FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2019928852> ?p ?o ?g. }

• W2019928852 endingPage "24650" @default.
• W2019928852 startingPage "24644" @default.
• W2019928852 abstract "Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression, through inactivation of the MCM complex. Inhibition of the progression of DNA replication prevents further initiation of DNA replication and allows cells to maintain arrested replication forks, but the proteins that are targets of the replication checkpoint system remain to be identified. We report here that human MCM4, a subunit of the putative DNA replicative helicase, is extensively phosphorylated in HeLa cells when they are incubated in the presence of inhibitors of DNA synthesis or are exposed to UV irradiation. The data presented here indicate that the consecutive actions of ATR-CHK1 and CDK2 kinases are involved in this phosphorylation in the presence of hydroxyurea. The phosphorylation sites in MCM4 were identified using specific anti-phosphoantibodies. Based on results that showed that the DNA helicase activity of the MCM4-6-7 complex is negatively regulated by CDK2 phosphorylation, we suggest that the phosphorylation of MCM4 in the checkpoint control inhibits DNA replication, which includes blockage of DNA fork progression, through inactivation of the MCM complex. The initiation of DNA replication in eukaryotes is triggered by activation of the MCM 1The abbreviations used are: MCM, minichromosome maintenance; ATM, ataxia telangiectasia-mutated; ATR, ATM-Rad3-related; CDK2, cyclin-dependent kinase 2; CHK, checkpoint kinase; HU, hydroxyurea; Pipes, 1,4-piperazinediethanesulfonic acid; aa, amino acid; pT, phospho-T. 1The abbreviations used are: MCM, minichromosome maintenance; ATM, ataxia telangiectasia-mutated; ATR, ATM-Rad3-related; CDK2, cyclin-dependent kinase 2; CHK, checkpoint kinase; HU, hydroxyurea; Pipes, 1,4-piperazinediethanesulfonic acid; aa, amino acid; pT, phospho-T. (2Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Google Scholar, 4Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Google Scholar, 5Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Google Scholar, 6Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Google Scholar, 7You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Google Scholar) complex, which is loaded with CDC6 and CDT1 onto replication origins to which ORCs are bound (1Tye B.K. Annu. Rev. Biochem. 1999; 68: 649-686Google Scholar, 2Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Google Scholar). The activation of the MCM complex requires phosphorylation of the MCM2 subunit by CDC7/DBF4 kinase and association of CDC45 with the origin region after the action of cyclin-dependent kinase. The MCM2-7 complex is the most likely candidate to act as the DNA replication helicase that catalyzes the unwinding of the DNA duplex during replication (4Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Google Scholar, 5Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Google Scholar). Although all MCM subunits possess a DNA-dependent ATPase motif in their central domain, DNA helicase activity is detected only with the MCM4-6-7 complex, which dimerizes to form a hexamer (6Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Google Scholar, 7You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Google Scholar, 8Lee J.-K. Hurwitz J. J. Biol. Chem. 2000; 275: 18871-18878Google Scholar, 9Lee J.-K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 54-59Google Scholar). Thus, it is possible that the MCM4-6-7 hexamer is an activated form of the MCM (2Kelly T.J. Brown G.W. Annu. Rev. Biochem. 2000; 69: 829-880Google Scholar, 3Bell S.P. Dutta A. Annu. Rev. Biochem. 2002; 71: 333-374Google Scholar, 4Aparicio O.M. Weinstein D.M. Bell S.P. Cell. 1997; 91: 59-69Google Scholar, 5Labib K. Tercero J.A. Diffley J.F. Science. 