DETERMINATION OF P53 PHOSPHORYLATION AND IDENTIFICATION OF P53-BINDING PROTEINS

(1) p53在x射线照射的正常人胚胎细胞中积累。研究了不同剂量x射线照射下正常人胚胎细胞中p53积累的动力学。照射1 h后p53水平升高，且剂量大于4 gy时呈双相积累，剂量小于2 gy时呈单相积累。我们观察到p53是通过链溶酶o引入细胞内的限制性内切酶pvu ii积累的。这些结果表明DNA双链断裂是p53蛋白积累的触发因素。(2) ATM对p53的位点特异性磷酸化，由于p53蛋白在at细胞中没有明显的积累，因此ATM蛋白可能参与了电离辐射对p53的磷酸化反应。在这里，我们使用了针对p53丝氨酸15磷酸化的多克隆抗体，发现p53丝氨酸15磷酸化在细胞中是缺乏的。通过DNA-纤维素拉下实验，我们发现ATM蛋白被招募到DNA双链断裂中。(3)用蛋白酶体抑制剂alln对细胞进行位点特异性磷酸化和p53稳定性调控，发现p53在控制状态下泛素化。我们发现，在x射线作用下，p53在丝氨酸15处被磷酸化，然而，磷酸化的p53也被泛素化，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的降解。

(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.