Inhibition of Mcm4,6,7 Helicase Activity by Phosphorylation with Cyclin A/Cdk2

Ishimi, Yukio; Komamura-Kohno, Yuki; You, Zhiying; Omori, Akira; Kitagawa, Masatoshi

doi:10.1074/jbc.m909040199

Cited by 89 publications

(78 citation statements)

References 49 publications

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“…Biochemical and genetic evidence which showed Cdc2-mediated phosphorylation of MCM4 was reported previously (27,28). Ishimi et al (43) recently reported that Cdk2-Cyclin A phosphorylates MCM4 and that this phosphorylation inhibits DNA helicase activity of MCM4-6-7 complex. Phosphorylation of MCM2 by Cdks did not cause any additional mobility shift on SDS-PAGE (Fig.…”

Section: Phosphorylation Of Mcm2 Complex and T-antigen By Hucdc7mentioning

confidence: 93%

Human Cdc7-related Kinase Complex

Masai¹,

Matsui²,

You³

et al. 2000

Journal of Biological Chemistry

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144

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huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

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Section: Phosphorylation Of Mcm2 Complex and T-antigen By Hucdc7mentioning

confidence: 93%

Human Cdc7-related Kinase Complex

Masai¹,

Matsui²,

You³

et al. 2000

Journal of Biological Chemistry

Self Cite

144

View full text Add to dashboard Cite

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“…It is likely that they act as a DNA-unwinding enzyme in DNA replication. We reported that the DNA helicase activity of the human and mouse MCM4-6-7 complex, a sub-complex of the MCM2-7 heterohexamer, is inhibited by the phosphorylation by CDK2-cyclin A (13,39). We identified six sites, including Ser-32, Ser-53, and Thr-109, in the amino-terminal region of mouse MCM4 that are required for the phosphorylation with CDK2-cyclin A.…”

Section: Fig 4 Phosphorylation Of Mcm4 By Cyclin-dependent Kinasementioning

confidence: 99%

Identification of MCM4 as a Target of the DNA Replication Block Checkpoint System

Ishimi

Komamura-Kohno

Kwon

et al. 2003

Journal of Biological Chemistry

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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 1 (2-7) complex, which is loaded with CDC6 and CDT1 onto replication origins to which ORCs are bound (1-3). 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 (4, 5). 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 (6 -9). Thus, it is possible that the MCM4-6-7 hexamer is an activated form of the MCM (2-7) hexamer, although other mechanisms leading to the activation of the MCM helicase activity have been proposed (10). MCM4 is phosphorylated in vivo at least in part by cyclin-dependent kinases, which probably leads to the inactivation of the MCM complex (11, 12). We reported previously (13) that the DNA helicase activity of the MCM4-6-7 complex is inhibited by the sitespecific 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 (14). Other targets of the cyclin-dependent kinase activity that contribute to the negative regulation of DNA replication include ORC, CDC6, and CDT1 (15).Cells normally protect the integrity of their genome from stresses such as ultraviolet light, ionizing radiation, alkylating reagents, and DNA replication blockage (16 -21). 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 ...

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“…We have recently shown that concerted action of Cdk is required for efficient phosphorylation of MCM complex by human Cdc7 in vitro (Masai et al, 2000). Phosphorylation of MCM by Cdk in vivo and in vitro and its effect on chromatin association and helicase activities have been reported (Hendrickson et al, 1996;Ishimi et al, 2000). It is likely that functions of MCM are under complex regulation of multiple sets of kinases during cell cycle.…”

Section: Hsk1 Functions For Initiation Of Dna Replication Through Phomentioning

confidence: 99%

Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

Takeda

Ogino

Tatebayashi

et al. 2001

MBoC

108

132

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Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase. INTRODUCTIONDNA replication needs to be stringently regulated for cell growth and cell division to occur in a coordinated manner (Stillman, 1996). Initiation of DNA replication requires assembly of multiprotein complexes at chromosomal replication origins during late M to early G1 phase Newlon, 1997). This complex, termed prereplicative complex (preRC), includes ORC (Bell and Stillman, 1992), Cdc6 (Cocker et al., 1996), and MCM proteins (Tye, 1994;Kearsey et al., 1995;Chong et al., 1996;Donovan et al., 1997), which are conserved from yeasts to human. After cells pass Start, the preRC is activated and DNA synthesis is initiated at replication origins. This process is accompanied with dissociation of Cdc6 (Cocker et al., 1996;Tanaka et al., 1997) and of at least some components of the MCM complex from origins Aparicio et al., 1997;Kubota et al., 1997;Tanaka et al., 1997), resulting in a postreplicative complex (postRC), which is inactive until the next cell cycle. The firing of origins requires actions of at least two distinct serine/threonine kinases, namely, G1/S-specific CDK-Cyclin (Nasmyth, 1996;Stillman, 1996) and Cdc7-Dbf4 (Hartwell, 1971, 1973.Saccharomyces cerevisiae CDC7 encodes a serine/threonine protein kinase required for the onset of DNA replication (Hollingsworth et al., 1992;Jackso...

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Inhibition of Mcm4,6,7 Helicase Activity by Phosphorylation with Cyclin A/Cdk2

Cited by 89 publications

References 49 publications

Human Cdc7-related Kinase Complex

Human Cdc7-related Kinase Complex

Identification of MCM4 as a Target of the DNA Replication Block Checkpoint System

Regulation of Initiation of S Phase, Replication Checkpoint Signaling, and Maintenance of Mitotic Chromosome Structures during S Phase by Hsk1 Kinase in the Fission Yeast

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