2000; 288: 1643-1647Google Scholar, 6Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Google Scholar, 7You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Google Scholar) hexamer, although other mechanisms leading to the activation of the MCM helicase activity have been proposed (10Tye B.K. Sawyer S. J. Biol. Chem. 2000; 275: 34833-34836Google Scholar). MCM4 is phosphorylated in vivo at least in part by cyclin-dependent kinases, which probably leads to the inactivation of the MCM complex (11Hendrickson M. Madine M. Dalton S. Gautier J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12223-12228Google Scholar, 12Fujita M. Yamada C. Tsurumi T. Hanaoka F. Matsuzawa K. Inagaki M. J. Biol. Chem. 1998; 273: 17095-17101Google Scholar). We reported previously (13Ishimi Y. Komamura-Kohno Y. J. Biol. Chem. 2001; 276: 34428-34433Google Scholar) that the DNA helicase activity of the MCM4-6-7 complex is inhibited by the site-specific phosphorylation of MCM4 with CDK2-cyclin A. In Saccharomyces cerevisiae, it has been shown that cyclin-dependent kinases play a role leading to the exclusion of MCM4 from the nucleus (14Labib K. Diffley J.F. Kearsey S.E. Nat. Cell Biol. 1999; 1: 415-422Google Scholar). Other targets of the cyclin-dependent kinase activity that contribute to the negative regulation of DNA replication include ORC, CDC6, and CDT1 (15Blow J.J. Hodgson B. Trends Cell Biol. 2002; 12: 72-78Google Scholar).Cells normally protect the integrity of their genome from stresses such as ultraviolet light, ionizing radiation, alkylating reagents, and DNA replication blockage (16Zhou B.-B. Elledge S.J. Nature. 2000; 408: 433-439Google Scholar, 17Rhind N. Russell P. J. Cell Sci. 2000; 113: 3889-3896Google Scholar, 18Abraham R.T. Genes Dev. 2001; 15: 2177-2196Google Scholar, 19Donaldson A.D. Blow J.J. Curr. Biol. 2001; 11: R979-R982Google Scholar, 20Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Google Scholar, 21Marchetti M.A. Kumar S. Hartsuiker E. Maftahi M. Carr A.M. Freyer G.A. Burhans W.C. Huberman J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7472-7477Google Scholar). Treatment with hydroxyurea (HU), which inhibits ribonucleotide reductase, not only blocks the progression of DNA replication but also activates a DNA replication checkpoint system that is required to maintain genomic integrity. In the presence of HU or methyl methanesulfonate, the initiation of DNA replication at late origins is prevented (22Santocanale C. Diffley J.F.X. Nature. 1998; 395: 615-618Google Scholar, 23Shirahige K. Hori Y. Shiraishi K. Yamashita M. Takahashi K. Obuse C. Tsurimoto T. Yoshikawa H. Nature. 1998; 395: 618-621Google Scholar, 24Tercero J.A. Diffley J.F.X. Nature. 2001; 412: 553-557Google Scholar), and the arrested replication fork structure is maintained (25Lopes M. Cotta-Ramusino C. Pellicioli A. Liberi G. Plevani P. Muzi-Falconi M. Newlon C.S. Foiani M. Nature. 2001; 412: 557-561Google Scholar) by an active process that includes protein phosphorylation by the Mec1 and Rad53 kinases in S. cerevisiae. Recently, Sogo et al. (26Sogo J.M. Lopes M. Foiani M. Science. 2002; 297: 599-602Google Scholar) showed that the accumulation of single-stranded DNA and replication fork reversal occur at stalled replication forks in the absence of checkpoint control. It is plausible that these abnormal DNA structures lead to a loss of genome integrity. Mec1 with Rad3-1-1 kinases are known to be sensors of the arrested fork structure, and Rad53 is an effector kinase that is activated by its phosphorylation by Mec1. Several target proteins in the replication checkpoint pathway have been identified. Rad53 phosphorylates Dbf4 to attenuate the Cdc7/Dbf4 kinase activity (27Weinreich M. Stillman B. EMBO J. 1999; 18: 5334-5346Google Scholar, 28Kihara M. Nakai W. Asano S. Suzuki A. Kitada K. Kawasaki Y. Johnston L.H. Sugino A. J. Biol. Chem. 2000; 275: 35051-35062Google Scholar, 29Snaith H.A. Brown G.W. Forsburg S.L. Mol. Cell. Biol. 2000; 20: 7922-7932Google Scholar), and replication protein A, a single-stranded DNA-binding protein, is phosphorylated by Mec1 in an HU-dependent manner (30Brush G.S. Kelly T.J. Nucleic Acids Res. 2000; 28: 3725-3732Google Scholar). The phosphorylation of DNA polymerase α is also regulated in this system (31Pellicioli A. Lucca C. Liberi G. Marini F. Lopes M. Plevani P. Romano A. Di Fiore P.P. Foiani M. EMBO J. 1999; 18: 6561-6572Google Scholar). However, it remains unclear whether these targets are necessary and sufficient for the checkpoint reactions (32Jares P. Donaldson A. Blow J.J. EMBO Rep. 2000; 1: 319-322Google Scholar). In higher eukaryotes, ATR (Mec1 homolog), which binds to the arrested fork structure with other sensor proteins, phosphorylates CHK1, an effector kinase, leading to its activation (33Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Google Scholar, 34Feijoo C. Hall-Jackson C. Wu R. Jenkins D. Leitch J. Gilbert D.M. J. Cell Biol. 2001; 154: 913-923Google Scholar). Chinese hamster ovary cells lacking CHK1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication (34Feijoo C. Hall-Jackson C. Wu R. Jenkins D. Leitch J. Gilbert D.M. J. Cell Biol. 2001; 154: 913-923Google Scholar). Target protein(s) in the replication checkpoint system remain to be identified.In this study we identified MCM4 as a target of the replication checkpoint system. The results suggest that the consecutive actions of ATR-CHK1 and CDK2 are required for the phosphorylation of MCM4. The phosphorylation of MCM4 should inhibit DNA replication through the inactivation of the MCM complex.EXPERIMENTAL PROCEDURESReagents—Caffeine, hydroxyurea, and 2-aminopurine were purchased from Sigma, and Gö6976 was from Calbiochem. Aphidicolin was purchased from Wako Pure Chemical. Antibodies against CHK1 were purchased from Santa Cruz Biochemicals, and CDK2-Thr-160 phosphoantibodies were from Cell Signaling. λ-Phosphatase was from New England Biolabs. Anti-MCM4 antibodies were obtained as reported (35Kimura H. Nozaki N. Sugimoto K. EMBO J. 1994; 13: 4311-4320Google Scholar).Preparation of Cell Fractions and Western Blotting—Cells were lysed at 2 × 106 cells/100 μl in modified CSK buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, 1 mm MgCl2, and 1 mm EGTA) containing 0.1% Triton X-100, 1 mm ATP, proteinase inhibitors (Pharmingen), and phosphatase inhibitors (10 mm sodium β-glycerophosphate, 5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 50 mm sodium fluoride) (solution A) and placed on ice for 15 min. The cell suspension was centrifuged (5000 rpm for 5 min in a microcentrifuge), and the supernatant (S1) was saved. The recovered precipitate was suspended in solution A, and the supernatant obtained after centrifugation was saved (S2). The precipitate (P) was suspended in a volume of solution A to yield 4 × 106 cells/100 μl. In some experiments, the suspended P fractions were incubated with 1.4 units/μl of DNase I (Takara) for 15 min at 30 °C. The solution was centrifuged to obtain the soluble S4 fraction and the residual P′ fraction. The Triton-soluble (S1 and S2) and chromatin-bound (S4, P′, and P) fractions were electrophoresed through 10 (for MCM4 and CHK1) or 15% (for CDK2) acrylamide gels containing SDS and then transferred to membranes (Immobilon, Millipore). Membranes were incubated at 37 °C for 1 h with primary antibodies in blocking solution (Blockace, Dai-nippon Pharmaceuticals) or 5% bovine serum albumin in TBS (50 mm Tris-HCl, pH 7.5, and 0.15 m NaCl) plus 0.1% Triton X-100. After washing with TBS plus 0.1% Triton X-100, membranes were incubated with peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Bio-Rad). The immunoreacted proteins were detected using a chemiluminescent detection system (Super-Signal West Pico or Femto Maximum Sensitivity Substrate, Pierce), and the level of reactivity was quantified (Cool Saver AE-6935, Atto Instruments).Preparation of Anti-phosphoantibodies—Antisera against phosphothreonine and phosphoserine at amino acids 7, 19, 32, 54, and 110 of human MCM4 were obtained by immunizing rabbits with a synthetic peptide of NH2-MSSPAS(pT)PSRRGSRRGRAC-COOH (for aa 7), NH2-SRRGRA(pT)PAQTPRSEC-COOH (for aa 19), NH2-CRSEDARS-(pS)PSQRRRG-COOH (for aa 32), NH2-CELQPMPT(pS)PGVDLQS-COOH (for aa 54), or NH2-CGTPRSGVRG(pT)PVRQRPDL (for aa 110); the antibodies were conjugated with the keyhole limpet at each amino terminus. Anti-phosphoantibodies were purified by phosphopeptide column chromatography. After the serum (10 ml) was loaded onto a phosphopeptide column (2 ml) that had been prepared by fixing the above peptides to CNBr-activated Sepharose, antibodies were eluted with 0.1 m glycine, pH 2.5, and 0.15 m NaCl. Eluted fractions were neutralized, dialyzed against phosphate-buffered saline, and then passed through a non-phosphopeptide-Sepharose column (2 ml). The material that passed through the column was concentrated and used as the anti-phosphoantibodies described below.In Vitro Phosphorylation—Human CDK2-cyclin A complex was prepared as reported (13Ishimi Y. Komamura-Kohno Y. J. Biol. Chem. 2001; 276: 34428-34433Google Scholar). Histidine-tagged human CHK1 and CHK2 proteins were expressed in insect cells using a baculovirus expression system (36Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Kaneko Y. Oncogene. 1999; 18: 3673-3681Google Scholar, 37Tominaga K. Morisaki H. Kaneko Y. Fujimoto A. Tanaka T. Ohtsubo M. Hirai M. Okayama H. Ikeda K. Nakanishi M. J. Biol. Chem. 1999; 274: 31463-31467Google Scholar) and purified by nickel-nitrilotriacetic acid column chromatography according to the manufacturer's protocol (Qiagen). Human MCM4 and -6 genes were cloned into a pAcUW31 vector (7You Z. Komamura Y. Ishimi Y. Mol. Cell. Biol. 1999; 19: 8003-8015Google Scholar), and the MCM7 gene was cloned into a pVL1392 vector for baculoviral expression. An MCM4-6-7 complex containing histidine-tagged MCM4 was purified from the viral infected High5 cells by nickel-nitrilotriacetic acid column chromatography and then MonoQ column chromatography. The human MCM4-6-7 complex (50 ng) was incubated with increasing levels of CHK1 (120 and 400 ng) in 50 mm Hepes-NaOH, pH 8, 10 mm MgCl2, 2.5 mm EGTA, 1 mm dithiothreitol, and 100 μm [γ-32P]ATP or incubated with CDK2-cyclin A (20, 60, and 200 ng) in 20 mm Tris-HCl, pH 7.5, 30 mm NaCl, 10 mm MgCl2, 0.01% Triton X-100, and 100 μm [γ-32P]ATP. The reaction mixtures were incubated for1hat37 °C, and the products were analyzed by electrophoresis on 10% polyacrylamide gel containing SDS.Purification of MCM4-6-7 Complex from HeLa Cells—HeLa cells were cultured in the presence or absence of HU (2 mm) for 24 h. The harvested cells (∼1 × 108 cells) were washed with phosphate-buffered saline and then stored at –80 °C. After thawing, the cells were suspended in modified CSK buffer containing phosphatase inhibitors (10 mm sodium β-glycerophosphate, 5 mm sodium pyrophosphate, 1 mm sodium orthovanadate, and 50 mm sodium fluoride) in the absence of Triton X-100 and kept at 0 °C for 15 min. The cell precipitate recovered after centrifugation was then suspended in CSK buffer containing 0.1% Triton X-100, 0.4 m NaCl, and phosphatase inhibitors and kept at 0 °C for 15 min. The recovered supernatant fraction contained about half the chromatin-bound MCM proteins. After centrifugation, the solubilized proteins were loaded onto a histone H3/H4 column (2 ml) that had been equilibrated with a buffer containing 0.3 m NaCl (6Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Google Scholar). The column was extensively washed with the same buffer, and proteins were eluted with a linear gradient of 0.3–2 m NaCl. The fractions containing MCM4, -6, and -7 proteins that were eluted with 0.6–0.9 m NaCl were pooled and concentrated with Centricon 30 (Amicon) in the presence of phosphatase inhibitors (Phosphatase Inhibitor Mixture 1 plus 2 from Sigma) and protease inhibitors. During the concentration, the solution was diluted to 0.15 m NaCl. The diluted and concentrated sample was further purified by glycerol gradient centrifugation. Fractions 3–6 of a total of 15 fractions were pooled and concentrated to 20 μl with Microcon (Amicon) after the addition of proteinase inhibitors. The concentrated sample, which contained ∼35 μg/ml proteins, was used for measuring the displacement of 17-mer oligonucleotide annealed to M13 single-stranded DNA by DNA helicase activity (6Ishimi Y. J. Biol. Chem. 1997; 272: 24508-24513Google Scholar).Another procedure was employed to obtain total cell extracts in the absence of phosphatase inhibitors. HeLa cells were washed with hypotonic buffer (20 mm Hepes, pH 7.5, 5 mm KCl, 1.5 mm MgCl2, and 1 mm dithiothreitol) and then homogenized with Dounce homogenizer (B pestle). Proteins extracted with 0.4 m NaCl in the same solution were loaded onto a histone H3/H4 column equilibrated with 0.3 m NaCl, and bound proteins were