Aurora B-dependent Regulation of Class IIa Histone Deacetylases by Mitotic Nuclear Localization Signal Phosphorylation

• DOI: 10.1074/mcp.m112.021030
• Publication Date: 2012-08-04T00:19:26Z
• Authors: Amanda J. Guise; Todd M. Greco; Irene Y. Zhang; Fang Yu; Ileana M. Cristea
• Keywords: Midbody; Histone deacetylase 5; HDAC4

Class IIa histone deacetylases (HDACs 4/5/7/9) are transcriptional regulators with critical roles in cardiac disease and cancer. HDAC inhibitors promising anticancer agents, although they known to disrupt mitotic progression, the underlying mechanisms of regulation by HDACs not fully understood. Here we provide first identification as substrates Aurora B kinase (AurB). Our study identifies class a novel family AurB targets provides evidence that temporally spatially regulated phosphorylation during cell cycle. We define precise site AurB-mediated conserved serine within nuclear localization signals HDAC4, HDAC5, HDAC9 at Ser265, Ser278, Ser242, respectively. establish interacts these vivo, this association increases upon disruption 14-3-3 binding. observe colocalization endogenous, phosphorylated midzone late anaphase midbody cytokinesis, complemented reduction interactions components corepressor complex. propose AurB-dependent induces sequestration gradient midzone, maintaining separation from re-forming nuclei contributing control. 1The abbreviations used are:HDAChistone deacetylaseAurBAurora kinaseNOCnocodazoleHUhydroxyureaNCoRnuclear complexTFtranscription factor binding domainNLSnuclear signalDACdeacetylase domainNESnuclear export sequenceCIDcollision induced association.1The association. 4, 5, 7 9) prominent epigenetic gene expression (1Bossuyt J. Helmstadter K. Wu X. Clements-Jewery H. Haworth R.S. Avkiran M. Martin J.L. Pogwizd S.M. Bers D.M. Ca2+/Calmodulin-dependent protein II delta D overexpression reinforce deacetylase 5 redistribution heart failure.Circ. Res. 2008; 102: 695-702Crossref PubMed Scopus (113) Google Scholar), response drug stimulus (2Renthal W. Maze I. Krishnan V. Covington 3rd, H.E. Xiao G. Kumar A. Russo S.J. Graham Tsankova N. Kippin T.E. Kerstetter K.A. Neve R.L. Haggarty McKinsey T.A. Bassel-Duby R. Olson E.N. Nestler E.J. Histone epigenetically controls behavioral adaptations chronic emotional stimuli.Neuron. 2007; 56: 517-529Abstract Full Text PDF (506) cancer (3Yang X.J. Grégoire S. deacetylases: sequence function, regulation, clinical implication.Mol. Cell. Biol. 2005; 25: 2873-2884Crossref (355) viral infection (4Murphy J.C. Fischle Verdin E. Sinclair J.H. Control cytomegalovirus lytic acetylation.EMBO 2002; 21: 1112-1120Crossref (183) Scholar). Distinct directly alter DNA sequences, is indirectly mediated through mechanism nucleo-cytoplasmic shuttling (5Yang Seto The Rpd3/Hda1 lysine bacteria yeast mice men.Nat. Rev. 9: 206-218Crossref (961) Alterations localizations critically linked human disease. Increased was shown failing heart, selective induction programs associated dilated cardiomyopathy (6Calalb M.B. Newkirk Huynh Sucharov C.C. Bristow M.R. phosphorylation-dependent heart.Clin. Translational Sci. 2009; 2: 325-332Crossref (23) Scholar, 7Harrison B.C. Kim M.S. van Rooij Plato C.F. Papst P.J. Vega R.B. McAnally J.A. Richardson Regulation stress signaling D1.Mol. 2006; 26: 3875-3888Crossref (135) Furthermore, HDAC5 levels were reported change dramatically following HIV-1 (8Keedy K.S. Archin N.M. Gates A.T. Espeseth Hazuda D.J. Margolis A limited group I acts repress immunodeficiency virus type 1 expression.J. Virol. 