DNA损伤生物学反应中ATM对p21^(WAF1/CIP1)蛋白的直接磷酸化

Direct Phosphorylation of p21^(WAF1/CIP1) with ATM in DNA Damage Response

毛细血管扩张性共济失调症突变蛋白 (mutatedinataxiatelangiectasia ,ATM)是直接感受DNA双链断裂损伤并起始诸多DNA损伤信号反应通路的主开关分子 .已有研究发现 ,DNA损伤生物学反应中 ,ATM可通过磷酸化活化p5 3,继而转录活化细胞周期检查点蛋白p2 1WAF1 CIP1的表达 ,而对于ATM是否直接参与p2 1WAF1 CIP1的早期活化迄今尚无实验证明 .通过免疫共沉淀反应 ,检测到细胞电离辐射 (ionizingradiation ,IR)反应早期ATM与p2 1WAF1 CIP1蛋白存在相互作用 .将p2 1WAF1 CIP1蛋白编码基因全长克隆入原核表达载体pGEX4T 2 ,经诱导表达及亲和层析纯化获取GST p2 1融合蛋白作为磷酸化底物 .体外磷酸化实验检测证明 ,IR活化的ATM具磷酸化p2 1WAF1 CIP1蛋白的功能 ,并且此磷酸化功能可被PI3K家族特异性抑制剂Wortmannin所抑制 .结果揭示了IR后ATM可通过直接磷酸化p2 1WAF1 CIP1蛋白 ,在IR致DNA损伤生物学反应早期调控p2 1WAF1

The ATM (mutated in ataxia telangiectasia) regulates the cell DNA damage response by phosphorylation of a series of proteins involved in cell checkpoints, DNA repair and apoptosis. It has been demonstrated that in DNA damage response, ATM was a bona fide protein kinase that was capable of phosphorylating and activating transcriptional activity of p53, and then resulting in transcriptional activation of p21 WAF1/CIP1 . To test whether ATM could phosphorylate and regulat p21 WAF1/CIP1 directly, the interaction between ATM and p21 WAF1/CIP1 was tested by coimmunoprecipitation. The interaction between these two proteins was identified in HeLa cells pretreated with 10 Gy γ radiation. Full length p21 WAF1/CIP1 gene was cloned into prokaryotic expression vector pGEX4T 2. The recombinant protein GST p21 was expressed and purified by affinity chromatography. ATM was immunoprecipitated from HeLa cells treated with 10 Gy γ radiation.The results of in vitro phosphoryiation assay showed that GST p21 was phosphorylated by ATM, and this phosphorylation reaction could be inhibited by Wortmannin. These results suggested that besides p53 induced transcriptional activation, p21 WAF1/CIP1 could be phosphorylated by ATM directly in cell treated with γ radiation,and this phosphorylation might participate in p21 activation in early stage of DNA damage response induced by IR .