PP2A regulates ionizing radiation–induced apoptosis through Ser46 phosphorylation of p53 Jun Mi, 1 Elzbieta Bolesta, 1 David L. Brautigan, 2 and James M. Larner 1 1 Department of Radiation Oncology and 2 Center for Cell Signaling, University of Virginia Health System, Charlottesville, Virginia Abstract In response to ionizing radiation, p53 plays a critical role in regulating DNA repair and apoptosis. Among multiple phosphorylation sites, evidence suggests that Ser46 promotes apoptotic cell death through mitochondrial outer membrane permeabilization (MOMP) and subsequent activation of the caspase 7–PARP pathway. Therefore, we investigated which phosphatase regulates Ser46 after ionizing radiation, reasoning that the responsible phosphatase should be a target for radiosensitization. We determined that both inhibition of PP2A by the cell-permeable inhibitor calyculin A and knockdown of PP2A by RNAi (a ) enhanced Ser46 phosphorylation in p53 and (b ) induced coincident caspase 7 and PARP cleavage in response to ionizing radiation. Furthermore, mutation of p53 Ser46 to Ala attenuated ionizing radiation–induced apoptotic signaling. Consequently, we concluded that PP2A regulates ionizing radiation–induced apoptotic signaling through dephosphorylation of p53 Ser46. [Mol Cancer Ther 2009;8(1):135–40] Introduction Apoptosis is a form of programmed cell death required for embryonic development and maintenance of tissue homeostasis, as well as for the removal of damaged cells. In response to DNA damage, apoptosis signaling is triggered by the tumor suppressor p53, which is a critical regulator of the apoptosis pathway and DNA repair signaling. p53 contains multiple serine (6, 9, 15, 20, 33, 37, 46, 315, 371, 376, 392) and threonine residues (18, 55, 81), which are phosphorylated in response to irradiation (1). Regulation of p53 by phosphorylation causes its stabilization and activation, which in turn, trigger p53-dependent transcrip- tion, growth arrest, and/or apoptosis. Posttranslational modifications of p53, including acetylation and phosphorylation, are critical in regulating radiation-induced apoptosis (2). The role of Ser46 phosphorylation in regulating gene transcription is controversial. Thompson et al. (3) have shown that p53 Ser46 phosphorylation is dispensable for transcriptional activation. However, the p53 Ser46 site is critical for apoptosis. The precise molecular mechanism by which p53 Ser46 regulates apoptosis is unknown. However, phosphorylation of p53 Ser46 is thought to promote apoptotic cell death through mitochondrial outer mem- brane permeabilization (MOMP) and subsequent activation of the caspase 7–PARP apoptotic cascade. In response to ionizing radiation, Ser46 phosphorylation promotes the expression of p53-regulated apoptosis-inducing protein 1 and its migration into the mitochondria (4). P53-regulated apoptosis-inducing protein 1 promotes the degradation of mitochondrial DCm and release of cytochrome c , causing apoptosis. p53 may select G 1 arrest pathways or apoptosis through this mechanism (5). Research continues to identify multiple kinases that phosphorylate Ser46. The ataxia-telangiectasia mutated gene (ATM) is a pivotal regulator of genotoxic stress response and directly or indirectly phosphorylates numer- ous downstream targets, including p53. Certain research indicates that the homeodomain-interacting protein kinase 2, a nuclear serine/threonine kinase, phosphorylates p53 Ser46 in response to UV-induced DNA damage. Depletion of homeodomain-interacting protein kinase 2 by small interfering RNA and inactivation of homeodomain- interacting protein kinase 2 by kinase-altering mutations attenuates the UV-induced phosphorylation of p53 Ser46, which is ATM-dependent (5, 6). However, other research indicates that dual-specificity tyrosine phosphorylation– regulated kinase 2 phosphorylates p53 Ser46 (1, 5) in vitro in response to Adriamycin (a genotoxic stress agent) treatment (7). These observations indicate that different kinases phosphorylate Ser46 in response to different types of DNA damage. Dephosphorylation of key sites on p53 limits apoptosis. The phosphatase responsible for dephosphorylation of the Ser46 site is unknown; one candidate, PP2A, is a major serine/threonine phosphatase involved in DNA damage signaling. For example, PP2A dephosphorylates Thr68 of checkpoint kinase 2 (8) and Ser1981 of ATM (9), both of which are phosphorylated in response to ionizing radia- tion. We have also shown that PP2A is regulated by ionizing radiation in an ATM-dependent manner (10–12). In our study, we investigated if PP2A (a ) is involved in the dephosphorylation of p53 Ser46 in response to ionizing radiation and (b ) if it triggers apoptotic signaling. Received 5/12/08; revised 9/16/08; accepted 10/26/08. Grant support: NIHgrantsES011975(J.M.Larner)andGM-56362 (D.L.Brautigan),andtheCharlesR.Burnett,Jr.andW.GriffinBurnett Fund. Thecostsofpublicationofthisarticleweredefrayedinpartbythe paymentofpagecharges.Thisarticlemustthereforebeherebymarked advertisement inaccordancewith18U.S.C.Section1734solelyto indicate this fact. Requests for reprints: James M. Larner, University of Virginia Health System, Box 800383 Charlottesville, VA. Phone: 434-924-5564; Fax: 434-243-9789. E-mail: jml2p@virginia.edu Copyright C 2009 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0457 135 Mol Cancer Ther 2009;8(1). January 2009 on May 29, 2020. © 2009 American Association for Cancer Research. mct.aacrjournals.org Downloaded from