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• W2012172422 abstract "Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations. Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations. In eukaryotic organisms during the G1 phase of the cell cycle a multiprotein complex known as the pre-replicative complex is formed around origin DNA. This contains the origin recognition complex, Cdc6, Cdt1, and minichromosome maintenance (MCM) proteins. Once in S-phase several components of the pre-replicative complex are phosphorylated by at least two kinases, a cyclin-dependent kinase (CDK) 3The abbreviations used are: CDK, cyclin-dependent kinase; DTT, dithiothreitol; CK2, casein kinase 2; Pipes, 1,4-piperazinediethanesulfonic acid; MS, mass spectrometry; ESI, electrospray ionization; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; NHDF, human dermal fibroblast; HA, hemagglutinin; HU, hydroxyurea; FACS, fluorescence-activated cell sorter; ATR, Ataxia telangiectasia and Rad3-related. and the Cdc7 kinase. This leads to the unwinding of double-stranded DNA and to the loading of several proteins such as Cdc45 and the GINS complex, which, together with DNA polymerases, participate in the semi-conservative synthesis of new DNA strands during 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. 2002; 71: 333-374Crossref PubMed Scopus (1394) Google Scholar, 3Kanemaki M. Sanchez-Diaz A. Gambus A. Labib K. Nature. 2003; 423: 720-724Crossref PubMed Scopus (215) Google Scholar, 4Blow J.J. Dutta A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 476-486Crossref PubMed Scopus (532) Google Scholar, 5Kubota Y. Takase Y. Komori Y. Hashimoto Y. Arata T. Kamimura Y. Araki H. Takisawa H. Genes Dev. 2003; 17: 1141-1152Crossref PubMed Scopus (169) Google Scholar). The MCM complex appears to be a crucial target of the S-phase-promoting kinases, and multiple subunits become phosphorylated at the time of origin activation (reviewed in Ref. 6Forsburg S.L. Microbiol. Mol. Biol. Rev. 2004; 68: 109-131Crossref PubMed Scopus (429) Google Scholar). Once initiation has occurred, the Mcm2-7 complex travels together with replicating enzymes on DNA, being required for replication fork progression possibly by acting as the replicative helicase (4Blow J.J. Dutta A. Nat. Rev. Mol. Cell. Biol. 2005; 6: 476-486Crossref PubMed Scopus (532) Google Scholar, 7Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Crossref PubMed Scopus (523) Google Scholar, 8Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Abstract Full Text Full Text PDF PubMed Scopus (460) Google Scholar). Several lines of evidence indicate that various subunits of Mcm2-7 complex are subject to multiple phosphorylations. In particular, phosphorylation of the Mcm2 protein, generally detected as altered mobility in SDS-PAGE (9Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 10Montagnoli A. Tenca P. Sola F. Carpani D. Brotherton D. Albanese C. Santocanale C. Cancer Res. 2004; 64: 7110-7116Crossref PubMed Scopus (115) Google Scholar), appears to occur in a cell cycle-dependent manner, being detected as cells enter S-phase until mitosis. Although Mcm2 is a good substrate for both CDKs and Cdc7 kinases, it is unclear whether these kinases act independently or cooperate in MCM phosphorylation. In budding yeast it was suggested that the only cyclin-dependent kinase, Cdk1, must act before Cdc7/Dbf4 (11Nougarede R. Della S.F. Zarzov P. Schwob E. Mol. Cell. Biol. 2000; 20: 3795-3806Crossref PubMed Scopus (106) Google Scholar), whereas in a Xenopus in vitro DNA replication system only chromatin that was exposed in a sequential manner to Cdc7 first, followed by Cdk2 kinase, can efficiently replicate. Reversing the order of the kinases appeared to be detrimental to DNA replication in this system (12Walter J.C. J. Biol. Chem. 2000; 275: 