Characterization of protein kinase activities associated with p53-large-T immune complexes from SV40-transformed rat cells.
Interaction of viral oncoproteins, such as SV40 large T, with cellular growth suppressor proteins Rb and p53 is presumed to inactive or modulate their growth suppression functions, thereby leading to transformation. An additional transformation-related activity of LT leads to hyperphosphorylation of p53. To search for kinases that might be responsible for this effect, p53-LT complexes were immunopurified from different SV40-transformed rat cell lines and assayed for associated kinase activities, in vitro. Protein kinase activity was readily observed in p53-LT immunecomplexes from wild-type transformed cells but was low or undetectable in p53 from mutant-transformed or normal cells. Optimal activity required the presence of Mn++. p53 was phosphorylated at all sites found in vivo. In contrast, LT was phosphorylated only at a subset of formerly identified sites and at additional sites not seen in vivo. The p53-LT-kinase complex was assayed for the presence of casein kinases, cdk like kinases, or DNA-activated kinase, using specific effectors, antibodies, or purified enzymes as tools. DNA-activated kinase or cdc2/cdk2 were not detectable, although the purified enzymes phosphorylated p53 in vitro. Casein kinase 2 represented the major activity, which on p53 phosphorylated not only the C-terminal Ser390 but also several sites in the N-terminal region. One additional activity, not identified so far, may represent an LT-induced or activated kinase. This kinase seems to enhance overall phosphorylation of p53 and, perhaps other substrates, and may thereby contribute to transformation.
Institut für Genetik, Universität Bonn, Germany.
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E
Müller
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B
Boldyreff
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KH
Scheidtmann
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex / Q000378:metabolism*; D000952:Antigens, Polyomavirus Transforming / Q000276:immunology / Q000378:metabolism*; D016203:CDC2 Protein Kinase / Q000378:metabolism; D047388:Casein Kinases; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D051379:Mice; D010766:Phosphorylation; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D000952:Antigens, Polyomavirus Transforming; D004268:DNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; D047388:Casein Kinases; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase