mM ZnCl,) or buffer B (0.15 M NaCl, 20 mM Tris-EDTA [pH 7], 1% Nonidet P-...

A noncatalytic domain conserved among cytoplasmic protein-tyrosine kinases modifies the kinase function and transforming activity of Fujinami sarcoma virus P130gag-fps.

“…It is of interest that the v-src residues 149 through 169 lost in the RSV deletion mutant NY320 correspond to a region in P130gag-fps (amino acids 821 through 839) within which the RX15m and AX9m linker insertion mutations are located. A second class of RSV mutants with structural alterations in the amino-terminal half of p60vsrc are temperature sensitive for transformation (6,52). The p60v-sc of one such mutant (LA32) showed no appreciable loss of kinase activity at the nonpermissive temperature (52), whereas another (tsCH119) with a deletion of p60vsrc residues 173 through 227 (consensus residues 38 through 98) had only a 50% decline in kinase activity at 41°C (6).…”

Isolation of temperature-sensitive tyrosine kinase mutants of v-abl oncogene by screening with antibodies for phosphotyrosine.

“…Traditionally, ts mutants of an oncogene have been isolated by selecting for ts defects in transformation. Many of the ts transformation-defective mutants of v-src have ts tyrosine kinase activity but at least one of these ts mutants (LA32) has normal kinase activity at the nonpermissive temperature, indicating that transformation-defective mutations can affect other aspects of the oncogene required for transformation (10). To select directly for mutations that alter the tyrosine kinase activity, we took a different approach.…”

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

“…These include the cytoskeletal protein vinculin (46,48), a 36,000-dalton (36-kDa) membrane-associated protein (pp36) (20,42,43), a 50-kDa cytoplasmic protein (6,7) of unknown function, and three non-rate-determining enzymes involved in the glycolytic pathway (16). The tyrosine phosphorylating activity correlates with transformation in most RSV temperature-sensitive mutants (28,53) and mutant revertants (37), although mutants and revertants exist which have tyrosine phosphorylating activity but which do not induce transformation (37,53). These facts suggest that src tyrosine kinase activity is required but not sufficient for transformation.…”