eluted by the linear gradient from 0.3 to 2 m NaCl. Fractions containing MCM4, -6, and -7 proteins were pooled and further purified by glycerol gradient centrifugation as described above, except that fractions obtained after glycerol gradient centrifugation were used without concentration for measuring DNA helicase activity.UV Irradiation of HeLa Cells—HeLa cells that were logarithmically growing in dishes (100 mm in diameter) were exposed to UV irradiation (100 J/m2) from a germicidal lamp (Toshiba GL15) at 2.5 J/m2 in the absence of growth medium. Fresh growth medium (Dulbecco's modified Eagle's medium plus 10% fetal bovine serum) was added to the cells, and they were cultured at 37 °C for the indicated periods. Cells harvested after incubation with trypsin/EDTA solution were washed with phosphate-buffered saline and stored at –80 °C before fractionation of the cells.RESULTSPhosphorylation of MCM4 in the Presence of HU—Logarithmically growing HeLa cells were cultured in the presence of HU for different periods (0, 4, 8, 16, and 24 h). After lysis, cells were fractionated into Triton-soluble (S1 and S2) and Triton-insoluble chromatin-bound fractions (P). The proteins in these fractions were reacted with anti-MCM4 antibodies (Fig. 1A, top panel). MCM4 was detected in both the soluble (S1) and the insoluble chromatin-bound (P) fractions isolated from cells cultured in the absence of HU. A portion of the MCM4 with slightly retarded mobility was detected in the P fraction. During incubation in the presence of HU, the level of MCM4 in the P fraction with retarded mobility gradually increased with maximal retardation observed after 16 h. In contrast, the mobility of the MCM4 in the S1 fraction did not change during the 24 h of incubation in the presence of HU. The alteration in the mobility of MCM4 appeared to be due to its phosphorylation because after λ-phosphatase treatment it migrated to a position identical to that observed for the MCM4 isolated from the S1 fraction (Fig. 1B). The phosphorylation of CHK1 was examined during incubation of cells with HU (Fig. 1A, middle panel). Retardation of its mobility, which is due to phosphorylation by ATR (34Feijoo C. Hall-Jackson C. Wu R. Jenkins D. Leitch J. Gilbert D.M. J. Cell Biol. 2001; 154: 913-923Google Scholar), was detected over a time course similar to that observed for the hyperphosphorylation of MCM4. It remains to be determined why the total amount of CHK1 protein decreases after 24 h of incubation with HU.Checkpoint-dependent Phosphorylation of MCM4 —To address the question of whether the observed phosphorylation of MCM4 is due to the action of the DNA replication checkpoint system, caffeine, an inhibitor of ATR/ATM, which plays a central role in the checkpoint system, was added to the growth medium containing HU. As shown in Fig. 2 (top panel), this addition markedly reduced the hyperphosphorylation of MCM4 isolated from the P fraction. The reduced level of hyperphosphorylated MCM4 in the presence of caffeine appears to be associated with the dephosphorylation of CHK1 protein, which is evident from the mobility shift of retarded CHK1 to the basal position (Fig. 2, middle panel). To clarify the role played by CHK1 kinase in the hyperphosphorylation of MCM4, Gö6976, an inhibitor of CHK1 kinase (38Kohn E.A. Yoo C.J. Eastman A. Cancer Res. 2003; 63: 31-35Google Scholar) but not CDK2 kinase or CHK2 kinase activity (Fig. 3, A and B), was added to the medium containing HU. The drug inhibited the hyperphosphorylation in a dose-dependent manner (Fig. 3C, top panel). At a concentration of 600 nm, hyperphosphorylation of MCM4 was largely prevented, whereas phosphorylation of CHK1 protein itself was unaffected by Gö6976 (Fig. 3C, middle panel), indicating that this drug does not affect ATR kinase activity. These findings suggest that an ATR-CHK1 pathway is involved in the hyperphosphorylation of MCM4.Fig. 2Caffeine blocks HU-induced hyperphosphorylation of MCM4. HeLa cells were cultured for 24 h in the presence or absence of 2 mm HU and 5 mm caffeine as indicated. Triton-soluble fraction (S1) and the chromatin fraction (P)(8 μl each) were examined for MCM4 (top panel), CHK1 (middle panel), and CDK2-Thr-160 phosphorylation (bottom panel) by immunoblotting analysis.View Large Image Figure ViewerDownload (PPT)Fig. 3Influence of Gö6976 on the in vitro and in vivo phosphorylation of key proteins.A, in vitro effects of Gö6976 on the phosphorylation of CDC25c and MCM4. GST-CDC25c (120 ng) (36Kaneko Y.S. Watanabe N. Morisaki H. Akita H. Fujimoto A. Tominaga K. Terasawa M. Tachibana A. Ikeda K. Nakanishi M. Kaneko Y. Oncogene. 