83: 4749-4756Crossref (162) though pathways phenomenon well Thus, exploring governing functions crucial uncovering their progression. nocodazole hydroxyurea complex transcription domain signal collision involves dynamic, site-specific (9Miska E.A. Karlsson C. Langley Nielsen Pines Kouzarides T. HDAC4 associates represses MEF2 factor.EMBO 1999; 18: 5099-5107Crossref (466) 10Grozinger C.M. Schreiber S.L. 4 activity 14-3-3-dependent cellular localization.Proc. Natl. Acad. U. 2000; 97: 7835-7840Crossref (501) 11Wang A.H. Kruhlak M.J. Bertos N.R. Vezmar Posner B.I. Bazett-Jones D.P. Yang proteins.Mol. 20: 6904-6912Crossref (229) 12McKinsey Zhang C.L. Lu Signal-dependent regulates muscle differentiation.Nature. 408: 106-111Crossref (875) 13Kao H.Y. Verdel Tsai Simon Juguilon Khochbin Mechanism for nucleocytoplasmic 7.J. Chem. 2001; 276: 47496-47507Abstract (198) 14Paroni Cernotta Dello Gallinari P. Pallaoro Foti Talamo F. Orsatti L. Steinkuhler Brancolini PP2A import.Mol. 19: 655-667Crossref (97) In two phosphorylations flanking (NLS) promote chaperone proteins cytoplasm, whereas de-phosphorylation facilitates import Recently, our lab numerous additional sites, several which present NLS, signal, deacetylation (15Greco T.M. Yu Guise A.J. Cristea I.M. Nuclear requisite phosphorylation.Mol. Proteomics. 2011; 10Abstract (76) particular, adjacent NLS (Ser278 Ser279) significant interest. showed Ser279 necessary efficient kidney cells (HEK293) osteosarcoma (U2OS) kinases responsible have recently been identified be PKA Cos-7 (16Ha C.H. J.Y. Zhao Wang Jhun B.S. Wong Jin Z.G. phosphorylates prevents its export, leading inhibition cardiomyocyte hypertrophy.Proc. 2010; 107: 15467-15472Crossref Scholar) Cdk5 neurons (17Taniguchi Carreira Smith L.N. Zirlin Cowan C.W. limits cocaine reward cAMP-induced import.Neuron. 2012; 73: 108-120Abstract (92) contrast, role neighboring Ser278 remains unexplored. Given wealth sites conservation multiple residues among HDACs, it likely enzymes broader spectrum than has previously documented. concerted actions acetyltransferases proposed dynamically cycle progression modulate acetylation mitosis. Specifically, global interferes (18Shin H.J. Baek K.H. Jeon Jang K.L. Sung Y.C. Lee Inhibition chromosomal instability aberrant checkpoint activation.Oncogene. 2003; 22: 3853-3858Crossref (75) 19Noh E.L. Lim D.S. Jeong J.S. An inhibitor, trichostatin A, delay G(2)/M transition, slippage spindle checkpoint, death transcription-dependent manner.Biochem. Biophys. Commun. 378: 326-331Crossref (66) triggering G2-phase (20Warrener Beamish Burgess Waterhouse N.J. Giles Fairlie D. Gabrielli B. Tumor cell-specific cytotoxicity targeting checkpoints.FASEB 17: 1550-1552Crossref 21Mikhailov Shinohara Rieder Topoisomerase G2/M transition p38 MAPK pathway.J. Cell 2004; 166: 517-526Crossref (129) defects kinetochore assembly (22Robbins A.R. Jablonski S.A. Yen T.J. Yoda Robey Bates S.E. Sackett D.L. Inhibitors disrupting pericentromeric heterochromatin.Cell Cycle. 4: 717-726Crossref (101) Moreover, HDAC3, interacting partner G1/S maintenance localize attachment (23Ishii Kurasawa Y. Yu-Lee L.Y. 3 localizes required kinetochore-microtubule attachment.Proc. 105: 4179-4184Crossref (71) Yet, contribution unknown. Here, determine (AurB), an essential kinase, vitro vivo single NLS. also demonstrate interaction augmented NLS-phosphorylated HDACs. show enriched mitosis, sequestered parallel loss members (NCoR). antibodies experiments immunoaffinity purifications, Western blotting, immunofluorescence studies in-house developed rabbit polyclonal antibody against