dCK与Cdk1相互作用对辐射诱导的细胞周期G2/M期检查点激活的调控机制

Deoxycytidine kinase(dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. Our preparatory work demonstrates that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR), and dCK interacts with cyclin-dependent kinase 1(Cdk1) after IR by means of mass spectrometry. In this study the colocalization of dCK and Cdk1 will be detected by immunofluorescence, the interaction between dCK and Cdk1 will be analyzed by GST-pull down and co-immunoprecipitation, the kinase activity assay in vitro will be used to verify the roles of dCK in phosphorylation of Cdk1, truncates analysis will be used to detect the binding sites between dCK and Cdk1 and the the relative mutants will be introduced to confirm the changes of binding activity. Finally the interaction between dCK and other regulators of Cdk1, such as weel, cdc25，cyclin B and pik1, will be detected by co-immunoprecipitation and Western blot. This study will clarify the new function of dCK in the regulation of G2/M checkpoint, the results will enrich the mechanism of cancer development and provide new treatment strategy for cancer.

脱氧胞苷激酶（dCK）是磷酸化天然脱氧核糖核苷酸和外源性核苷类抗肿瘤药物的关键酶，在DNA合成及药物活化方面起重要作用。我们前期研究首次发现电离辐射导致ATM激活后，dCK Ser74位点发生磷酸化，进而参与G2/M期检查点的调控，质谱分析表明周期蛋白依赖性激酶1（Cdk1）是dCK结合蛋白。本课题运用免疫荧光共定位、GST-Pulldown和 Co-IP方法验证dCK与Cdk1相互作用；体外激酶活性分析验证dCK磷酸化Cdk1；通过截短体实验确定dCK与Cdk1结合位点；通过对结合位点进行突变，观察dCK与Cdk1结合功能的变化；通过 Co-IP和 Western blot确定dCK与其它Cdk1调控蛋白的关系。本研究将揭示dCK参与调控细胞周期G2/M检查点的新功能，对丰富肿瘤发生机制、指导肿瘤治疗具有重要意义。

脱氧胞苷激酶（dCK）是磷酸化天然脱氧核糖核苷酸和外源性核苷类抗肿瘤药物的关键酶，在DNA合成及药物活化方面起重要作用，本研究主要探讨dCK参与调控细胞周期G2/M检查点以及细胞死亡的新功能。.研究发现：1）dCK基因沉默导致电离辐射诱导的DNA损伤增多、G2/M检查点缺失，提示dCK参与DNA 损伤后G2/M期检查点的激活；2）辐射诱导ATM+/+细胞GM0536发生dCK激活，而ATM-/-细胞GM1526则无此反应，沉默ATM后细胞对Ara-C抗性增加，提示dCK活性下降，激酶活性分析实验发现dCK Ser74被ATM直接磷酸化；3）dCK Ser-74磷酸化是辐射诱导G2/M检查点激活必需过程：将野生型wt、S74A、S74E表达载体分别转染到dCK或ATM沉默细胞中，发现野生型dCK可恢复dCK沉默所致的检查点缺失，野生型和S74E都可以恢复ATM沉默细胞中G2/M检查点的功能缺失；4）dCK与CDK1、mTOR三者在细胞内存在结合：免疫共沉淀结果发现dCK与CDK1有结合；进而构建不同的dCK和CDK1截短子检测二者的结合部位，结果发现dCK1-130和dCK96-253与CDK1均有不同程度结合，mTOR和dCK1-130有结合，与dCK96-253无结合，从而判定mTOR与dCK的结合区域在dCK1-96；dCK与野生型CDK1和CDK1-170均有结合，与CDK1-100无结合，从而确定了dCK与CDK100-170二者结合区域；5)dCK与CDK1其它调节蛋白的关系：磷酸化dCK-S74E显著增加G2 /M检查点回复的效应蛋白PIK1和细胞周期蛋白CDC25C表达，降低G2/M检查点抑制性激酶wee1和细胞周期蛋白Cyclin B1表达；6）沉默dCK基因增加Hela细胞的辐射敏感性，过表达dCK-WT和S74E能够挽救dCK基因沉默引起的细胞死亡；7)沉默dCK 促进电离辐射诱导细胞凋亡，过表达磷酸化 dCK则起抑制作用；dCK 可以增加辐射诱导的自噬，dCK S74位点的磷酸化参与了该过程；8）确定了dCK-mTOR-AKT、dCK-HSP90α通路参与了辐射诱导自噬的调控。.本研究揭示了dCK参与调控细胞周期G2/M检查点和细胞死亡的新功能，对丰富肿瘤机制有重要的意义，并对协同放化疗敏感性起指导作用。