1999; 18: 3673-3681Google Scholar) was phosphorylated with human CHK1 (200 ng) (left panel) or human CHK2 (150 ng) (right panel) in the absence or presence of increasing levels of Gö6976 under the conditions described under “Experimental Procedures.” Human MCM2-4-6-7 (90 ng) prepared from HeLa cells was incubated with CDK2-cyclin A (35 ng) under the same conditions (middle panel). Proteins were analyzed on 15–25% polyacrylamide gel containing SDS followed by autoradiography. The positions of phosphorylated GST-CDC25c, CHK1, CHK2, cyclin A, MCM2 and -4 are indicated. Reactions with kinases as substrates were electrophoresed in the left-hand lanes of each gel. B, quantification of the inhibitory effect of Gö6976 on the phosphorylation of GST-CDC25c by CHK1 (Xes), CHK2 (open circles), and the phosphorylation of MCM2 and -4 by CDK2-cyclin A (filled circles). Radioactivity incorporated into each substrate in the absence of Gö6976 was taken as 100%. C, in vivo effect of Gö6976. HeLa cells were cultured for 24 h in the presence or absence of HU and increasing concentrations of Gö6976. Specific fractions prepared from the treated HeLa cells, as described under “Experimental Procedures,” were examined for MCM4 (P fraction only, top), CHK1 (S1 only, middle), and CDK2-Thr-160 (S1 and S2, bottom) phosphorylation.View Large Image Figure ViewerDownload (PPT)Involvement of Cyclin-dependent Kinase in MCM4 Phosphorylation—In the presence of HU, ATR kinase phosphorylates CHK1 kinase, an effector kinase in the checkpoint system, to activate it in HeLa cells (Fig. 1A) (34Feijoo C. Hall-Jackson C. Wu R. Jenkins D. Leitch J. Gilbert D.M. J. Cell Biol. 2001; 154: 913-923Google Scholar). To identify the kinase responsible for MCM4 hyperphosphorylation in the presence of HU, we first phosphorylated the human MCM4-6-7 complex with a human CHK1 kinase preparation in vitro. Both MCM4 and -6 proteins in the complex appeared to be phosphorylated (Fig. 4A, right panel), but phosphorylation with CHK1 did not lead to the generation of highly phosphorylated forms of MCM4 (Fig. 4B, 1st column on the left panel). In contrast, MCM4 products with retarded mobility were generated by phosphorylation with CDK2-cyclin A (Fig. 4, A, left panel, and B, 1st column on the left panel). We reported previously (39Ishimi Y. Komamura-Kohno Y. You Z. Omori A. Kitagawa M. J. Biol. Chem. 2000; 275: 16235-16241Google Scholar) that mouse MCM4 in an MCM4-6-7 complex was extensively phosphorylated with CDK2-cyclin A and that this phosphorylation inhibited the DNA helicase activity of the complex. Phosphorylation in this system of mutant mouse MCM4-6-7 complexes, where amino acids of MCM4 were altered in a site-specific manner, indicated that six sites (Ser-3, Thr-7, Thr-19, Ser-32, Ser-53, and Thr-109) in the amino-terminal region of mouse MCM4 were required for the phosphorylation of MCM4 (13Ishimi Y. Komamura-Kohno Y. J. Biol. Chem. 2001; 276: 34428-34433Google Scholar). Specific phosphoantibodies against three (Ser-32, Ser-54, and Thr-110) of these sites in human MCM4 were raised, and they were examined for binding to the phosphorylated MCM4 (Fig. 4B). They interacted with the MCM4-phosphorylated product formed with CDK2-cyclin A but not with the phosphorylated MCM4 product formed with CHK1 (Fig. 4B, 2nd to 4th columns on the left panel). Next, we examined the interaction of these phosphoantibodies with MCM4-hyperphosphorylated products produced in the presence of HU (Fig. 4B, 2nd to 4th columns on the right panel). These phosphoantibodies recognized the hyperphosphorylated MCM4 products that were isolated from the chromatin fractions (S4 and P′) of HU-treated HeLa cells. Quantification of the level of binding of these phosphoantibodies indicates that phosphorylation of MCM4 in the chromatin fractions increased by 2.5–6-fold in the presence of HU. These results strongly suggest that cyclin-dependent kinase activity is involved directly in the HU-induced hyperphosphorylation of MCM4.Fig. 4Phosphorylation of MCM4 by cyclin-dependent kinase.A, human MCM4-6-7 complex (50 ng) was incubated with increasing amounts of CDK2-cyclin A (left panel) or CHK1 (right panel). Phosphorylated proteins were analyzed by electrophoresis on 10% polyacrylamide gel, followed by autoradiography. The maximal level of 32P incorporated into MCM4 by CDK2 was 2.3 mol/mol of MCM4 and that of 32P incorporated into MCM4 + MCM6 by CHK1 was 4 mol/mol of MCM4 + MCM6. B, left panel, human MCM4-6-7 complex was phosphorylated by CHK1 or CDK2-cyclin A in vitro, and products were analyzed by SDS-PAGE. Proteins were analyzed by Western blotting using MCM4 antibodies (1st column) and phosphoantibodies against amino acids Ser-32, Ser-54, and Thr-110 of MCM4 (2nd to 4th columns), as indicated. Right panel, the soluble (S1) and chromatin-bound fractions (S4 and P′) prepared from HeLa cells treated with HU were analyzed by Western blotting using MCM4 and the anti-phosphoantibodies. Bars at the left of gels indicate the normal position of unphosphorylated MCM4.View Large Image Figure ViewerDownload (PPT)To gain more insight on the mechanism by which MCM4 is hyperphosphorylated by a cyclin-dependent kinase, we monitored the activation of CDK2 by examining its phosphorylation at Thr-160 (40Gu Y. Rosenblatt J. Morgan D.O. EMBO J. 1992; 11: 3995-4005Google Scholar) during the incubation of HeLa cells with HU (Fig. 1A, bottom panel). Phosphorylation of Thr-160 increased 2–3-fold after 16 and 24 h of the incubation with HU. The addition of caffeine (Fig. 2, bottom panel) or Gö6976 (Fig. 3C, bottom panel) decreased the level of phosphorylated CDK2 produced in the presence of HU. These results indicate that the activation of CDK2 kinase activity correlates with the increased level of MCM4 hyperphosphorylation induced by HU.Functional Significance of Phosphorylation of MCM4 —To understand the functional significance of phosphorylation of MCM4, DNA helicase activity was compared between MCM4-6-7 complexes prepared from HU-treated and non-treated HeLa cells. After extraction of a large portion of the soluble forms of the MCM complex, approximately half the chromatin-bound MCM complex was recovered by extraction of the remaining insoluble fraction with a buffer containing 0.4 m NaCl (data not shown). The MCM4-6-7 complex was purified from the 0.4 m NaCl-soluble fraction of HU-treated HeLa cells and non-treated cells in the presence phosphatase inhibitors, and it was also purified from total cell extracts of non-treated cells in the absence of phosphatase inhibitors as described under “Experimental Procedures.” As shown in Fig. 5A, these three MCM4-6-7 complexes exhibited similar protein compositions and concentration, although only a slight amount of MCM2 was detectable in the complexes prepared from HU-treated cells in the presence of phosphatase inhibitors and from non-treated cells in the absence of phosphatase inhibitors. Two MCM4 bands that migrated closely on a gel were detected from the complex prepared from non-treated cells in the absence or presence of phosphatase inhibitors, although the ratio of these two bands varied between the two complexes. Only one MCM4 band with retarded mobil" @default.
• W2019928852 created "2016-06-24" @default.
• W2019928852 creator A5008475857 @default.
• W2019928852 creator A5034145502 @default.
• W2019928852 creator A5053138435 @default.
• W2019928852 creator A5080169128 @default.
• W2019928852 creator A5090691633 @default.
• W2019928852 date "2003-07-01" @default.
• W2019928852 modified "2023-10-17" @default.
• W2019928852 title "Identification of MCM4 as a Target of the DNA Replication Block Checkpoint System" @default.
• W2019928852 cites W1529783836 @default.
• W2019928852 cites W1566625041 @default.
• W2019928852 cites W1597133026 @default.
• W2019928852 cites W1752089945 @default.
• W2019928852 cites W1887764107 @default.
• W2019928852 cites W1965596350 @default.
• W2019928852 cites W1966432984 @default.
• W2019928852 cites W1967849546 @default.
• W2019928852 cites W1968074226 @default.
• W2019928852 cites W1973565188 @default.
• W2019928852 cites W1991539934 @default.
• W2019928852 cites W1997266913 @default.
• W2019928852 cites W2005727369 @default.
• W2019928852 cites W2005814109 @default.
• W2019928852 cites W2010841813 @default.
• W2019928852 cites W2026169763 @default.
• W2019928852 cites W2038762172 @default.
• W2019928852 cites W2042732211 @default.
• W2019928852 cites W2046005298 @default.
• W2019928852 cites W2046066638 @default.
• W2019928852 cites W2055604308 @default.
• W2019928852 cites W2056590571 @default.
• W2019928852 cites W2061065780 @default.
• W2019928852 cites W2092506986 @default.
• W2019928852 cites W2093703127 @default.
• W2019928852 cites W2103251875 @default.
• W2019928852 cites W2106130068 @default.
• W2019928852 cites W2108606404 @default.
• W2019928852 cites W2110513450 @default.
• W2019928852 cites W2110902629 @default.
• W2019928852 cites W2111403239 @default.
• W2019928852 cites W2116468279 @default.
• W2019928852 cites W2126517753 @default.
• W2019928852 cites W2128252882 @default.
• W2019928852 cites W2143028761 @default.
• W2019928852 cites W2143611225 @default.
• W2019928852 cites W2146830682 @default.
• W2019928852 cites W2151180466 @default.
• W2019928852 cites W2159536716 @default.
• W2019928852 cites W2160193607 @default.
• W2019928852 cites W2167211051 @default.
• W2019928852 cites W2168215139 @default.
• W2019928852 cites W2329294870 @default.
• W2019928852 doi "https://doi.org/10.1074/jbc.m213252200" @default.
• W2019928852 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12714602" @default.
• W2019928852 hasPublicationYear "2003" @default.
• W2019928852 type Work @default.
• W2019928852 sameAs 2019928852 @default.
• W2019928852 citedByCount "93" @default.
• W2019928852 countsByYear W20199288522012 @default.
• W2019928852 countsByYear W20199288522013 @default.
• W2019928852 countsByYear W20199288522014 @default.
• W2019928852 countsByYear W20199288522015 @default.
• W2019928852 countsByYear W20199288522016 @default.
• W2019928852 countsByYear W20199288522018 @default.
• W2019928852 countsByYear W20199288522019 @default.
• W2019928852 countsByYear W20199288522021 @default.
• W2019928852 countsByYear W20199288522023 @default.
• W2019928852 crossrefType "journal-article" @default.
• W2019928852 hasAuthorship W2019928852A5008475857 @default.
• W2019928852 hasAuthorship W2019928852A5034145502 @default.
• W2019928852 hasAuthorship W2019928852A5053138435 @default.
• W2019928852 hasAuthorship W2019928852A5080169128 @default.
• W2019928852 hasAuthorship W2019928852A5090691633 @default.
• W2019928852 hasBestOaLocation W20199288521 @default.
• W2019928852 hasConcept C104317684 @default.
• W2019928852 hasConcept C105696609 @default.
• W2019928852 hasConcept C116834253 @default.
• W2019928852 hasConcept C12590798 @default.
• W2019928852 hasConcept C159047783 @default.
• W2019928852 hasConcept C185592680 @default.
• W2019928852 hasConcept C2524010 @default.
• W2019928852 hasConcept C2777210771 @default.
• W2019928852 hasConcept C29537977 @default.
• W2019928852 hasConcept C33923547 @default.
• W2019928852 hasConcept C41008148 @default.
• W2019928852 hasConcept C54086632 @default.
• W2019928852 hasConcept C54355233 @default.
• W2019928852 hasConcept C552990157 @default.
• W2019928852 hasConcept C59822182 @default.
• W2019928852 hasConcept C70721500 @default.
• W2019928852 hasConcept C73573662 @default.
• W2019928852 hasConcept C86803240 @default.
• W2019928852 hasConceptScore W2019928852C104317684 @default.
• W2019928852 hasConceptScore W2019928852C105696609 @default.
• W2019928852 hasConceptScore W2019928852C116834253 @default.
• W2019928852 hasConceptScore W2019928852C12590798 @default.
• W2019928852 hasConceptScore W2019928852C159047783 @default.

• next