CDC7 Kinase Phosphorylates Serine Residues Adjacent to Acidic Amino Acids in the Minichromosome Maintenance 2 Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 103; no. 31; pp. 11521 - 11526
• Main Authors: Cho, Won-Ho, Lee, Young-Joo, Kong, Soo-Im, Hurwitz, Jerard, Lee, Joon-Kyu
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 01.08.2006; National Acad Sciences
• Subjects: Acidic amino acids; Amino Acid Sequence; Amino acids; Amino Acids, Acidic - metabolism; Animals; Autoradiography; Biological Sciences; Cell Cycle Proteins - genetics; Cell Cycle Proteins - metabolism; Chromosomes; DNA; DNA replication; Enzymes; Eukaryotes; Humans; Interphase; Mice; Minichromosome Maintenance Complex Component 2; Molecular Sequence Data; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Phosphorus; Phosphorylation; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Replication origin; Serine - metabolism; Substrate specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0604990103

Cdc7 is an essential kinase required for the initiation of eukaryotic DNA replication. Previous studies in many species showed that the minichromosome maintenance complex is a major physiological target of this kinase. In this study, we have mapped the sites in human Mcm2 protein that are phosphorylated by Cdc7. The in vitro phosphorylation of several Mcm2 truncated proteins and peptides revealed that Mcm2 contains two major ($^{5}S$ and $^{53}S$) and at least three minor phosphorylation sites ($^{4}S$, $^{7}S$, and $^{59}T$) located at the N-terminal region. Alanine substitution experiments with Mcm2 peptides showed that the phosphorylation of $5^{S}$ and $^{53}S$ by Cdc7 required the presence of an acidic amino acid adjacent to a serine residue. Furthermore, although Cdc7 was unable to phosphorylate a Mcm2 peptide (spanning amino acids 19-30 and containing $^{26}S$ and $^{27}S$), it phosphorylated 265 efficiently when this peptide contained a chemically synthesized $phospho-^{27}$ modification. Hence, additional Cdc7 phosphorylation sites could be generated in Mcm2 by its prior phosphorylation by a cyclin-dependent kinase. This finding may explain why the sequential action of cyclindependent and Cdc7 kinases is essential for the initiation of DNA replication.