Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity.
Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues of Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared from HeLa cells at mitotic phase but did not bind to those at G(1) and G(1)/S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.
Ishimi
Y
Y
Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan. yukio@libra.ls.m-kagaku.co.jp
Komamura-Kohno
Y
Y
D000595:Amino Acid Sequence; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins / Q000378:metabolism*; D019925:Cyclin A / Q000502:physiology*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000502:physiology*; D004265:DNA Helicases / Q000037:antagonists & inhibitors*; D004261:DNA Replication; D004268:DNA-Binding Proteins; D016195:G2 Phase; D064148:Minichromosome Maintenance Complex Component 4; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D018797:Cell Cycle Proteins; D019925:Cyclin A; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004265:DNA Helicases; C092802:MCM4 protein, S cerevisiae; D064148:Minichromosome Maintenance Complex Component 4