The Role of Irradiation/ATM Activated Protein Phosphatase1 in the Damage Response

描述（由申请人提供）：已知治疗性辐照可诱导DNA损伤，从而激活多种信号通路、细胞周期检查点和基因转录。细胞周期检查点的诱导是细胞响应辐射的损伤和细胞毒性效应并阻止其生长的关键机制。新出现的证据表明，组蛋白去乙酰化酶1（HDAC 1）在Pol-II介导的基因转录调控中起着关键作用。蛋白磷酸酶-1（PP 1）是一种丝氨酸/苏氨酸蛋白磷酸酶，控制包括转录和细胞周期进程在内的多种细胞过程。我们实验室的新数据不仅将PP 1与转录调节因子HDAC 1联系起来，还将其与关键的细胞周期调节因子Rb（G1期进展）和Nek 2（调节中心体分离的关键激酶）联系起来。我们的数据清楚地表明：（1）在没有辐射的情况下，作为IR激活的ATM依赖性信号通路的结果，PP 1通过T320的去磷酸化而被激活，（2）PP 1与Rb和HDAC 1以及与Nek 2物理缔合以形成两个离散的复合物，（3）IR激活这两个复合物中的PP 1，（4）IR激活HDAC 1，并诱导Rb和HDAC 1从PP 1上解离;（5）IR通过PPI抑制Nek 2。 该项目的长期目标是通过描绘由PPI：RB：HDAC 1和PP 1：Nek 2组成的多蛋白复合物在诱导IR细胞周期阻滞和基因转录中的作用，开发化学/辐射增敏剂的新靶点。我们假设IR导致PPlc的T320磷酸化减少，导致PP 1的活化和Rb和HDAC 1从ATM依赖性过程中的预形成复合物中的释放以及Nek 2的抑制（已知PP 1是Nek 2的负调节剂）。IR激活的PP 1通过Rb和Nek 2的去磷酸化对检查点激活至关重要，并且通过其对HDAC 1激活的调节对基因转录至关重要。我们将通过执行以下具体目标来检验这一假设： 目标1。确定PP 1用于调节损伤反应的分子机制。 目标二。确定IR是否诱导从由Rb、PP 1和HDAC 1组成的多蛋白复合物中募集或解离蛋白质。 目标3：确定IR激活的PP 1在HDAC 1调节中的作用。

DESCRIPTION (provided by applicant): Therapeutic irradiation is known to induce DNA damage, which activates multiple signaling pathways, cell cycle checkpoints and gene transcription. Induction of a cell-cycle checkpoint is a critical mechanism by which cells respond to the damaging and cytotoxic effects of irradiation and arrest their growth. Emerging evidence suggests that histone deacetylase 1 (HDAC1) plays a critical role in the regulation of Pol-II mediated gene transcription. Protein phosphatase-1 (PP1) is a serine/threonine protein phosphatase that controls diverse cellular processes including transcription and cell cycle progression. New data from our laboratory link PP1 not only to the transcriptional regulator HDAC1 but also the key cell-cycle regulators Rb (G1 progression) and Nek2 (a key kinase regulating centrosome separation). Our data clearly establish that: (1) PP1 is activated by dephosphorylation of T320 as a result of an IR-activated, ATM-dependent signaling pathway in the absence of radiation, (2) PP1 physically associates with Rb and HDAC1 as well as with Nek2 to form two discrete complexes, (3) IR activates PP1 in both of these complexes, (4) IR activates HDAC1 and also induces dissociation of both Rb and HDAC1 from PP1, and (5) IR inhibits Nek2 through PPI. The long-term goal of this project is to develop novel targets for chemo/radiation sensitizers by delineating the role of the multiprotein complexes consisting of PPI:RB:HDAC1 and PP1 :Nek2 in the induction of IR cell-cycle arrest and gene transcription. We hypothesize that IR causes reduction in T320 phosphorylation of PPlc, resulting in activation of PP1 and release of Rb and HDAC1 from a pre-formed complex in an ATM-dependent process as well as inhibition of Nek2 (PP1 is known to be a negative regulator of Nek2). IR-activated PP1 is critical for checkpoint activation through dephosphorylation of Rb and Nek2 and gene transcription through its regulation of HDAC1 activation. We will test this hypothesis by performing the following specific aims: Aim 1. Identify the molecular mechanism used by PP1 to regulate the damage response. Aim 2. Determine if IR induces the recruitment or dissociation of proteins from the multiprotein complex consisting of Rb, PP1 and HDAC1. Aim 3. Determine the role of IR-activated PP1 in the regulation of HDAC1.