Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases.

Department of Biology, Nerviano Medical Sciences-Oncology, Via Pasteur 10, 20014 Nerviano, Italy.
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.
Mesh Terms:
Amino Acid Sequence, Binding Sites, Blotting, Western, CDC2 Protein Kinase, Casein Kinase II, Cell Cycle, Cell Cycle Proteins, Chromatin, Chromatography, Liquid, Cyclin-Dependent Kinase 2, Cyclin-Dependent Kinases, DNA Helicases, Electrophoresis, Polyacrylamide Gel, Fibroblasts, HeLa Cells, Humans, Ions, Luciferases, Mass Spectrometry, Microscopy, Fluorescence, Molecular Sequence Data, Nuclear Proteins, Peptides, Phosphorylation, Proline, Protein Isoforms, Protein Structure, Tertiary, Recombinant Fusion Proteins, S-Phase Kinase-Associated Proteins, Serine, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thymidine, Transfection, Trypsin
J. Biol. Chem. Apr. 14, 2006; 281(15);10281-90 [PUBMED:16446360]
Download 2 Interactions For This Publication
131621
Switch View:
  • Interactions (2)