The Role of IR/ATM Activated PP1 in the Damage Response

描述（由申请人提供）：已知治疗性照射可诱导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。诱导细胞周期检查点是细胞对辐照的损伤和细胞毒性作用作出反应并阻止其生长的关键机制。新的证据表明，组蛋白去乙酰化酶1 （HDAC1）在调节Pol-II介导的基因转录中起着关键作用。蛋白磷酸酶-1 （PP1）是一种丝氨酸/苏氨酸蛋白磷酸酶，控制多种细胞过程，包括转录和细胞周期进程。我们实验室的新数据表明，PP1不仅与转录调节剂HDAC1有关，还与关键的细胞周期调节剂Rb （G1进展）和Nek2（调节中心体分离的关键激酶）有关。我们的数据清楚地表明：(1)在没有辐射的情况下，由于IR激活的atm依赖信号通路，PP1被T320的去磷酸化激活，(2)PP1与Rb、HDAC1以及Nek2物理结合形成两个独立的复合物，(3)IR激活这两个复合物中的PP1， (4) IR激活HDAC1并诱导Rb和HDAC1与PP1分离，(5)IR通过PPI抑制Nek2。

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.