The long term objective of this study is to identify the mechanism by Glycogen Synthase Kinase-3beta (GSK-3beta) and the ATM kinase activates the p53 protein following exposure to ionizing radiation. The hypothesis to be tested is that ionizing radiation activates p53 through consecutive phosphorylation of p53 by ATM and GSK-3beta.
3 specific aims will be undertaken. (1) We will determine if ATM regulates phosphorylation of serines 15 and 37 of p53. Using in vitro kinase assays, we will determine if purified ATM protein directly phosphorylates p53. AT cells will be transfected with ATM cDNA to determine if this restores the radiation-activation of the p53 protein. Phospho- specific antibodies which recognize serines in the N-terminal of p53 will be used to monitor p53 phosphorylation after ionizing radiation. (2) GSK-3beta phosphorylates serine 33 of p53 only if serine 37 is already phosphorylated. This phosphorylation increases the transcriptional activity of the p53 protein. GSK- 3beta binds to p53 phosphorylated at serine 37. The site of interaction between p53 and GSK-3beta will be identified using peptides, by PCR generated deletions of the p53 protein and by immunoprecipitation. The regulation of GSK-3beta kinase activity following irradiation will be examined. Cellular GSK-3beta kinase activity will be suppressed using dominant negatives to determine GSK-3beta's role in the activation of p53 by radiation. (3) Cell lines with mutations in the key p53 phosphorylation sites will be prepared using the inducible rTetR expression system. The role of N-terminal phosphorylation of p53 in regulation of p53 activation by Ionizing Radiation will be determined. An understanding of the mechanisms which help cells survive radiation, including the role of the ATM kinase in regulating the p53 response, may allow new therapeutic approaches to be applied to radiation therapy.
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