Inhibition of Aurora-B kinase activity by poly(ADP-ribosyl)ation in response to DNA damage Lucia Monaco*†, Ullas Kolthur-Seetharam*†, Romain Loury*, Josiane Me´ nissier-de Murcia‡, Gilbert de Murcia‡, and Paolo Sassone-Corsi*§ *Institut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Centre National de la Recherche Scientifique, Institut National de la Sante´et de la Recherche Me´dicale, Universite´Louis Pasteur, B.P. 10142, 67404 Illkirch, Strasbourg, France; and ‡Ecole Supe´rieure de Biotechnologie de Strasbourg, Unite´9003 du Centre National de la Recherche Scientifique, Boulevard Se´bastien Brant, B.P. 10413, 67412 Illkirch Cedex, France Communicated by Pierre Chambon, Institut de Ge´ne´ tique et de Biologie Mole´culaire et Cellulaire, Strasbourg, France, July 22, 2005 (received for review July 1, 2005) The cell cycle-regulated Aurora-B kinase is a chromosomal passen- and cell death. The identification of specific mediators of DNA ger protein that is implicated in fundamental mitotic events, damaging signals, such as ATM (ataxia telangiectasia mutated) including chromosome alignment and segregation and spindle and ATR (ATM- and Rad3-related) (24), and the deciphering of checkpoint function. Aurora-B phosphorylates serine 10 of histone how they influence the progression through the cell cycle has H3, a function that has been associated with mitotic chromatin greatly improved our understanding of the cellular response to condensation. We find that activation of poly(ADP-ribose) poly- genotoxic agents. merase (PARP) 1 by DNA damage results in a rapid block of H3 Poly(ADP-ribosyl)ation is an immediate cellular response to ϩ phosphorylation. PARP-1 is a NAD؉-dependent enzyme that plays DNA strand breaks that is catalyzed by NAD -dependent a multifunctional role in DNA damage detection and repair and enzymes, poly(ADP-ribose) polymerases (PARPs) (25). PARPs maintenance of genomic stability. Here, we show that Aurora-B catalyze the stepwise addition of ADP-ribose moeities to sub- ϩ physically and specifically associates with the BRCT (BRCA-1 C- strate proteins by using intracellular NAD as source of ADP- terminal) domain of PARP-1. Aurora-B becomes highly poly(ADP- ribose. The PARP family contains several members, although ribosyl)ated in response to DNA damage, a modification that leads Ͼ90% of cellular poly(ADP-ribosyl)ation is ascribed to PARP-1 to a striking inhibition of its kinase activity. The highly similar (26), the most active and best-characterized member of the Aurora-A kinase is not regulated by PARP-1. We propose that the PARP family. It is a nuclear protein with DNA damage scanning specific inhibition of Aurora-B kinase activity by PARP-1 contributes activity that is implicated in the maintenance of genomic integ- to the physiological response to DNA damage. rity (27–29), leading to the control of cellular proliferation and carcinogenesis (30). mitosis ͉ poly(ADP-ribose) polymerase-1 ͉ histone H3 ͉ phosphorylation Here, we report that mitotic P-H3͞Ser-10 phosphorylation is drastically reduced upon DNA damage. This event is coupled to the inhibition of the Aurora-B kinase by PARP-1-mediated itosis is a highly orchestrated process that entails a pleth- poly(ADP-ribosyl)ation. Our findings establish an intriguing link ora of control mechanisms. Signaling events that coordi- M among DNA damage, chromatin modifications, and regulation nate mitosis induce a wave of protein phosphorylation governed of mitotic events. The NADϩ dependence of PARP-1 enzymatic by the cyclin-dependent kinase Cdc2. After Cdc2 activation, activity may extend the physiological implications of these downstream mitotic controllers are recognized within the fam- observations to the control of cellular metabolism. ilies of Aurora, Polo, and NIMA-related kinases (1). Mitotic ͞ phosphorylation of histone H3 at serine 10 (P-H3 Ser-10) Materials and Methods occurs highly synchronously and is associated with the initiation Cell Cultures. NIH 3T3 mouse fibroblast cells were cultured in of chromosome condensation (2, 3). The kinase implicated in DMEM supplemented with 10% serum and treated with H2O2 this event is Aurora-B, one of the three members of the Aurora (1 mM) for different times. After incubation, cells were collected family of kinases (4–6). and lysed in Laemmli buffer; equal amounts of proteins, deter- The cell cycle-regulated Aurora-B kinase is a chromosomal mined by Coomassie staining, were loaded onto a SDS- passenger protein that has been shown to play essential roles in acrylamide gel and processed for Western blot analysis. When mitosis (7–9). A number of critical proteins have been shown to the PARP-1 inhibitor 3-aminobenzamide (3-AB) (Sigma) was be substrates of Aurora-B, including the myosin II regulatory used, a 3-h pretreatment was performed before adding H2O2. light chain, vimentin, desmin, and GFAP (glial fibrillary acidic Mouse embryonic fibroblasts (MEFs) were isolated by micro- protein) (10–12), which suggests a role for Aurora-B at the dissection