title: Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells.
abstract: The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.
authors.affiliation: Novartis Institute of Biomedical Research, Oncology, Emeryville, California 94608, USA. deb.charych@fiveprime.com
authors.forename: Deborah H
authors.initials: DH
authors.lastname: Charych
authors.forename: Mazie
authors.initials: M
authors.lastname: Coyne
authors.forename: Asha
authors.initials: A
authors.lastname: Yabannavar
authors.forename: Jamie
authors.initials: J
authors.lastname: Narberes
authors.forename: Sylvia
authors.initials: S
authors.lastname: Chow
authors.forename: Marco
authors.initials: M
authors.lastname: Wallroth
authors.forename: Cynthia
authors.initials: C
authors.lastname: Shafer
authors.forename: Annette O
authors.initials: AO
authors.lastname: Walter
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D064111:Minichromosome Maintenance Complex Component 2; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; C485678:DBF4 protein, human; D009687:Nuclear Proteins; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; C084859:MCM2 protein, human; D064111:Minichromosome Maintenance Complex Component 2