title: Cdc7-dependent and -independent phosphorylation of Claspin in the induction of the DNA replication checkpoint.
abstract: Claspin is a critical mediator protein in the DNA replication checkpoint, responsible for ATR-dependent activation of the effector kinase Chk1. Cdc7, an essential kinase required for the initiation of DNA replication, can also interact with and phosphorylate Claspin. In this study we use small-molecule inhibitors of Cdc7 kinase to further understand the relationship between Cdc7, Claspin and Chk1 activation. We demonstrate that inhibition of Cdc7 kinase delays HU-induced phosphorylation of Chk1 but does not affect the maintenance of the replication checkpoint once it is established. We find that while chromatin association of Claspin is not affected by Cdc7 inhibition, Claspin phosphorylation is attenuated following HU treatment, which may be responsible for the altered kinetics of HU-induced Chk1 phosphorylation. We demonstrate that Claspin is an in vitro substrate of Cdc7 kinase, and using mass-spectrometry, we identify multiple phosphorylation sites that help to define a Cdc7 phosphorylation motif. Finally, we show that the interaction between Claspin and Cdc7 is not dependent on Cdc7 kinase activity, but Claspin interaction with the DNA helicase subunit Mcm2 is lost upon Cdc7 inhibition. We propose Cdc7-dependent phosphorylation regulates critical protein-protein interactions and modulates Claspin's function in the DNA replication checkpoint.
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Rainey
authors.forename: Brendan
authors.initials: B
authors.lastname: Harhen
authors.forename: Guan-Nan
authors.initials: GN
authors.lastname: Wang
authors.forename: Paul V
authors.initials: PV
authors.lastname: Murphy
authors.forename: Corrado
authors.initials: C
authors.lastname: Santocanale
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002851:Chromatography, High Pressure Liquid; D004261:DNA Replication; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D013058:Mass Spectrometry; D064111:Minichromosome Maintenance Complex Component 2 / Q000378:metabolism; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D010881:Piperidones / Q000494:pharmacology; D060066:Protein Interaction Maps; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011758:Pyrroles / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D018797:Cell Cycle Proteins; C531237:PHA 767491; D010748:Phosphopeptides; D010881:Piperidones; D011758:Pyrroles; D034741:RNA, Small Interfering; D011494:Protein Kinases; C068619:CDC7 protein, human; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2; D006918:Hydroxyurea
keywords: Cdc7; Claspin; DNA replication; checkpoint; hydroxyurea; kinase; phosphorylation