39773-39778Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Finally, in vitro phosphorylation assays using purified human recombinant proteins have indicated that pre-phosphorylation of MCMs with CDKs can stimulate Cdc7 activity, suggesting that CDK phosphorylation may be a prerequisite for Cdc7 function at origins (9Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Recently, the checkpoint kinase ataxia telangiectasia and Rad3-related was shown to interact with Mcm7 through its regulatory subunit ATRIP and to phosphorylate Mcm2 specifically at Ser-108, suggesting that MCM is a target of the S-phase checkpoint pathway (14Cortez D. Glick G. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10078-10083Crossref PubMed Scopus (264) Google Scholar, 15Yoo H.Y. Shevchenko A. Shevchenko A. Dunphy W.G. J. Biol. Chem. 2004; 279: 53353-53364Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This is consistent with the finding that Ser-108 Mcm2 phosphorylation is increased in cells challenged with DNA-damaging agents. However, because basal levels of Mcm2 Ser-108 phosphorylation are detected in the absence of damage or in cells in which ATR activity is impaired, it is likely that a different kinase exists that is able to phosphorylate Mcm2 at the same site (14Cortez D. Glick G. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10078-10083Crossref PubMed Scopus (264) Google Scholar, 15Yoo H.Y. Shevchenko A. Shevchenko A. Dunphy W.G. J. Biol. Chem. 2004; 279: 53353-53364Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Finally, Mcm2 contains a canonical consensus site for casein kinase 2 (CK2), a kinase with pleiotropic functions that has also been implicated in regulating DNA replication proteins (16Kulartz M. Hiller E. Kappes F. Pinna L.A. Knippers R. Biochem. Biophys. Res. Commun. 2004; 315: 1011-1017Crossref PubMed Scopus (23) Google Scholar, 17Pinna L.A. J. Cell Sci. 2002; 115: 3873-3878Crossref PubMed Scopus (412) Google Scholar). At present it is not known whether all these kinases indeed phosphorylate Mcm2 in cells, what is the temporal sequence of different phosphorylation events, and, ultimately, how phosphorylation at different sites contributes to MCM function. To begin to address these questions, we set out to map specific phosphosites in the human Mcm2 protein. By generating specific immunological reagents we found that the Mcm2 N-terminal tail is phosphorylated in at least seven different sites and that the observed cell cycle-dependent phosphorylation of Mcm2 occurs almost exclusively at Cdc7-dependent sites. We also found that Cdc7 kinase can phosphorylate Mcm2 in the same site that is also recognized by ATR and that Cdc7 and Cdk can phosphorylate adjacent serines. Recombinant Proteins and Synthetic Peptides—Recombinant human N-terminal Mcm2 protein, corresponding to residues 10-294, was produced as described in Ref. 18Montagnoli A. Bosotti R. Villa F. Rialland M. Brotherton D. Mercurio C. Berthelsen J. Santocanale C. EMBO J. 2002; 21: 3171-3181Crossref PubMed Scopus (69) Google Scholar. Mcm2-HA for cell expression was obtained by cloning the full-length Mcm2 into the mammalian expression vector pCDNA-HA. Point mutations described under “Results” were generated by oligonucleotide-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene). All mutants were sequenced in their entirety. Cdc7/Dbf4 kinase was obtained as described in Ref. 18Montagnoli A. Bosotti R. Villa F. Rialland M. Brotherton D. Mercurio C. Berthelsen J. Santocanale C. EMBO J. 2002; 21: 3171-3181Crossref PubMed Scopus (69) Google Scholar. Cdk2/A, Cdk1/B were produced as described elsewhere (19Lolli G. Thaler F. Valsasina B. Roletto F. Knapp S. Uggeri M. Bachi A. Matafora V. Storici P. Stewart A. Kalisz H.M. Isacchi A. Proteomics. 