RADIATION MODULATION OF THE P53 GENE

本研究的长期目标是确定暴露于电离辐射后糖原合成酶激酶-3 β（GSK-3 β）和ATM激酶激活p53蛋白的机制。 待检验的假设是电离辐射通过ATM和GSK-3 β连续磷酸化p53激活p53。 将实现三个具体目标。(1)我们将确定ATM是否调节p53的丝氨酸15和37的磷酸化。 使用体外激酶测定，我们将确定纯化的ATM蛋白是否直接磷酸化p53。用ATM cDNA转染AT细胞，以确定这是否恢复了p53蛋白的辐射活化。 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的p53磷酸化。 (2)GSK-3 β仅在p53的丝氨酸37已被磷酸化的情况下磷酸化丝氨酸33。 这种磷酸化增加了p53蛋白的转录活性。 GSK-3 β与丝氨酸37处磷酸化的p53结合。 p53和GSK-3 β之间的相互作用位点将使用肽、通过PCR产生的p53蛋白缺失和通过免疫沉淀来鉴定。 将检查照射后GSK-3 β激酶活性的调节。 细胞GSK-3 β激酶活性将使用显性阴性来抑制，以确定GSK-3 β在辐射激活p53中的作用。(3)将使用诱导型rTetR表达系统制备在关键p53磷酸化位点具有突变的细胞系。 将确定p53的N-末端磷酸化在电离辐射调节p53活化中的作用。 了解有助于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.