of embryos at 13.5 days of gestation resulting from cleavage furrow during cytokinesis. Other targets of Aurora-B in intercrosses between wild-type or mutant mice. The spermato- cytokinetic processes are INCENP (inner-centromere protein) gonial cell line GC-spg1 was purchased from American Type ͞ and ZEN-4 CeMKLP1, as reported in Caenorhabditis elegans Culture Collection. For immunofluorescence analysis, cells were and mammalian cells (13, 14). Interference of Aurora-B activity fixed with 4% paraformaldehyde in PBS, permeabilized with causes defects in chromosome congression because of its in- 0.2% Triton X-100, blocked with 5% BSA, and then incubated volvement in the regulation of the kinetochore–microtubule overnight with primary antibodies, washed, and revealed with interactions (15–17). secondary FITC- or Cy3-conjugated antibodies. Treatment of Aurora-B and its related kinase Aurora-A (7) are overexpressed cells with 0.1 ␮g͞ml nocodazole was extended for various periods in a number of human tumors, and their ectopic overexpression in as indicated. cultured cells results in cellular transformation, centrosome abnor- malities, and aneuploidy (18–21). Failure of mitotic chromosome segregation leads to aneuploidy that may contribute to cancer Abbreviations: PARP, poly(ADP-ribose) polymerase; MEF, mouse embryonic fibroblast; onset (22). Therefore, identification of the pathways that control BRCT, BRCA-1 C-terminal; 3-AB, 3-aminobenzamide; IR, ionizing radiation. Aurora-B function is of central importance. †L.M. and U.K.-S. contributed equally to this work. Genotoxic stress causes activation of cellular checkpoints (23), §To whom correspondence should be addressed. E-mail: [email protected]. leading to diverse responses such as cell cycle arrest, DNA repair, © 2005 by The National Academy of Sciences of the USA 14244–14248 ͉ PNAS ͉ October 4, 2005 ͉ vol. 102 ͉ no. 40 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0506252102 Downloaded by guest on September 26, 2021 Immunoprecipitation. GC-spg1 cells and testis from adult mice were homogenized in a lysis buffer (50 mM Hepes, pH 7.4͞100 mM KCl͞0.5% Nonidet P-40͞proteinase and phosphatase in- hibitors) and immunoprecipitated overnight with the following antibodies: anti-rabbit Aurora-A, anti-rat or rabbit Aurora-B, anti-mouse PARP-1, and anti-rabbit IgG. The immunocom- plexes were collected by protein A- or G- Sepharose beads, and bound proteins were washed several times with lysis buffer and eluted with Laemmli buffer to be processed for Western blot. Western Blot Analysis. Protein extracts were separated by SDS- acrylamide and transferred to nitrocellulose membrane; blots were probed with the indicated antibodies and visualized by using the Pierce chemiluminescence detection system. Poly(ADP-ribosyl)ation Assay. Recombinant Myc-tagged Aurora-B or Aurora-A was expressed in Cos-1 cells and immunoprecipi- tated with anti-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology). Poly(ADP-ribosyl)ation assays were performed on the immunoprecipitated proteins in 40 ␮l of reaction buffer (50 mM Tris⅐HCl, pH 8.0͞4 mM MgCl2͞0.2 mM DTT͞2 ␮g/ml DNaseI-activated calf thymus DNA) containing 1 ␮g of purified PARP-1 and 0.3 ␮Ci (1 Ci ϭ 37 GBq) of [32P]-NADϩ for 10 min at 25°C. Proteins were separated by 10% SDS͞PAGE, trans- ferred to nitrocellulose membranes, and analyzed by autoradiog- Fig. 1. Effect of DNA damage on mitotic histone H3 phosphorylation. NIH 3T3 mouse fibroblasts were cultured in DMEM supplemented with 10% raphy. Subsequently, to evaluate the kinase activity of the ϩ serum, and DNA damage was induced by various means. (A) Western analyses modified Aurora-B, cold NAD was used in the poly(ADP- of Ser-10 H3 phosphorylation in cells treated with H2O2 (1 mM) at different ribosyl)ation assay. times. The protein levels of Aurora-A, Aurora-B, and cyclin B1 were assayed as controls. (B) Cells exposed to IR (6 Gy) or treated with N-nitroso-N-methylurea Aurora-B Kinase Assay. Immunoprecipitated Aurora-B from (2 mM) for 45 min and allowed to recover. (C and D) Analysis of various histone treated and control cells or in vitro poly(ADP-ribosyl)ated modifications upon DNA damage by using 1 mM H2O2 or 6-Gy IR. Antibodies Aurora-B was used in the presence of 5 ␮Ci of [32P]-␥-ATP. One directed against the indicated specific histone modifications were used in microgram of total histones or GST-H3 was used as a substrate Western assays. (E) DNA damage does not influence EGF-induced H3 phos- as indicated. The reaction was carried out for 30 min at 30°C and phorylation. Cells were serum-starved for 24 h and then stimulated with EGF for1hasdescribed in refs. 23–27, in conjunction with 1 mM H2O2 treatment stopped by the addition of Laemmli buffer. Proteins were or not. analyzed by SDS͞PAGE followed by autoradiography. BIOCHEMISTRY Results activated protein kinase 1), and I␬B kinase-␣ (31–33), depending Specific Reduction of Histone H3 Ser-10 Mitotic Phosphorylation in on the cell type and signaling event.