2003; 3: 1287-1298Crossref PubMed Scopus (34) Google Scholar), and CK2 was purchased from Calbiochem. Synthetic biotinylated peptides with sequence RTDALTSSPGRDLPPFG unphosphorylated or phosphorylated in residues Ser-40, Ser-41, or Ser-40 and Ser-41 were custom synthesized (Tufts University). Mcm2 biotinylated peptides 49-64 and 36-44 were synthesized in-house. Kinase and Phosphatase Assays—For in vitro kinase assay 6 μm N-terminal recombinant Mcm2 (wild type or mutants) was incubated for 30 min at 37 °C in the presence of 50 mm Tris, pH 7.5, 10 mm MgCl2, 2 mm DTT, 100 μm ATP (kinase buffer), and 10 nm Cdc7/Dbf4 or Cdk2/cyclin E or CK2. For kinase assay after immunoprecipitation, the beads were equilibrated in kinase buffer and incubated in the same buffer containing 6 μm Mcm2 for 30 min at 37 °C. For liquid chromatography/MS analysis, enzyme substrate ratio was decreased to 1:10, and the reaction was performed for 2 h in 50 mm Hepes, pH 7.9, 10 mm MgCl2, 2 mm DTT. Peptide phosphorylation in Fig. 2E was performed with 50 μm peptide as substrate, 4 nm enzyme, 10 μm ATP traced with radiolabeled [γ-33P]ATP in 40-μl reaction in a 96-well plate. Dowex resin (Supelco) was used to capture residual ATP before counting radioactivity. For Dot Blot analysis 50 μm peptide substrate were incubated for 2 h at 37°C in the presence of 50 mm Tris, pH 7.5, 10 mm MgCl2, 2 mm DTT, 500 μm ATP, and 50 nm Cdc7/Dbf4 or Cdk2/CycA. For the phosphatase experiment, 15 μg of HeLa extract prepared without phosphatase inhibitors was incubated with 100 units of λ-phosphatase (Calbiochem) for 30 min at 30 °C. Liquid Chromatography/MS Analysis—20 μg of Mcm2-(10-294) protein was analyzed before or after phosphorylation with the different kinases. The chromatographic separations were performed on a 1100 Agilent instrument using a Vydac C-4 column (2.1-mm i.d._25-cm length, particle size 5 μ, pore size 300 A). After column equilibration by 10% aqueous acetonitrile containing 0.05% trifluoroacetic acid, an eluent program was performed with a linear gradient from 10 to 75% ACN containing 0.05% trifluoroacetic acid in 35 min. The flow rate was set to 0.2 ml/min. The eluate from the column was sent directly to the MS instrument. Positive ion ESI mass spectra were obtained using 1946 single quadrupole mass spectrometer (Agilent) with an orthogonal ESI source. The needle voltage was set at 3000 V. The nebulizer as well as drying gas (nitrogen) were maintained at 30 psi and a flow rate of 10 liters/min, respectively. The mass range was set to m/z 600-2000. The resulting final ESI mass spectrum of intact protein samples was deconvoluted automatically using Agilent chemstation deconvolution software. For peptide analysis the chromatography separation was performed on a Vydac C-18 column (2.1-mm i.d. 25-cm length, particle size 5 μ, pore size 300 A) with an elution gradient from 5 to 75% ACN containing 0.05% trifluoroacetic acid in 60 min. In-gel Tryptic Digestion—Protein digestion was performed with trypsin by using the Digest Pro system (Intavis, Koeln, Germany) following the standard protocol. The elution mixture was then dried in a speed vacuum and redissolved in 10 μl of aqueous 0.1% trifluoroacetic acid. Sample desalting was performed by ZipTip C18 (Millipore) with fractionated elution of 10 μl of 10% acetonitrile, 0.1% trifluoroacetic acid in water, followed by 10 μl of 30% acetonitrile, 0.1% trifluoroacetic acid in water and finally 10 μl of 60% acetonitrile, 0.1% trifluoroacetic acid in water. 