In vivo effect of sodium orthovanadate on pp60c-src kinase.

Abstract

We have compared the tyrosine kinase activity of pp60cs`isolated from intact chicken embryo fibroblasts treated with micromolar sodium orthovanadate for 4 h and from untreated cells. We found an approximate 50% reduction in both autophosphorylation of pp604-' and phosphorylation of casein when examined in the immune complex kinase assay. The reduction of in vitro enzymatic activity correlated with a vanadate-induced increase in in vivo phosphorylation of pp60`Csrc at the major site of tyrosine phosphorylation in the carboxyl-terminal half of the molecule and at serine in the amino-terminal half of the molecule. Our observations in vivo and those of Courtneidge in vitro (EMBO J. 4:1471(EMBO J. 4: -1477(EMBO J. 4: , 1985 Transformation of cells by Rous sarcoma virus (RSV) is correlated with an 8-to 10-fold increase in cellular phosphotyrosine (44) distributed among numerous cellular proteins (36). This increase is attributable to the expression of a functional (44), 60-kilodalton (kDa), virus-encoded (5, 38) cyclic AMP-independent (21), tyrosine-specific (12, 25, 33) protein kinase (pp60v-src). Several specific intracellular substrates for pp60v-src have been identified (for a review, see reference 24), although the role of tyrosine phosphorylation in transformation has not been elucidated. However, the consistent correlation between tyrosine phosphorylation and transformation indicates that expression of pp60v-src kinase activity is necessary for transformation (44).The primary structure of the normal cellular homolog of pp60v-src, pp604-src, is very similar but not identical to that of pp6jv-src* The 19 carboxyl-terminal amino acids of chicken pp60c-src are replaced by a new set of 12 amino acids at the carboxyl-terminal end of pp60v-src encoded by the SchmidtRuppin A strain RSV, and 8 additional amino acid substitutions are found throughout the pp60v-src molecule (48). In the intact cell pp6&c-src and pp60v-src are each phosphorylated at a serine residue in the amino-terminal half of the molecule, serine 17 (11, 19, 46), whereas pp6Ocsrc is phosphorylated in the carboxyl-terminal half of the molecule at a tyrosine residue distinct from tyrosine 416, which is phosphorylated in pp60v-src (10,29,45). Recent studies have indicated that phosphorylation of pp6&-src in the intact cell occurs at tyrosine 527, located in the 19 carboxyl-terminal amino acids unique to pp6Ocsrc (14).Earlier experiments showed that although the range of exogenous substrates and other aspects of the in vitro tyrosine kinase activities of pp60c-src and pp6OV-src are similar, the specific activities of pp60csrc for exogenous substrates and autophosphorylation are lower than those of * Corresponding author. pp60v-src (18). pp6O -,rc in normal cells occurs at a level 30 to 50 times lower than pp60vsrc in cells infected and transformed by RSV (10). To test the hypothesis that cell transformation comes about as a result of a general overexpression of the src-related tyrosine kinase activity, pp6O-src was expressed in rodent fibroblasts at a leve...

Growth cone enrichment and cytoskeletal association of non‐receptor tyrosine kinases

“…Our protocol of short, non-equilibrium labeling in whole GCPs, resulted in very little phosphorylation of the 3 I-kDa N-terminal fragment or of the 9-kDa fragment containing the autophosphorylation site [Bolen et al, 19871. It should be noted that, in earlier studies, vanadate-induced increases in N-terminal phosphorylation were observed only after several hours of incubation [Ryder and Gordon, 1987;Gould and Hunter, 19881; furthermore, other investigators have reported previously that c-src autophosphorylation may not be detected if radiolabeling is performed in a quasi-physiological environment [Ryder and Gordon, 1987;Zheng et al, 1992;Kmiecik and Shalloway, 19871. However, the 4-kDa C-terminal fragment, known to contain Tyr-527 [Bolen et al, 19871 was labeled significantly.…”

Characterization of avian and viral p60src proteins expressed in yeast.

“…This hypothesis is supported by experiments showing an increase in the in vitro kinase activity of p60c-src when orthovanadate (a phosphatase inhibitor) was included in the cell lysis buffer (10) or when p6Oc-src was extracted from cells treated with orthovanadate (12) and an elevation of p60c-src kinase activity upon phosphatase treatment in vitro (13). Furthermore, single amino acid substitutions in p60c-src that activate its tyrosine kinase activity and convert it to a transforming protein result in a shift of the phosphorylation site to tyrosine-416 (14,15).…”

Suppressive Effect of Orthovanadate on Hepatic Stellate Cell Activation and Liver Fibrosis in Rats

“…Our data showing that the effect of OV on HSCs was partly abrogated by the Src family inhibitors support the importance of Src activation in the action of OV. Although incubation of fibroblastic cells with OV has been shown to inhibit Src activity by increasing tyrosine 527 phosphorylation, 29 the apparently contradictory results may be attributable to shortterm incubation in the study, since the effect of OV on protein tyrosine phosphorylation in viable cells in culture proceeds slowly and steadily for more than 6 hours 18 and continues for at least several days. 8,9 Alternatively, because it has been shown that distinct protein tyrosine phosphatases regulate the phosphorylation status of these two tyrosine residues, 16 the preferential effect on tyrosine phosphorylation may be attributable to a specific protein tyrosine phosphatase expression profile in HSCs suggesting that Src was activated in the presence of OV.…”