DETERMINATION OF P53 PHOSPHORYLATION AND IDENTIFICATION OF P53-BINDING PROTEINS

P53 磷酸化的测定和 P53 结合蛋白的鉴定

• 批准号: 10680518
• 负责人: SUZUKI Keiji
• 金额: $ 2.11万
• 依托单位: NAGASAKI UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10680518/
• 关键词: IONIZING RADIATION; PHOSPHORYLATION; P53; ATM; SIGNAL TRANSDUCTION; MDM2; 分子生物学; 放射線生物学

(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.

(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。

项目成果

Keiji Suzuki: "Suppressive effect of low-close preirradiation on genetic instability induced by X rays in normal human embryonic cells" Radiation Research. 150. 656-662 (1998)

Keiji Suzuki：“低近距离预照射对正常人胚胎细胞中 X 射线诱导的遗传不稳定性的抑制作用”辐射研究。

Jagadish C.Ghosh,et al.: "Effects of protein kianse inhibitors on the accumulation kinetics of p53 protein in normal human embryo cells following X-irradiation"J.Radiat.Res.. 40(1). 23-37 (1999)

Jagadish C.Ghosh 等人：“蛋白激酶抑制剂对 X 射线照射后正常人胚胎细胞中 p53 蛋白积累动力学的影响”J.Radiat.Res.. 40(1)。

Jagadish C.Ghosh,et al.: "Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation"Radiat.Res.. (2000)

Jagadish C.Ghosh 等人：“X 照射后正常人胚胎细胞中 TP53 蛋白的剂量依赖性双相积累”Radiat.Res.. (2000)

Keiji Suzuki, et al.: "Recruitment of ATM protein to double strand DNA irradiated with ionizing radiation"J. Biol. Chem.. 274(36). 25571-25575 (1999)

Keiji Suzuki 等人：“将 ATM 蛋白招募到电离辐射照射的双链 DNA 中”J.

Jagadish C. Ghosh, et al.: "Dose-dependent biphasic accumulation of TP53 protein in normal human embryo cells after X irradiation"Radiat. Res.. (2000)

Jagadish C. Ghosh 等人：“X 辐射后正常人胚胎细胞中 TP53 蛋白的剂量依赖性双相积累”Radiat。