RADIATION MODULATION OF THE P53 GENE

这项研究的长期目标是确定糖原合成酶-3β(GSK-3β)和ATM激酶在电离辐射后激活P53蛋白的机制。需要检验的假设是，电离辐射通过ATM和GSK-3β对P53的连续磷酸化来激活P53。将采取3个具体目标。(1)我们将确定ATM是否调节P53的丝氨酸15和37的磷酸化。我们将使用体外激酶分析来确定纯化的ATM蛋白是否直接磷酸化P53。AT细胞将被转染ATM基因，以确定这是否恢复了P53蛋白的辐射激活。识别P53 N-末端丝氨酸的磷酸特异性抗体将用于监测电离辐射后P53的磷酸化。(2)只有当P53的丝氨酸37已经被磷酸化时，GSK-3β才能使P53的丝氨酸33磷酸化。这种磷酸化增加了P53蛋白的转录活性。GSK-3β与丝氨酸37位磷酸化的P53结合。P53和GSK-3β之间的相互作用部位将通过多肽、通过聚合酶链式反应产生的P53蛋白缺失和免疫沉淀来确定。辐射后GSK-3β激酶活性的调节将被检测。细胞GSK-3β激酶活性将被抑制，利用显性负值来确定GSK-3β在辐射激活P53中的作用。(3)利用可诱导的rTetR表达系统制备p53关键磷酸化位点突变的细胞系。P53的N-末端磷酸化在电离辐射调节P53激活中的作用将被确定。了解帮助细胞在辐射中存活的机制，包括ATM激酶在调节P53反应中的作用，可能会使新的治疗方法应用于放射治疗。

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.