Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro.
Two species of polyoma virus middle T antigen were detected in both lytically infected and transformed cells by in vitro kinase assay of immunoprecipitates. A minor species with an apparent molecular weight of 58,000 (58K) represented less than 10% of the total middle T protein. This species was roughly 10 times more active as a phosphate acceptor than was the predominant 56K form. Partial proteolytic mapping experiments showed that the same site was phosphorylated in both species. Mapping of the middle T antigens from a series of deletion mutants suggested that the major site of phosphorylation is tyrosine residue 315. Phosphorylation occurred on both middle T species in vivo, involving sites predominantly other than the tyrosine labeled in vitro. The 56K and 58K middle T forms differed from each other in their in vivo phosphorylation patterns. Some phosphate was incorporated into the 58K species in a region of the molecule to which at least part of the apparent molecular weight different could be mapped. hr-t mutant NG-59, which codes for a slightly altered middle T, produced only a single species (56K) which was inactive in the in vitro kinase reaction. Moreover, no 58K species appeared in vivo with this mutant. hr-t mutants are therefore defective in both aspects of phosphorylation. Phenotypically normal revertant cells of a polyoma transformed line failed to express any middle T antigens or associated kinase activity.
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D000956:Antigens, Viral / Q000378:metabolism*; D000957:Antigens, Viral, Tumor; D002460:Cell Line; D002472:Cell Transformation, Viral; D002474:Cell-Free System; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011120:Polyomavirus / Q000276:immunology / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D000957:Antigens, Viral, Tumor; D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases