The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme.
We have observed that intact wild type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF), when incubated with micromolar sodium orthovanadate for as little as 4 h, results in a 2-3-fold increase in pp60v-src kinase activity as judged by the IgG kinase assay. The addition of sodium vanadate or vanadyl sulfate to transformed cell lysates or the immunoprecipitate assay was without effect on the activity of isolated pp60v-src kinase activity. The increased kinase activity is reflected intracellularly by an increased phosphotyrosine content of a known substrate of pp60v-src, a 36-kDa phosphoprotein, without a change in total phosphate incorporation into cellular proteins. Pulse and pulse-chase experiments with [35S]methionine indicated that there was no change in the net or rate of biosynthesis of pp60v-src which might have accounted for the elevated kinase activity. Along with this stimulation of pp60v-src kinase activity, we found a parallel increase in the phosphate content of the enzyme and the appearance of an electrophoretic variant after vanadate treatment. The increased phosphorylation of the kinase is accounted for by an increase in phosphotyrosine content without a change in phosphoserine content. The increased phosphotyrosine content is shown to be resident in the carboxyl-terminal fragment and, unexpectedly, in the amino-terminal fragment of the kinase. The phosphotyrosine present in the amino-terminal portion may be a de novo phosphorylation, but could also represent a tyrosine site(s) not easily detected under unstimulated conditions in the absence of vanadate.
D000818:Animals; D001358:Avian Sarcoma Viruses; D002472:Cell Transformation, Viral; D002642:Chick Embryo; D005347:Fibroblasts / Q000201:enzymology; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014639:Vanadium / Q000494:pharmacology*; D014764:Viral Proteins / Q000378:metabolism*
D007143:Immunoglobulin Heavy Chains; D014764:Viral Proteins; D014639:Vanadium; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D015688:Oncogene Protein pp60(v-src)