Characterization of middle T antigen expressed by using an adenovirus expression system.
The adenovirus Ad5(pymT) has been used to express middle T antigen at very high levels in 293 cells. The middle T antigen produced was localized to membranes and was modified in the same way as that expressed in polyoma virus-infected mouse cells. It was phosphorylated in vivo on serine residues and in vitro on tyrosine residues. The in vivo phosphorylations occurred between residues 223 and 275. The middle T antigen encoded by A d5(pymT) was phosphorylated in vitro in a complex with human pp60c-src. Interestingly, the extreme overexpression of middle T antigen did not cause a parallel increase in the amount of complex; most of the pp60c-src remained unassociated. Immunoaffinity purification resulted in approximately 100 micrograms of middle T antigen from a 100-mm tissue culture dish. Several cell proteins copurified with the Ad5(pymT)-derived middle T antigen. Two of these, the 74- and 63-kilodalton species, are of particular interest because they were also purified from mouse tumors expressing middle T antigen.
D000260:Adenoviruses, Human / Q000235:genetics*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics* / Q000302:isolation & purification; D002460:Cell Line; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000302:isolation & purification; D010766:Phosphorylation; D011120:Polyomavirus / Q000201:enzymology / Q000235:genetics*; D011494:Protein Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016392:Proto-Oncogene Proteins pp60(c-src); D014158:Transcription, Genetic
D000596:Amino Acids; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D009856:Oncogene Proteins, Viral; D011518:Proto-Oncogene Proteins; D011494:Protein Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)