DETERMINATION OF P53 PHOSPHORYLATION AND IDENTIFICATION OF P53-BINDING PROTEINS

(1)X射线照射正常人胚细胞P_（53）积聚的研究本文报道了不同剂量X射线照射正常人胚细胞P_（53）积聚的动力学。照射后1小时检测到P_（53）水平升高，剂量大于4戈伊时为双相蓄积，剂量小于2戈伊时为单相蓄积。我们观察到P53在链溶素-O的帮助下被引入细胞的限制酶PVU II积聚。这些结果表明DNA双链断裂是P53蛋白积聚的触发因素。(2)ATM引起的P53位点特异性磷酸化由于P53蛋白在AT细胞中无明显蓄积，推测ATM蛋白参与了电离辐射引起的P53磷酸化反应。我们用多克隆抗体抗P53丝氨酸15位磷酸化，发现AT细胞中P53丝氨酸15位磷酸化缺乏。使用DNA-纤维素下拉试验，我们表明ATM蛋白质被标记为DNA双链断裂。(3)用蛋白酶体抑制剂ALLN处理细胞，发现P53在对照状态下是普遍存在的。我们发现，P53在丝氨酸15处磷酸化对X射线无反应，然而，磷酸化的P53也是普遍存在的，这表明P53在丝氨酸15处的磷酸化通过抑制蛋白酶体识别来抑制P53降解。

(1) P53 ACCUMULATION IN X-IRRADIATED NORMAL HUMAN EMBRYONIC CELLS.THE KINETICS OF P53 ACCUMULATION IN NORMAL HUMAN EMBRYO CELLS IRRADIATED WITH VARIOUS DOSES OF X-RAYS WAS EXAMINED. THE INCREASE OF THE P53 LEVEL WAS DETECTED 1 HOUR AFTER IRRADIATION, AND THE ACCUMULATION WAS BI-PHASIC WITH DOSES MORE THAN 4 GY, WHEREAS THE ACCUMULATION WAS MONO-PHASIC WITH DOSES LESS THAN 2 GY. WE OBSERVED THAT P53 WAS ACCUMULATED WITH RESTRICTION ENZYME PVU II INTRODUCED INTO CELLS BY THE AID OF STREPTLYSIN-O. THESE RESULTS INDICATE THAT DNA DOUBLE STRAND BREAKS ARE THE TRIGGER FOR ACCUMULATION OF P53 PROTEIN.(2) SITE-SPECIFIC PHOSPHORYLATION OF P53 BY ATMBECAUSE P53 PROTEIN DOES NOT ACCUMULATED SIGNIFICANTLY IN AT CELLS, ATM PROTEIN IS SUGGESTED TO BE INVOLVED IN P53 PHOSPHORYLATION IN RESPONSE TO IOZING RADIATION. HERE WE HAVE USED POLYCLONAL ANTIBODY AGINST PHOSPHORYLATED P53 AT SERINE 15, AND FOUND THAT PHSOPHORYLATION OF P53 AT SERINE 15 WAS DEFICIENT IN AT CELLS. USING DNA-CELLULOSE PULL-DOWN ASSAY, WE SHOWED THAT ATM PROTEIN WAS RECRUITED TO DNA DOUBLE STRAND BREAKS.(3) SITE-SPECIFIC PHOSPHORYALTION AND REGULATION OF P53 STABILITYTREATMENT OF CELLS WITH THE PROTEASOME INHIBITOR, ALLN, REVEALED THAT P53 WAS UBIQUITINATED UNDER THE CONTROL STATE. WE FOUND THAT P53 WAS PHOSPHORYLATED AT SERINE 15 INRESPONSE TO X-RAYS, HOWEVER, PHOSPHORYLATED P53 WAS ALSO UBIQUITINATED, SUGGESTING THAT PHOSPHORYLATION OF P53 AT SERINE 15 SUPPRESSED P53 DEGRADATION THROUGH AN INHIBITION OF PROTEASOME RECOGNITION.