0.5 μl of each sample was used for MALDI-MS analysis, and the remaining material was dried down and redissolved in 0.1% formic acid, 50% acetonitrile for nano-ESI MS/MS analysis. Additional digestion with AspN on ZipTip eluates was performed, redissolving dried ZipTip eluate in 10 μl of NH4HCO3 50 mm and adding 0.1 μg of AspN protease and incubating at 37 °C. The digestion was monitored by MALDI MS, mixing 0.5 μl of digestion mixture with 0.5 μl of matrix and analyzing it in reflector mode. MALDI-MS—Samples for matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were prepared by spotting 0.5 μl of peptide mixture with 0.5 μl of α-cyano-4-hydroxycinnamic acid (10 mg/ml in 50:50 acetonitrile:water containing 0.1% trifluoroacetic acid) and analyzed on a Voyager DE-PRO (Applied Biosystems). All spectra were collected in reflector mode using four peptides of known mass as external calibration standards. Nano-ESI-MS/MS—Tandem mass spectrometry was performed on a hybrid quadrupole-time of flight instrument (Q-ToF2; Micromass, Manchester, UK) equipped with a Z-spray source and calibrated by injecting a solution of Glu-Fibrinopeptide (Sigma-Aldrich) (0.5 pmol/μl) at 0.5 μl/min, applying 3.2 kV to the spraying capillary, and a collision energy of 29 V. For phosphosite assignment by MS/MS, the desalted samples were dried down in a speed vacuum, redissolved in a 1:1 acetonitrile/0.1% formic acid mixture, and loaded directly into nanoflow probe tips (Micromass). Each fraction was subjected to MS analysis over the 100-2000 m/z scan range; MS/MS analyses were performed by manually selecting the phosphorylated peptides and fragmenting them under user-defined parameters of collision energy. Antibodies—The anti-pSer-13, -pSer-40/41, -pSer-108, and -pSer-139 Mcm2-specific antibodies were generated in collaboration with Zymed Laboratories Inc. by immunizing rabbits with the phosphopeptides MAS-pS-PAQRRR, APLT-pS-pS-PGR, EELTA-pS-QRE, and LLYD-pS-DEEDE, respectively. The antibodies were then purified from serum with two rounds of affinity chromatography using both phospho- and nonphosphopeptide affinity columns. The phospho-Ser-53 Mcm2-specific antibody was generated in collaboration with BIOSOURCE by injecting the rabbits with the phosphopeptide FEDE-pS-EGLLG. In this case crude antiserum was used in the experiments. Antibodies against pSer-41 and pSer-27 Mcm2 were purchased from Bethyl. Antibody to total Mcm2 was from BD Biosciences; anti-Cyclin A and Cyclin B were from Santa Cruz. For Cdc7 Western blots an antibody from Neomarker was used. Immunoprecipitation of HA-Mcm2 was performed using anti-HA affinity matrix (Roche Applied Science). Cdc7 immunoprecipitations were performed with the monoclonal antibody 12A10 that was developed together with ARETA (www.Areta.com), while an anti-ATR antibody from Santa Cruz (N19) was used for ATR immunoprecipitation. For peptide competition experiments, the anti-phospho Mcm2 antibodies were preincubated for 1 h at room temperature with the corresponding peptides before adding to the membranes. The peptides used were MAS-pS-PAQRRR, FEDE-pS-EGLLG, EELTA-pS-QRE, LLYD-pS-DEEDE (and the corresponding unphosphorylated peptides) for anti-pSer-13, pSer-53, pSer-108 and -pSer-139 antibodies, respectively. Synthetic peptides with sequence RTDALTSSPGRDLPPFG unphosphorylated or phosphorylated in residues Ser-41 or Ser-40 plus Ser-41 were used for anti-pSer-41 and anti-pSer-40/41 competition experiments. A ratio between peptides and purified antibodies of 5:1 (w/w) was used, while for anti-pSer-53 serum, 0.5 μg of peptides were used per μl of crude serum. Cell Synchronization and Protein Preparations—HeLa cells were grown in modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum. Human dermal fibroblasts (NHDF) were grown in fibroblast basal medium (Promocell) supplemented with 10% fetal bovine serum and growth factors. NHDF were synchronized in G0 by culturing the cells in medium without serum for 48 h. Thymidine synchronization was obtained by adding thymidine (2 mm) for 14 h. Synchronization was monitored by flow cytometry using FACScan (BD Biosciences). For total protein extraction, cells were lysed in SDS buffer (125 mm Tris, pH 6.8, 5% SDS, 1 mm DTT) and sonicated. For