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Characterization of protein kinase activities associated with p53-large-T immune complexes from SV40-transformed rat
E Müller1, B Boldyreff, K H Scheidtmann
1Institut für Genetik, Universität Bonn, Germany.
Abstract:
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.
Insights
Viral oncoproteins like SV40 large T antigen (LT) interact with tumor suppressors. This study identifies casein kinase 2 as a major kinase activity in p53-LT complexes, contributing to cell transformation.
Area of Science:
- Molecular Biology
- Virology
- Oncology
Background:
- Viral oncoproteins, such as SV40 large T antigen (LT), interact with cellular tumor suppressor proteins like Rb and p53.
- This interaction is believed to inactivate or modulate the tumor suppressors' functions, promoting cellular transformation.
- LT possesses an additional transformation-related activity involving the hyperphosphorylation of p53.
Purpose of the Study:
- To identify the specific kinases responsible for the hyperphosphorylation of p53 induced by SV40 large T antigen (LT).
- To investigate the kinase activities associated with p53-LT complexes in SV40-transformed cells.
- To elucidate the role of these kinases in the transformation process.
Main Methods:
- Immunopurification of p53-LT complexes from SV40-transformed rat cell lines.
- In vitro kinase assays using immunoprecipitated complexes.
- Phosphorylation site analysis of p53 and LT.
- Characterization of associated kinase activities using specific inhibitors, antibodies, and purified enzymes (e.g., casein kinase 2, DNA-activated kinase, cdc2/cdk2).
Main Results:
- Significant protein kinase activity was detected in p53-LT immune complexes from wild-type transformed cells, but not from mutant or normal cells.
- Casein kinase 2 was identified as the major kinase activity within the p53-LT complexes, phosphorylating multiple sites on p53.
- An additional, unidentified kinase activity was observed, potentially LT-induced or activated, which enhances p53 phosphorylation and may contribute to transformation.
Conclusions:
- Casein kinase 2 is a key component of the p53-LT kinase complex and contributes to p53 hyperphosphorylation.
- An uncharacterized kinase activity associated with LT may also play a role in enhancing p53 phosphorylation and promoting cellular transformation.
- Understanding these kinase interactions is crucial for deciphering the mechanisms of viral oncogenesis.