Abstract
MCM7 is one of the subunits of the MCM2-7 complex that plays a critical role in DNA replication initiation and cell proliferation of eukaryotic cells. After forming the pre-replication complex (pre-RC) with other components, the MCM2-7 complex is activated by DDK/cyclin-dependent kinase to initiate DNA replication. Each subunit of the MCM2-7 complex functions differently under regulation of various kinases on the specific site, which needs to be investigated in detail. In this study, we demonstrated that MCM7 is a substrate of cyclin E/Cdk2 and can be phosphorylated on Ser-121. We found that the distribution of MCM7-S121A is different from wild-type MCM7 and that the MCM7-S121A mutant is much less efficient to form a pre-RC complex with MCM3/MCM5/cdc45 compared with wild-type MCM7. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Further analysis indicates that the Chk1 pathway is activated in MCM7-overexpressed cells in a p53-dependent manner. We performed experiments with the human normal cell line HL-7702 and also observed that overexpression of MCM7 can cause S phase block through checkpoint activation. In addition, we found that MCM7 could also be phosphorylated by cyclin B/Cdk1 on Ser-121 both in vitro and in vivo. Furthermore, overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser-121 in M phase is very important for a proper mitotic exit. These data suggest that the phosphorylation of MCM7 on Ser-121 by cyclin/Cdks is involved in preventing DNA rereplication as well as in regulation of the mitotic exit.
Highlights
Cyclin/cyclin-dependent kinase (CDK) play an important role in cell cycle regulation
We took the approach of immunoprecipitation to confirm the association between MCM7 and Cdk2. 293T cells were transfected with pCMV FLAG-MCM7 and pCMV myc-cyclin E or Cdk2
The data indicated that GST-MCM7 can interact with myc-cyclin E and myc-Cdk2 expressed in 293T cells (Fig. 1C)
Summary
Cyclin/CDKs play an important role in cell cycle regulation. Results: MCM7 can be phosphorylated at Serine-121 by cyclin E/Cdk and cyclin B/Cdk. By using the Tet-On inducible HeLa cell line, we revealed that overexpression of wild-type MCM7 but not MCM7-S121A can block S phase entry, suggesting that an excess of the pre-RC complex may activate the cell cycle checkpoint. Overexpression of MCM7-S121A causes an obvious M phase exit delay, which suggests that phosphorylation of MCM7 on Ser121 in M phase is very important for a proper mitotic exit. Cell Cycle Functions of MCM7 phase checkpoint activation under regulation of cyclin E/Cdk2 [10]. We demonstrated that MCM7 is the substrate of cyclin E/Cdk that phosphorylates MCM7 at Ser-121 and regulates its distribution in cells. Phosphorylations of MCM7 by cyclin/Cdks plays an important role in S phase checkpoint activation as well as in the regulation of proper M phase progression
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.