chromatin binding experiments, cells were lysed in CSK buffer (10 mm Pipes, 100 mm NaCl, 300 mm sucrose, 1 mm MgCl2, 1 mm DTT, 1 mm EGTA, 0.1% Triton X-100), protease (Roche Applied Science) and phosphatase inhibitors (Sigma cocktails I and II) and centrifuged at 3000 rpm for 10 min (sup fraction). The pellet was washed and resuspended in SDS lysis buffer (pellet fraction). For immunoprecipitation purposes, cells were lysed in Triton buffer (50 mm Tris, pH 7.5, 250 mm NaCl, 1 mm EDTA, 1 mm DTT, 0.1% Triton X-100, protease and phosphatase inhibitors). Immunofluorescence—HeLa cells were fixed with 3.7% paraformaldehyde and permeabilized with 0.5% Triton. The antibodies were used at the following dilutions: anti-Mcm2 (BD Biosciences) 1:300, anti-Ser-41 Mcm2 (Bethyl) 1:1000, anti-Ser-40 Mcm2 1:200, anti-Ser-108 Mcm2 1:200. As secondary antibody, anti-rabbit IgG Cy3 conjugate was used (Sigma). In the indicated cases, cells were incubated in CSK buffer for 10 min on ice before fixation. For competition experiments the antibodies were preincubated with the corresponding peptides as previously indicated before adding to the coverslips. Transfections—HeLa cells were transfected using FuGENE (Roche Applied Science) following the manufacturer's instructions. Small interference RNA experiments were as previously described (10Montagnoli A. Tenca P. Sola F. Carpani D. Brotherton D. Albanese C. Santocanale C. Cancer Res. 2004; 64: 7110-7116Crossref PubMed Scopus (115) Google Scholar). Identification of Mcm2 Phosphosites in Vitro—To address the regulation of MCMs by phosphorylation, we began to identify phosphorylated sites on the Mcm2 protein. Annotation of the human Mcm2 amino acid sequence has been recently updated in its N terminus, and here we refer to its new Swiss Prot accession number, P49736. We and others have previously shown that an Mcm2 N-terminal fragment, corresponding to amino acids 10-294, is a good substrate for both Cdc7 and CDKs (9Masai H. Matsui E. You Z. Ishimi Y. Tamai K. Arai K. J. Biol. Chem. 2000; 275: 29042-29052Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 18Montagnoli A. Bosotti R. Villa F. Rialland M. Brotherton D. Mercurio C. Berthelsen J. Santocanale C. EMBO J. 2002; 21: 3171-3181Crossref PubMed Scopus (69) Google Scholar, 20Ishimi Y. Komamura-Kohno Y. Arai K. Masai H. J. Biol. Chem. 2001; 276: 42744-42752Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We extended these observations and also found that CK2 kinase, which has been proposed to modulate DNA replication by phosphorylating multiple substrates (17Pinna L.A. J. Cell Sci. 2002; 115: 3873-3878Crossref PubMed Scopus (412) Google Scholar), efficiently uses the Mcm2 N-terminal fragment as a substrate. Incubation of Mcm2-(10-294) with recombinant Cdk2/A, Cdc7/Dbf4, or CK2 in the presence of radiolabeled ATP resulted in a highly labeled Mcm2-(10-294) (Fig. 1A) after SDS-PAGE separation and autoradiography. LC/MS analysis of Mcm2-(10-294) after incubation with the kinases of interest and cold ATP clearly showed the presence of several Mcm2-(10-294) species differing by 80 Da and phosphorylated in up to three sites in the reactions performed either with Cdk2 or Cdc7. Similar results were obtained using Cdk1/CycB kinase (data not shown). In contrast, only one phosphate group was added by CK2 (Fig. 1B). For all three samples, phosphorylated Mcm2-(10-294) was digested with trypsin, and the resulting peptides were analyzed by MALDI-TOF mass spectrometry. Comparison of spectra obtained before and after phosphatase treatment allowed us to determine that Cdk2/CycA and Cdk1/CycB kinase complexes showed a completely superimposable pattern with three phosphorylated peptides (regions 10-17, 20-30, 34-44). Nano-ESI MS/MS analysis on the selected peptides identified Ser-13, Ser-27, and Ser-41 as phosphorylated residues (supplemental Fig. S1 and data not shown). Two of three of these sites, Ser-27 and Ser-41, correspond to canonical S/TPXR/K CDK consensus sites (21Endicott J.A. Noble M.E. Tucker J.A. Curr. Opin. Struct. Biol. 1999; 9: 738-744Crossref PubMed Scopus (101) Google Scholar), whereas just the partial consensus sequence S/TP was observed in Ser-13. Using the same procedure we found that the peptide 134-149 was stoichiometrically phosphorylated (MW 1973.84) by CK2, and again by nano-ESI MS/MS we confirmed that CK2 can phosphorylate Mcm2 at serine 139 (data not shown) in a context that corresponds to the canonical casein kinase II phosphorylation motif (17Pinna L.A. J. Cell Sci. 2002; 115: 3873-3878Crossref PubMed Scopus (412) Google Scholar). Mutation of serine 139 into alanine completely abolished CK2 phosphorylation, clearly indicating that Ser-139 is the only amino acid that can be modified by the kinase in the Mcm2-(10-294) fragment (supplemental Fig. S2A). Upon tryptic digestion of MCM2-(10-294) phosphorylated by Cdc7, three different phosphopeptides were detected (34-44, 45-79, and 83-115). The phosphorylated form of peptide 83-115 after partial digestion with AspN endoprotease could be analyzed by nano-ESI MS/MS, and phosphorylation on Ser-108 was clearly detected (Fig. 2A). The nano-ESI MS/MS analysis of the other two peptides was not successful due to the large molecular weight of peptide 45-79 and to the poor ionization properties and low stoichiometry of peptide 36-44 after Cdc7 phosphorylation. Therefore the corresponding synthetic peptides (36-44 and 49-64) were produced, and upon in vitro phosphorylation with Cdc7/Dbf4, pSer-53 (Fig. 2B), and pSer-40 (Fig. 2C) were identified as specific phosphoamino acids. Mcm2 Ser-40, Mcm2 Ser-53, and Mcm2 Ser-108 are the first specific sites reported for Cdc7 kinase. Alignment of the sequences surrounding phosphorylated serines did not reveal an obvious consensus motif, although we observed that acidic amino acids surround both Ser-53 and Ser-108. Intriguingly, Ser-108 is the same residue that was found phosphorylated by ATR kinase (14Cortez D. Glick G. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10078-10083Crossref PubMed Scopus (264) Google Scholar) by two separate groups. We then independently confirmed that immunoprecipitated ATR phosphorylates Mcm2 N terminus at Ser-108 and that Ser-108 is the only residue phosphorylated by this kinase in this Mcm2 fragment (supplemental Fig. S2B). We also noticed that Cdc7 phosphorylation on Ser-40 immediately precedes Ser-41, which in the previous experiment was found phosphorylated by Cdk2. If surrounding negative charges are needed for better substrate recognition by Cdc7, then the introduction of a phosphate group in position +1 (Ser-41) may facilitate phosphorylation by Cdc7 on Ser-40. This observation prompted us to further investigate the relationships between Cdk2 and Cdc7 in the phosphorylation of a MCM2 peptide spanning this region (amino acids 36-44). As expected, both kinases were able to independently phosphorylate the peptide, and MS/MS analysis confirmed that only Ser-40 was phosphorylated by Cdc7 (Fig. 2C) and only Ser-41 was phosphorylated by Cdk2 (supplemental Fig. S1). We then tested whether the same peptide could be phosphorylated on both residues. Peptide 36-44 was first incubated with Cdk2, and products of the reaction were isolated by high performance liquid chromatography as a single phosphorylated peptide on Ser-41. This was then incubated with Cdc7 kinase, and MALDI analysis indicated that a second phosphate group was transferred. MS/MS analysis confirmed that both Ser-40 and Ser-41 were modified (Fig. 2D). Thus, under these experimental conditions Cdc7 and Cdk2 phosphorylation of this peptide are not mutually exclusive. By MALDI-MS we observed that pre-phosphorylation