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

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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.