12-O-tetradecanoylphorbol-13-acetate stimulates phosphorylation of the 58,000-Mr form of polyomavirus middle T antigen in vivo: implications for a possible role of protein kinase C in middle T function.
The 58,000-Mr form (58K form) of the polyomavirus middle T antigen (mT) is a minor species distinguished by its phosphorylation in vivo on serine and by its efficient phosphorylation on tyrosine in immune complexes (B.S. Schaffhausen and T.L. Benjamin, J. Virol. 40:184-196, 1981). Here we report that the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, rapidly stimulates phosphorylation of this mT species when added to cultures of wild-type polyomavirus-infected or polyomavirus-transformed 3T3 cells. Incubation with TPA leads to an accumulation of the 58K mT species to levels 1.5- to 5-fold higher than that in untreated cells within 15 min. TPA specifically stimulates phosphorylation of the 58K mT species without affecting that of the 56K species. Mapping by partial proteolysis shows that TPA-stimulated phosphorylation occurs at or near the site in 58K mT that is normally phosphorylated in the absence of TPA. A synthetic diacyl glycerol, 1-oleoyl-2-acetyl-glycerol, also specifically stimulates phosphorylation of 58K mT in vivo, while an inactive phorbol analog does not. TPA fails to induce phosphorylation of a 58K mT species encoded by certain nontransforming virus mutants with altered mT proteins that normally fail to undergo phosphorylation at the 58K site. These results indicate that the 58K form of mT is phosphorylated by or through the action of protein kinase C. TPA treatment of infected cells also leads to increased levels of 58K mT as measured in the immune complex kinase reaction, in which mT becomes phosphorylated on tyrosine by pp60c-src. These results are discussed in terms of a possible role for protein kinase C in activating mT function(s), including the formation of stable complexes with pp60c-src.
J T
JT
Matthews
T L
TL
Benjamin
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000378:metabolism*; D002460:Cell Line; D002472:Cell Transformation, Viral; D004075:Diglycerides / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009154:Mutation; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D011120:Polyomavirus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src); D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D004075:Diglycerides; D009856:Oncogene Proteins, Viral; D010704:Phorbols; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D012694:Serine; C038237:1-oleoyl-2-acetylglycerol; D016392:Proto-Oncogene Proteins pp60(c-src); D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate