Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

• DOI: 10.1074/jbc.m512921200
• Publication Date: 2006-01-31T01:48:45Z
• Authors: Alessia Montagnoli; Barbara Valsasina; Deborah Brotherton; Sonia Troiani; Sonia Rainoldi; Pierluigi Tenca; Antonio Molinari; Corrado Santocanale
• Keywords: Identification

Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome eukaryotic organisms. Upon entry into S-phase several subunits MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates intrinsic DNA helicase activity, thus allowing formation active forks. Cdc7, Cdk2, and ataxia telangiectasia Rad3-related kinases regulate progression known to phosphorylate Mcm2 subunit. In this work, by vitro kinase reactions mass spectrometry analysis products, we have mapped sites N terminus Cdk1, CK2. We found Cdc7 phosphorylates at least three different sites, one which corresponds site also reported be phosphorylated Rad3-related. Three serine/proline were identified for Cdk2 unique was raised specific anti-phosphopeptide antibodies all cells. Importantly, although Cdc7-dependent phosphosites fluctuate during cell cycle with kinetics similar activity protein levels, putative cyclin-dependent (Cdk) consensus constant cycle. Furthermore, our indicates majority isoforms not stably associated chromatin. This study forms basis understanding how functions regulated multiple within response external perturbations. organisms G1 phase multiprotein as pre-replicative formed around origin DNA. contains recognition complex, Cdc6, Cdt1, minichromosome (MCM) proteins. Once components two kinases, (CDK) 3The abbreviations used are: CDK, kinase; DTT, dithiothreitol; CK2, casein 2; Pipes, 1,4-piperazinediethanesulfonic acid; MS, spectrometry; ESI, electrospray ionization; MALDI-TOF, matrix-assisted laser desorption/ionization-time flight; NHDF, human dermal fibroblast; HA, hemagglutinin; HU, hydroxyurea; FACS, fluorescence-activated sorter; ATR, Ataxia kinase. leads unwinding double-stranded loading such Cdc45 GINS which, together polymerases, participate semi-conservative synthesis new strands chain elongation (1Diffley J.F. Labib K. J. Cell Sci. 2002; 115: 869-872Crossref PubMed Google Scholar, 2Bell S.P. Dutta A. Annu. Rev. Biochem. 71: 333-374Crossref Scopus (1394) 3Kanemaki M. Sanchez-Diaz Gambus Nature. 2003; 423: 720-724Crossref (215) 4Blow J.J. Nat. Mol. Cell. Biol. 2005; 6: 476-486Crossref (532) 5Kubota Y. Takase Komori Hashimoto Arata T. Kamimura Araki H. Takisawa Genes Dev. 17: 1141-1152Crossref (169) Scholar). The appears crucial target S-phase-promoting become time activation (reviewed Ref. 6Forsburg S.L. Microbiol. 2004; 68: 109-131Crossref (429) initiation has occurred, Mcm2-7 travels replicating enzymes on DNA, being required fork possibly acting replicative (4Blow 7Labib Tercero J.A. Diffley Science. 2000; 288: 1643-1647Crossref (523) 8Ishimi Chem. 1997; 272: 24508-24513Abstract Full Text PDF (460) Several lines evidence indicate various subject phosphorylations. particular, protein, generally detected altered mobility SDS-PAGE (9Masai Matsui E. You Z. Ishimi Tamai Arai 275: 29042-29052Abstract (129) 10Montagnoli Tenca P. Sola F. Carpani D. Brotherton Albanese C. Santocanale Cancer Res. 64: 7110-7116Crossref (115) Scholar), occur cycle-dependent manner, cells enter until mitosis. Although good substrate both CDKs it unclear whether these act independently or cooperate phosphorylation. budding yeast suggested only kinase, must before Cdc7/Dbf4 (11Nougarede R. Della S.F. Zarzov Schwob 20: 3795-3806Crossref (106) whereas Xenopus system chromatin exposed sequential manner first, followed can efficiently replicate. Reversing order appeared detrimental (12Walter J.C. 39773-39778Abstract (130) Finally, assays using purified recombinant indicated pre-phosphorylation MCMs stimulate suggesting CDK may prerequisite function origins Recently, checkpoint shown interact Mcm7 through its regulatory subunit ATRIP specifically Ser-108, pathway (14Cortez Glick G. Elledge S.J. Proc. Natl. Acad. U. S. 101: 10078-10083Crossref (264) 15Yoo H.Y. Shevchenko Dunphy W.G. 279: 53353-53364Abstract (109) consistent finding Ser-108 increased challenged DNA-damaging agents. However, because basal levels absence damage ATR impaired, likely exists able same canonical 2 (CK2), pleiotropic been implicated regulating (16Kulartz Hiller Kappes Pinna L.A. Knippers Biophys. Commun. 315: 1011-1017Crossref (23) 17Pinna 3873-3878Crossref (412) At present indeed cells, what temporal sequence events, and, ultimately, contributes function. To begin address questions, set out map protein. By generating immunological reagents N-terminal tail seven observed occurs almost exclusively sites. recognized Cdk adjacent serines. Recombinant Proteins Synthetic Peptides—Recombinant corresponding residues 10-294, produced described 18Montagnoli Bosotti Villa Rialland Mercurio Berthelsen EMBO 21: 3171-3181Crossref (69) Scholar. Mcm2-HA expression obtained cloning full-length mammalian vector pCDNA-HA. Point mutations under “Results” generated oligonucleotide-directed mutagenesis QuikChange site-directed kit (Stratagene). All mutants sequenced their entirety. Cdk2/A, Cdk1/B elsewhere (19Lolli Thaler Valsasina B. Roletto Knapp Uggeri Bachi Matafora V. Storici Stewart Kalisz H.M. Isacchi Proteomics. 3: 1287-1298Crossref (34) CK2 purchased from Calbiochem. biotinylated peptides RTDALTSSPGRDLPPFG unphosphorylated Ser-40, Ser-41, Ser-40 Ser-41 custom synthesized (Tufts University). 49-64 36-44 in-house. Kinase Phosphatase Assays—For assay 6 μm (wild type mutants) incubated 30 min 37 °C presence 50 mm Tris, pH 7.5, 10 MgCl2, 100 ATP (kinase buffer), nm Cdk2/cyclin E For after immunoprecipitation, beads equilibrated buffer containing °C. liquid chromatography/MS analysis, enzyme ratio decreased 1:10, reaction performed h Hepes, 7.9, DTT. Peptide Fig. 2E peptide substrate, 4 enzyme, traced radiolabeled [γ-33P]ATP 40-μl 96-well plate. Dowex resin (Supelco) capture residual counting radioactivity. Dot Blot 37°C 500 ATP, Cdk2/CycA. phosphatase experiment, 15 μg HeLa extract prepared without inhibitors units λ-phosphatase (Calbiochem) Liquid Chromatography/MS Analysis—20 Mcm2-(10-294) analyzed kinases. chromatographic separations 1100 Agilent instrument Vydac C-4 column (2.1-mm i.d._25-cm length, particle size 5 μ, pore 300 A). After equilibration 10% aqueous acetonitrile 0.05% trifluoroacetic acid, an eluent program linear gradient 75% ACN acid 35 min. flow rate 0.2 ml/min. eluate sent directly MS instrument. Positive ion ESI spectra 1946 single quadrupole spectrometer (Agilent) orthogonal source. needle voltage 3000 nebulizer well drying gas (nitrogen) maintained psi liters/min, respectively. range m/z 600-2000. resulting final spectrum intact samples deconvoluted automatically chemstation deconvolution software. chromatography separation C-18 i.d. 25-cm A) elution 60 In-gel Tryptic Digestion—Protein digestion trypsin Digest Pro (Intavis, Koeln, Germany) following standard protocol. mixture then dried speed vacuum redissolved μl 0.1% acid. Sample desalting ZipTip C18 (Millipore) fractionated acetonitrile, water, 30% water finally 60% water. 0.5 each sample MALDI-MS remaining material down formic 50% nano-ESI MS/MS analysis. Additional AspN eluates performed, redissolving NH4HCO3 adding 0.1 protease incubating monitored MALDI mixing matrix analyzing reflector mode. MALDI-MS—Samples desorption ionization-time flight (MALDI-TOF) spotting α-cyano-4-hydroxycinnamic (10 mg/ml 50:50 acetonitrile:water acid) Voyager DE-PRO (Applied Biosystems). collected mode four calibration standards. Nano-ESI-MS/MS—Tandem hybrid quadrupole-time (Q-ToF2; Micromass, Manchester, UK) equipped Z-spray source calibrated injecting solution Glu-Fibrinopeptide (Sigma-Aldrich) (0.5 pmol/μl) μl/min, applying 3.2 kV spraying capillary, collision energy 29 phosphosite assignment MS/MS, desalted vacuum, 1:1 acetonitrile/0.1% mixture, loaded nanoflow probe tips (Micromass). Each fraction subjected over 100-2000 scan range; analyses manually selecting fragmenting them user-defined parameters energy. Antibodies—The anti-pSer-13, -pSer-40/41, -pSer-108, -pSer-139 Mcm2-specific collaboration Zymed Laboratories Inc. immunizing rabbits phosphopeptides MAS-pS-PAQRRR, APLT-pS-pS-PGR, EELTA-pS-QRE, LLYD-pS-DEEDE, serum rounds affinity phospho- nonphosphopeptide columns. phospho-Ser-53 antibody BIOSOURCE phosphopeptide FEDE-pS-EGLLG. case crude antiserum experiments. Antibodies against pSer-41 pSer-27 Bethyl. Antibody total BD Biosciences; anti-Cyclin A Cyclin B Santa Cruz. Western blots Neomarker used. Immunoprecipitation HA-Mcm2 anti-HA (Roche Applied Science). immunoprecipitations monoclonal 12A10 developed ARETA (www.Areta.com), while anti-ATR Cruz (N19) immunoprecipitation. competition experiments, anti-phospho preincubated 1 room temperature membranes. FEDE-pS-EGLLG, LLYD-pS-DEEDE (and peptides) pSer-53, pSer-108 antibodies, plus anti-pSer-41 anti-pSer-40/41 between 5:1 (w/w) used, anti-pSer-53 serum, per serum. Synchronization Protein Preparations—HeLa grown modified Eagle's medium (Invitrogen) supplemented fetal bovine Human fibroblasts (NHDF) fibroblast (Promocell) growth factors. NHDF synchronized G0 culturing 48 h. Thymidine synchronization thymidine (2 mm) 14 cytometry FACScan (BD Biosciences). extraction, lysed SDS (125 6.8, 5% SDS, DTT) sonicated. binding CSK NaCl, sucrose, EGTA, Triton X-100), Science) (Sigma cocktails I II) centrifuged rpm (sup fraction). pellet washed resuspended lysis (pellet immunoprecipitation purposes, (50 250 EDTA, X-100, inhibitors). Immunofluorescence—HeLa fixed 3.7% paraformaldehyde permeabilized 0.5% Triton. dilutions: anti-Mcm2 Biosciences) 1:300, anti-Ser-41 (Bethyl) 1:1000, anti-Ser-40 1:200, anti-Ser-108 1:200. As secondary antibody, anti-rabbit IgG Cy3 conjugate (Sigma). cases, ice fixation. experiments previously coverslips. Transfections—HeLa transfected FuGENE manufacturer's instructions. Small interference RNA (10Montagnoli Identification Phosphosites Vitro—To regulation phosphorylation, began identify Annotation amino recently updated terminus, here refer Swiss Prot accession number, P49736. others fragment, acids 20Ishimi Komamura-Kohno Masai 2001; 276: 42744-42752Abstract (44) extended observations proposed modulate phosphorylating substrates (17Pinna uses fragment substrate. Incubation Cdc7/Dbf4, resulted highly labeled (Fig. 1A) autoradiography. LC/MS incubation interest cold clearly showed species differing 80 Da up either Cdc7. Similar results Cdk1/CycB (data shown). contrast, phosphate group added 1B). samples, digested trypsin, MALDI-TOF spectrometry. Comparison treatment allowed us determine Cdk2/CycA complexes completely superimposable pattern (regions 10-17, 20-30, 34-44). Nano-ESI selected Ser-13, Ser-27, (supplemental S1 data Two Ser-27 correspond S/TPXR/K (21Endicott Noble M.E. Tucker Curr. Opin. Struct. 1999; 9: 738-744Crossref (101) just partial S/TP Ser-13. Using procedure 134-149 stoichiometrically (MW 1973.84) again confirmed serine 139 shown) context II motif Mutation alanine abolished indicating Ser-139 S2A). tryptic MCM2-(10-294) (34-44, 45-79, 83-115). form 83-115 endoprotease could 2A). other successful due large molecular weight 45-79 poor ionization properties low stoichiometry Therefore synthetic (36-44 49-64) produced, upon pSer-53 2B), pSer-40 2C) phosphoamino acids. Ser-53, first Alignment sequences surrounding serines did reveal obvious motif, acidic surround Ser-53 Ser-108. Intriguingly, residue Scholar) separate groups. immunoprecipitated S2B). noticed immediately precedes previous experiment Cdk2. If negative charges needed better introduction position +1 (Ser-41) facilitate Ser-40. observation prompted further investigate relationships MCM2 spanning region (amino 36-44). expected, peptide, S1). tested residues. products isolated high performance Ser-41. second transferred. 2D). Thus, experimental conditions mutually exclusive. increase efficiency explore phenomenon compared region, monophosphorylated Indeed, ∼3-fold when 2E). result supports hypothesis negatively charged tails human, mouse, conserved, 3). Characterization Anti-phospho-Mcm2 Antibodies—With goal verifying vivo, rabbit polyclonal Ser-139. immunized animals carrying double-phosphorylated Specific antigen Anti-pSer-27 commercial check specificity reagents, probes blot mock treated relevant anti-pSer-13 recognizes Cdk1 4A), anti-pSer-139 but 4B), anti-pSer-108 recognize 4C). characterize immunization panel 34 49 unphosphorylated, mono-phosphorylated double 4D, top panel), therefore reagent will defined anti-Ser-40/41 throughout entire work. Consistent easily middle panel). Importantly cannot strongly prevents bottom antibody. equally 4E, pane