Temperature-sensitive membrane association of pp60src in tsNY68-infected cells correlates with increased tyrosine phosphorylation of membrane-associated proteins.
Incubation of membrane vesicles from normal and Rous sarcoma virus-transformed chick embryo fibroblasts (CEF) with [gamma-32P]ATP resulted in the phosphorylation of a large number of proteins. The major differences observed between the membrane vesicles of untransformed and transformed cells were: (1) a 5- to 10-fold increase in the proportion of labeled phosphotyrosine in transformed vesicles and (2) the phosphorylation of pp60src in vesicles from transformed cells. Of the many proteins labeled in vitro, only pp60src was immunoprecipitated by TBR serum. Phosphorylation of the immunoprecipitated pp60src occurred on tyrosine in the 26-kDa carboxy-terminal Staphylococcus aureus V8 protease fragment. pp60src was not phosphorylated in vitro in membrane vesicles prepared from tsNY68-infected cells grown at the nonpermissive temperature. The proportion of labeled phosphotyrosine in membrane proteins from tsNY68-infected cells grown at the nonpermissive temperature was only slightly increased relative to that observed in membranes prepared from normal cells. Subcellular fractionation indicated that while pp60src was membrane associated in tsNY68-infected cells grown at the permissive temperature, pp60src was chiefly soluble in tsNY68-infected cells grown at the nonpermissive temperature. Temperature-sensitive membrane association of pp60src in tsNY68-infected cells was also observed by indirect immunofluorescence microscopy. When membranes were prepared from tsNY68-infected cells that had been downshifted from the nonpermissive to the permissive temperature, the reappearance of in vitro phosphorylated pp60src and the increase in the proportion of labeled phosphotyrosine in membrane vesicles correlated with the kinetics of src immune complex kinase reactivation and membrane association of pp60src.
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000502:physiology*; D002462:Cell Membrane / Q000032:analysis; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D002642:Chick Embryo; D008565:Membrane Proteins / Q000378:metabolism*; D009154:Mutation; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D019000:Phosphotyrosine; D011494:Protein Kinases / Q000378:metabolism; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism*
D008565:Membrane Proteins; D014764:Viral Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D015688:Oncogene Protein pp60(v-src)