Phosphorylation of polyomavirus large T antigen: effects of viral mutations and cell growth state.
Phosphorylation is responsible for the shift in electrophoretic mobility of polyomavirus large T antigen observed in pulse-chase or continuous-labeling experiments. Phosphorylated forms migrated more slowly than newly synthesized [35S]methionine large T antigen, and alkaline phosphatase treatment reversed the mobility shift. Analysis of phosphopeptides with Staphylococcus aureus V8 protease showed that large T antigen forms of intermediate mobility were enriched in peptides 1 to 4, 8, and 9, while the slower migrating species had all nine phosphopeptides, including peptides 5 and 7. The phosphorylations represented by phosphopeptides 5 and 7 were of particular interest. These phosphopeptides were entirely lacking in large T antigen from tsa mutants such as ts616 labeled at the nonpermissive temperature. Also, the phosphorylation of peptides 5 and 7 depends on the growth state of the cell. Early in infection of quiescent cells intermediate mobility forms of large T antigen with little or no phosphorylation, particularly of peptides 5 and 7, were seen, whereas peptides 5 and 7 were well represented at the same time in patterns from growing cells. Later in infection of growth-arrested cells, these phosphorylations were observed, suggesting that infection stimulates the relevant kinase. Because large T antigen of hrt mutants, which lack middle and small T antigens, showed phosphorylation of peptides 5 and 7, large T antigen was apparently responsible for the stimulation. Because some differences in the distribution of phosphopeptides were noted between hrt mutants and the wild type, middle T antigen, small T antigen, or both may play a modulating role in large T antigen phosphorylation.
D000469:Alkaline Phosphatase; D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics* / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D051379:Mice; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011120:Polyomavirus / Q000201:enzymology / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D009856:Oncogene Proteins, Viral; D011494:Protein Kinases; D000469:Alkaline Phosphatase