蛋白磷酸酶5(PP5)对p53功能的调节及其在p53相关肿瘤发生中作用机理的研究

The p53 tumor suppressor plays a central role in protecting mammalian cells from neoplasia and tumorigenesis in response to various cellular stresses. In unstressed cells, p53 is maintained at lower levels as a result of MDM2-mediated ubiquitination and degradation. When cells are confronted by genotoxic stress, kinases, e.g. ATM/ATR and Chk1/2 become activated to phosphorylate p53 and/or MDM2. These phosphorylations, along with other post-translational modifications (e.g. acetylation and methylation), inhibit MDM2-mediated p53 degradation and subsequently induce p53 level and activity. Dephosphorylation plays an opposite role in regulating p53 function. Protein phosphatase 5 (PP5 or Ppp5) has been shown in regulating cellular growth, proliferation, differentiation, migration, and survival in response to various stresses using cultured cells. We have generated PP5-deficient mice. Our initial analyses of PP5-null cells reveal that p53 stability and activity are upregulated. In vitro biochemical analyses further demonstrate that PP5 is able to directly interact with p53 and dephosphorylate phosphop53 at multiple sites. Also, two potential p53 responsive DNA sequences have been identified in the PP5 promoter and intron 1 regions. Thus, we hypothesize that PP5 may serve as an important negative feedback serine/threonine phosphatase in regulating p53 activity. To test this hypothesis, we propose two specific aims: 1) We will determine if PP5 plays a role in p53-related tumorigenesis employing genetically manipulated mouse models.2）We will determine if PP5 acts as a feedback phosphatase for regulating p53 activity using biochemical approaches; 3)We will screen PP5 target small molecular drugs. This proposed study will unveil a novel role for PP5 in regulating the p53 pathway via a negative feedback mechanism in mammalian cells. In addition, our animal work will shed light on the biological role of PP5 in regulating p53 function as well as in p53-related tumorigenesis. The information gained from this study will also provide a molecule target for anti-cancer drug development

p53蛋白磷酸化和去磷酸化的协调作用在p53的稳定性和转录活性调控方面起着重要作用。相对于磷酸化，p53的去磷酸化研究相对较为薄弱。蛋白磷酸酶5（PP5）是一个广泛表达的丝氨酸/苏氨酸磷酸酶。通过创建PP5基因敲除小鼠模型，我们发现PP5 KO小鼠中，p53的总蛋白和磷酸化蛋白水平显着升高，体外生化分析证明PP5能够在多个位点使p53去磷酸化，此外还发现在PP5基因中有两个p53结合位点。本项目将通过已有的PP5基因敲除和新创建的PP5过表达小鼠模型与p53基因敲除小鼠杂交，研究PP5在p53相关肿瘤发生中的作用，同时结合体外生化试验，从体内、体外两个层面验证我们的假说"p53介导的细胞信号和肿瘤发生受控于PP5活性，而且p53能够反馈调节PP5的表达，二者之间存在一个反馈调控环路"。在此基础上通过筛选抑制和激活PP5靶点的小分子，为探寻p53相关肿瘤的发生、预防和治疗提供新的思路。

利用PP5和p53 KO小鼠动物模型，分析PP5或p53 KO后不同组织p53或PP5的表达情况。分离野生型和PP5 KO小鼠原代成纤维细胞，用实时定量PCR和免疫印迹分析p53及p53调节基因的表达情况，研究PP5对p53功能的调节。分离野生型和PP5 KO原代骨髓低密度单核细胞，用流式细胞仪分析遗传毒性试剂DOX处理后PP5 KO对细胞凋亡的影响。用逆转录病毒pMSCV-sh-p53介导的RNA干扰下调PP5 KO原代骨髓低密度单核细胞p53的表达来研究p53在DOX处理后对细胞凋亡的影响，通过这些研究结果分析p53在PP5生物功能中的作用。组织化学研究PP5 KO小鼠模型和野生对照小鼠在DOX给药后心脏病理的变化，进一步分析在病理上p53对PP5功能的调节作用。体外用HEK293T细胞系进行PP5对p53的去磷酸化分析。体外用PP5对DNA-PK磷酸化的p53进行去磷酸化分析，研究PP5是否可以直接使p53去磷酸化。利用H1299细胞系共转染p53和PP5表达质粒，用免疫共沉淀（Co-IP）和GST pull-down技术研究PP5和p53的相互作用。用H1299细胞系进行染色质共沉淀（ChIP）分析p53和PP5启动子区保守结合位点的结合情况，并进一步用荧光素酶报告基因分析p53对PP5表达情况的影响。PP5 KO小鼠不同组织p53的蛋白表达水平及下游调节基因表达上调，相似的结果也从原代分离的骨髓低密度单核细胞和原代成纤维细胞中得到，而p53 KO小鼠不同组织及分离的原代成纤维细胞中PP5蛋白表达水平也上调。遗传毒性试剂DOX对PP5 KO骨髓低密度单核细胞处理及PP5 KO小鼠给药的心脏病理学研究表明PP5 KO细胞细胞凋亡增加、心脏病理加重，且PP5的功能由p53介导。免疫共沉淀和GST pull-down分析表明PP5和p53有一个直接的相互作用，磷酸化和去磷酸实验分析表明PP5可以使p53去磷酸化。染色共沉淀技术分析表明p53直接和PP5基因启动子区的保守结合位点相结合，而荧光素酶报告基因进一步分析表明p53对PP5基因表达具有抑制作用。结论：PP5可以和p53直接结合，调节p53的稳定性和活性，而且p53也参与PP5的相关生物学功能，另一方面p53可以和PP5启动子区的保守结合位点结合抑制PP5基因的表达。