Identification of Mcm2 Phosphorylation Sites by S-phase-regulating Kinases

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 allow...

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Bibliographic Details
Published in:The Journal of biological chemistry 2006-04, Vol.281 (15), p.10281-10290
Main Authors: Montagnoli, Alessia, Valsasina, Barbara, Brotherton, Deborah, Troiani, Sonia, Rainoldi, Sonia, Tenca, Pierluigi, Molinari, Antonio, Santocanale, Corrado
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Blotting, Western
Casein Kinase II - metabolism
CDC2 Protein Kinase - metabolism
Cell Cycle
Cell Cycle Proteins - metabolism
Cell Cycle Proteins - physiology
Chromatin - chemistry
Chromatography, Liquid
Cyclin-Dependent Kinase 2 - metabolism
Cyclin-Dependent Kinases - metabolism
DNA Helicases - chemistry
Electrophoresis, Polyacrylamide Gel
Fibroblasts - metabolism
HeLa Cells
Humans
Ions
Luciferases - metabolism
Mass Spectrometry
Microscopy, Fluorescence
Minichromosome Maintenance Complex Component 2
Molecular Sequence Data
Nuclear Proteins - metabolism
Nuclear Proteins - physiology
Peptides - chemistry
Phosphorylation
Proline - chemistry
Protein Isoforms
Protein Structure, Tertiary
Recombinant Fusion Proteins - chemistry
S-Phase Kinase-Associated Proteins - metabolism
Serine - chemistry
Spectrometry, Mass, Electrospray Ionization
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Thymidine - chemistry
Transfection
Trypsin - pharmacology
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M512921200