GFP, described (24Cristea Williams Chait B.T. Rout M.P. Fluorescent proteomic probes.Mol. 1933-1941Abstract (203) monoclonal anti-GFP (Roche), MAb414 (Covance, gift Glavy, Stevens Institute), (Abcam, Cambridge, MA), GAPDH (Abcam), 14-3-3ε (T-16, Santa Cruz, CA), H3pS10 (Millipore), Tubulin NCOR1 (Fisher Scientific), TBL1X (Santa Cruz Biotechnology, TBL1XR1 Biotechnology), HDAC3 Biotechnology). phosphospecific HDAC5-pS278 generated using synthetic peptide containing residue (QKVAERR-pS-SPLLRRC) affinity purified YenZym Antibodies, LLC (San Francisco, CA). Secondary peroxidase-conjugated goat-anti-mouse IgG (Jackson ImmunoResearch Laboratories, Westgrove, PA), mouse-anti-rabbit (Fisher, Rockford, IL), donkey-anti-goat Laboratories) blotting. For experiments, U2OS or HEK293 lines stably expressing EGFP-FLAG-tagged wild-type phosphomutant (S278A, S278/279A, S259/498A), constructed PhoenixTM retrovirus system (Orbigen, San Diego, CA) CEM T study, procedure plasmids kind (25Franco Farooqui Wei orphan receptor TR2 both deacetylases.Mol. Endocrinol. 15: 1318-1328Crossref (27) EGFP obtained pEGFP-N1 (Clontech, Mountain View, Cells tagged selected 300 mg/L G418 (EMD, Gibbstown, NJ) sorted FACS (Vantage TurboSort II, Becton Dickinson, Franklin Lakes, site-directed mutagenesis existing tagged-HDAC5 constructs. list primers provided supplementary material, supplemental Table S1. EGFP-tagged (wild-type phosphomutants) control isolated purification via tag on magnetic beads conjugated anti-GFP, 26Cristea Conjugation immunopurification complexes.Cold Spring Harb. Protoc. (2011): 534-537Google mass spectrometry analyses immunoisolates between 0.5 g 2.0 starting material account differential (0.5 1.5 2 HDAC9). 0.2 IP-Western experiments. Briefly, and/or tagged-HDACs harvested, frozen, cryogenically lysed Retch MM301 Mixer Mill (Retch, Newtown, isolation resulting frozen powder homogenized lysis buffers individually optimized effective isolations each HDAC. suspended 20 mm HEPES-KOH, pH 7.4, 0.1 m potassium acetate, MgCl2, 0.1% Tween-20, μm ZnCl2, CaCl2, 0.5% Triton X-100, 250 NaCl, μg/ml DNase I, 1/100 (v/v) protease, phosphatase inhibitor cocktails (Sigma). performed buffer 1.0% sodium deoxycholate, 200 protease 0.3% 150 Dnase cocktails. All subjected homogenization Polytron (Kinematica), if initial equal greater g, vortexing followed centrifugation 8000 × 10 min °C. Affinity incubation (M-270 epoxy beads, Invitrogen) h (HDAC4 isolations) 2.5 (HDAC9 °C, (27Cristea 538-541Google Immunoisolates eluted 30–50 μl 1X LDS sample 70 LDS-eluted complexes processed immediately in-gel in-solution digestion complementary analyses, (28Kramer Greco Enquist L.W. Proteomic Characterization Pseudorabies Virus Extracellular Virions.J. 85: 6427-6441Crossref (77) either digested in-gel, reduced alkylated 100 trypsin (5 ng/μl) ammonium bicarbonate (ABC) Vivacon 500 centrifugal filters (10 kDa MWCO; Sartorius Stedim Biotech, Goettingen, Germany), (29Tsai Y.-C. Boonmee Miteva Functional proteomics establishes SIRT7 chromatin remodeling expands RNA polymerase transcription.Mol. 11 (10.1074/mcp.M111.015156)Abstract digestion, resolved SDS-PAGE ∼3 cm (12.5 50 ABC 6 37 Peptides concentrated vacuum ∼10 analyzed nLC-MS/MS Dionex Ultimate 3000 RSLC coupled LTQ-Orbitrap Velos ETD spectrometer (ThermoFisher Scientific, Jose, desalted online (trap column; Magic C18 AQ, μm, cm, Michrom Bioresources, Inc.) offline StageTips (30Rappsilber Mann Ishihama Protocol micro-purification, enrichment, pre-fractionation storage peptides StageTips.Nat. Protocols. 