Characterization of pp60src phosphorylation in vitro in Rous sarcoma virus-transformed cell membranes.
Phosphorylation of the src gene product pp60v-src was studied in plasma membrane fractions prepared from Rous sarcoma virus-transformed vole cells. Upon addition of [gamma-32P]ATP to isolated membrane vesicles, phosphate was incorporated into a 60,000-dalton polypeptide identified as pp60v-src. In the presence of vanadate, pp60v-src phosphorylation was stimulated ca. 30-fold. At low concentrations of ATP (1 microM), this reaction occurred almost exclusively on the carboxy-terminal 26,000-dalton region of pp60v-src. However, at higher ATP concentrations (100 microM), additional sites of phosphorylation were evident in the amino-terminal 34,000-dalton region. Kinetic analyses, performed under conditions in which ATP hydrolysis was minimal, revealed that the phosphorylation reaction at the carboxy terminus exhibited a higher Vmax and a lower Km for ATP than those occurring at the amino terminus. In addition, the amino-terminal region of pp60v-src was more rapidly dephosphorylated than the carboxy-terminal region. These results indicate that interaction of pp60v-src with the plasma membrane may limit the extent of amino-terminal phosphorylation by lowering the rate of the reaction and the affinity for the substrate while increasing its susceptibility to phosphoprotein phosphatases. We suggest that the use of transformed-cell membrane preparations provides a model system for studying the possible regulatory roles of phosphorylation and dephosphorylation on pp60v-src function.
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D000818:Animals; D003411:Arvicolinae; D001358:Avian Sarcoma Viruses; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D008565:Membrane Proteins / Q000378:metabolism*; D015688:Oncogene Protein pp60(v-src); D010446:Peptide Fragments / Q000032:analysis; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014639:Vanadium / Q000494:pharmacology; D014764:Viral Proteins / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D008565:Membrane Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D014361:Trypsin Inhibitors; D014764:Viral Proteins; D014639:Vanadium; D015688:Oncogene Protein pp60(v-src); D015032:Zinc