of Mcm2 peptide 36-44 on Ser-41 by Cdk2 appears to increase the efficiency of Cdc7 phosphorylation on Ser-40 (data not shown). To further explore this phenomenon we compared the efficiency of Cdc7 kinase in the phosphorylation of two synthetic peptides spanning this region, either unphosphorylated or monophosphorylated in Ser-41. Indeed, we found that Cdc7 activity was increased ∼3-fold when the phosphorylated pSer-41 peptide was used as substrate (Fig. 2E). This result supports the hypothesis that the negatively charged residues may facilitate phosphorylation by Cdc7. Alignment of N-terminal tails of Mcm2 proteins from human, mouse, and Xenopus indicates that the serines phosphorylated by Cdc7 and CK2 are highly conserved, whereas only two of three sites identified for Cdk phosphorylation are found in all three species (Fig. 3). Characterization of Anti-phospho-Mcm2 Antibodies—With the goal of verifying whether the phosphosites that were mapped in Mcm2 protein in vitro with purified kinases were also phosphorylated in vivo, we raised rabbit polyclonal antibodies against synthetic phosphopeptides spanning Ser-13, Ser-53, Ser-108, and Ser-139. We also immunized animals with a peptide carrying double-phosphorylated Ser-40 and Ser-41. Specific anti-pSer-13, -pSer-108, and -pSer-139 antibodies were then affinity purified on antigen columns. Anti-pSer-27 and anti-pSer-41 antibodies were purchased from a commercial source. To check the specificity of the immunological reagents, purified antibodies were used as probes in Western blot experiments against recombinant Mcm2 mock treated or phosphorylated with the relevant kinase. We found that anti-pSer-13 antibody specifically recognizes Mcm2 phosphorylated by either Cdk2 or Cdk1 kinases (Fig. 4A), that anti-pSer-139 recognizes Mcm2 phosphorylated by CK2 but not by Cdc7 (Fig. 4B), and that anti-pSer-108 and anti-pSer-53 recognize Mcm2 phosphorylated by Cdc7 (Fig. 4C). Finally, to characterize the antibodies obtained by immunization with the double-phosphorylated Ser-40 and Ser-41 peptide, we tested them against a panel of four synthetic peptides spanning amino acids 34 to 49 either unphosphorylated, mono-phosphorylated on Ser-40 or Ser-41, or double phosphorylated on Ser-40 plus Ser-41. We found that these antibodies only recognized the double-phosphorylated peptide (Fig. 4D, top panel), and therefore this reagent will be defined as anti-Ser-40/41 antibody throughout the entire work. Consistent with MS data when mono-phosphorylated peptide on Ser-41 was incubated with Cdc7 kinase, phosphorylation at Ser-40 was also easily detected using the anti-pSer-40/41 antibody (Fig. 4D, middle panel). Importantly a double-phosphorylated peptide cannot be detected after incubation of mono-phosphorylated Ser-40 peptide with Cdk2, strongly suggesting that previous phosphorylation in Ser-40 prevents substrate recognition by this kinase (Fig. 4D, bottom panel). Using the same set of peptides we tested the specificity of the commercial anti-pSer-41 antibody. We found that this reagent equally recognizes the mono-phosphorylated peptide in Ser-41 and the double-phosphorylated peptides in Ser-40 and Ser-41 (Fig. 4E, top pane" @default.
• W2012172422 created "2016-06-24" @default.
• W2012172422 creator A5011360407 @default.
• W2012172422 creator A5018690973 @default.
• W2012172422 creator A5040015786 @default.
• W2012172422 creator A5040236379 @default.
• W2012172422 creator A5062095629 @default.
• W2012172422 creator A5063451090 @default.
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• W2012172422 creator A5088097104 @default.
• W2012172422 date "2006-04-01" @default.
• W2012172422 modified "2023-10-01" @default.
• W2012172422 title "Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases" @default.
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