ATM介导自噬分子Beclin1磷酸化修饰的新功能解析

自噬在肿瘤的发生发展中起着重要作用，Beclin1是参与自噬调控的中心分子,它与III型PI3K（hVsp34）形成复合物在自噬体形成中起着关键作用。我们近期研究发现，抑制肿瘤细胞内ATM蛋白激酶活性能够促进细胞自噬发生；通过生物信息学分析发现在Beclin1蛋白序列中存在ATM的潜在磷酸化位点,并可能同时也是14-3-3蛋白的结合位点；免疫共沉淀实验显示ATM的活化可以促进ATM和Beclin1的结合。基于此,我们提出了科学假设: ATM可能磷酸化Beclin1,14-3-3蛋白则与磷酸化后的Beclin1结合,从而抑制Beclin1通过调节hVsp34所介导的细胞自噬,以实现ATM对细胞自噬的负调控。本课题将应用透射电镜、免疫荧光、免疫共沉淀、GST pull-down、体外激酶活性分析等技术来验证这一科学假说，将为以Beclin1为核心的自噬调控的分子机制提供新的认识。

Autophagy plays an important role in the occurrence and development of malignant tumor, and the Beclin1 is a key molecule which involved in the regulation of autophagy. The complex of Beclin1 and III type PI3K (hVsp34) plays a critical role in autophagosome formation. Our recent study found that, inhibtion of the activity of ATM could induce the autophagy in cancer cells. By the bioinformatics analysis, it is revealed the presence of potential ATM phosphorylation motif in the Beclin1 protein, and this motif may be the binding site of 14-3-3 protein which is the important serine/threonine phosphorylation binding-protein. Our experimental results of co-immunoprecipitation showed that the activation of ATM could increase the combination of ATM and Beclin1 in cancer cells. Thus,our hypothesized that, ATM may be negatively regulated the autophagy through phosphorylation on Beclin1, and induce the binding of 14-3-3 protein with this phosphorylated motif, then inhibit the hVsp34 activity and autophagy.In this project, we will confirm this hypothesis by using transmission electron microscopy, immunofluorescence, co-immunoprecipitation, GST pull-down and in vitro kinase activity analysis etc.It would provide a new understanding of the molecular mechanism for the regulation of autophagy through Beclin1.