Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen.
D2 hybrid T antigen is a protein closely related to simian virus 40 large T antigen and is synthesized in large quantities in cells infected with Ad2+D2, an adenovirus-simian virus 40 hybrid. We have analyzed the effects of phosphorylation on the structure and DNA binding of this protein. On nondenaturing pore-gradient gels, the purified protein migrated with an apparent molecular weight of 135,000, with a minor band at 330,000 molecular weight. In vitro phosphorylation catalyzed by the protein kinase activity associated with the protein resulted in a structural change so that most of it migrated with an apparent molecular weight of 740,000. Treatment of the phosphorylated form of the protein with alkaline phosphatase (which removed 95% of the phosphate) caused the disappearance of the 740,000-molecular-weight form and reappearance of the smaller forms. Partial tryptic digestion showed that D2 T antigen has two major regions of phosphorylation, only one of which was phosphorylated in vitro. The region phosphorylated in vitro was responsible for the aggregation of D2 T antigen and was tentatively assigned to the N-terminal part of the protein. As shown by protein blotting onto nitrocellulose filters, it was mainly the form of 740,000 molecular weight that bound to simian virus 40 DNA. However, sucrose gradient analyses showed that only a fraction of the in vitro-phosphorylated protein bound to DNA, suggesting that aggregation alone is not sufficient for binding.
D000260:Adenoviruses, Human / Q000276:immunology*; D000956:Antigens, Viral; D000957:Antigens, Viral, Tumor; D055598:Chemical Phenomena; D002621:Chemistry; D004279:DNA, Viral / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013539:Simian virus 40 / Q000276:immunology*
D000956:Antigens, Viral; D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D046911:Macromolecular Substances; D011494:Protein Kinases