1896-1906Crossref (2588) separated reverse phase chromatography (Acclaim PepMap RSLC, 1.8 75 25 cm) flow rate nl/min 90 180 samples, operated data-dependent acquisition mode FT preview scan disabled predictive AGC dynamic exclusion enabled (repeat count: 1, duration: s). comprised full-scan (m/z = 350–1700) Orbitrap (r 30,000 m/z 400), collision-induced dissociation (CID) fragmentation top most intense precursor ions (min 1E3) dual-pressure linear ion trap. full MS IT MS2 target values 1E6 5E3, maximum injection times set ms, CID width Th, normalized energy 30, activation time ms. MS/MS spectra extracted, filtered, searched Proteome Discoverer/SEQUEST (v1.3 ThermoFisher Scientific) database (UniProt-SwissProt, 2010-11) appended common contaminants (21,570 entries), automatically reversed concatenated forward sequences. Spectra criteria: enzyme specificity; missed cleavages; tolerance: ppm; fragment Da; static modification carbamidomethylcysteine (+57 Da), variable modifications methionine oxidation (+16 phosphoserine, threonine, tyrosine (+80 acetyl-lysine (+42 Da). comparative SEQUEST search results Scaffold (v3.3.1; Software) refinement X!Tandem (Beavis Informatics). Probabilities spectral matches calculated PeptideProphet (31Keller Nesvizhskii A.I. Kolker Aebersold Empirical statistical model estimate accuracy identifications made search.Anal. 74: 5383-5392Crossref (3896) Scaffold, Percolator (32Käll Canterbury J.D. Weston Noble W.S. MacCoss Semi-supervised learning shotgun datasets.Nat. Methods. 923-925Crossref (1373) Discoverer. Probability empirically reduce false discovery less 1%. Phosphorylation confirmed manual inspection evaluation phosphoRS algorithm (Proteome Discoverer, v1.3). As facilitate label-free counting analysis assess relative abundance changes well-established after arrest. minimum five total biological replicate least one treatment condition (DMSO (n 4), NOC 5), HU 3)) quantification. To correct, differences immunoisolated bait, prey counts replicates respective HDAC5-EGFP DMSO controls. Spectrum count fold ratio averaged individual condition. Protein tables HDAC5-interacting partners Tables S2 S3, substrate corresponding region, KVAERRSSPLLRRK, specificity phosphorylation. Kinase assays TrisHCl, 7.6, (1:100, Sigma), dithiothreitol, substrate. reactions initiated addition ATP ng recombinant (Cell Signaling Technology, Boston, MA) room temperature 30 min. absence staurosporine, general serine/threonine inhibitor. Reactions terminated trifluoroacetic acid 0.5%. 50% acetonitrile formic acid, infused into ESI μl/min. Peptide determined tandem electron transfer (ETD) 4+ charge state detection analyzer (resolution 7500). Wild-type HDAC5-EGFP-FLAG cultured glass coverslips, pretreated poly-d-Lys (Sigma, St. Louis, MO). After 24 h, fixed 2% paraformaldehyde, washed glycine/d-PBS, permeabilized X-100 d-PBS (PBST) 15 blocked (w/v) bovine serum albumin, 0.2% Tween-20 RT 60 Incubation primary blocking buffer. then PBST incubated goat anti-mouse anti-rabbit secondary Alexa 488, 568, 633 (Invitrogen/Invitrogen). DAPI PBS visualized confocal microscopy Zeiss LSM510 (Zeiss, Dublin, 63× oil-immersion lens 100× lens. Coverslips mounted slides Aqua-Poly/Mount media (Polysciences). seeded 30% confluence, Hesperadin (Selleck Chemicals, Houston, TX) added standard (Dulbecco's modified Eagle's medium (Invitrogen), 10% fetal (Gemini MedSupply Partners, Atlanta, GA), 1% Pen/Strep Partners)) final concentrations 20, 40, nm. Hesperadin-treated (DMSO-treated) harvested trypsinization centrifugation. Whole lysates prepared samples bl