Transforming activity of polyoma virus middle-T antigen probed by site-directed mutagenesis.
The ability of polyoma virus to transform cells results primarily from the action of one of the virus-coded early proteins, called middle-T antigen. Middle-T has an associated tyrosine-specific protein kinase activity that can be measured in vitro and results in the phosphorylation of middle-T itself. Almost all mutants so far tested that lack the ability to transform cells, also lack associated kinase activity. Attempts to map within middle-T the tyrosine residue(s) that are phosphorylated in vitro suggest that a likely site of phosphorylation is tyrosine 315 (refs 8-10 and unpublished results). The amino acid sequence preceding Tyr 315 includes a tract of six contiguous glutamic acid residues and bears some homology with that preceding the tyrosine phosphorylated in vivo in pp60v-src, the transforming protein of Rous sarcoma virus, and with a region in the polypeptide hormone, gastrin, preceding a tyrosine that is sulphated. Furthermore, although surprisingly large tracts of middle-T may be removed without affecting its transforming activity, mutants that lack the sequences corresponding to amino acids 311-318 inclusive are transformation defective. Because the likely site of phosphorylation, the homology with pp60v-src and gastrin and the sequence apparently required for transformation all overlap, it has generally been accepted that this region of middle-T may form part of an essential region, possibly an active site on the protein. Here we have used techniques of site-directed and site-specific mutagenesis to probe the sequence requirements in more detail. Contrary to expectation, the results obtained strongly suggest that Tyr 315 and conservation of the surrounding amino acid sequence are not essential for transformation.
D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming; D001483:Base Sequence; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005814:Genes, Viral; D009154:Mutation; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics* / Q000276:immunology; D011494:Protein Kinases / Q000235:genetics*; D011505:Protein-Tyrosine Kinases; D014764:Viral Proteins / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D014764:Viral Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D004262:DNA Restriction Enzymes