Phosphorylation of MCM3 protein by cyclin E/cyclin-dependent kinase 2 (Cdk2) regulates its function in cell cycle

Abstract

MCM2-7 proteins form a stable heterohexamer with DNA helicase activity functioning in the DNA replication of eukaryotic cells. The MCM2-7 complex is loaded onto chromatin in a cell cycle-dependent manner. The phosphorylation of MCM2-7 proteins contributes to the formation of the MCM2-7 complex. However, the regulation of specific MCM phosphorylation still needs to be elucidated. In this study, we demonstrate that MCM3 is a substrate of cyclin E/Cdk2 and can be phosphorylated by cyclin E/Cdk2 at Thr-722. We find that the MCM3 T722A mutant binds chromatin much less efficiently when compared with wild type MCM3, suggesting that this phosphorylation site is involved in MCM3 loading onto chromatin. Interestingly, overexpression of MCM3, but not MCM3 T722A mutant, inhibits the S phase entry, whereas it does not affect the exit from mitosis. Knockdown of MCM3 does not affect S phase entry and progression, indicating that a small fraction of MCM3 is sufficient for normal S phase completion. These results suggest that excess accumulation of MCM3 protein onto chromatin may inhibit DNA replication. Other studies indicate that excess of MCM3 up-regulates the phosphorylation of CHK1 Ser-345 and CDK2 Thr-14. These data reveal that the phosphorylation of MCM3 contributes to its function in controlling the S phase checkpoint of cell cycle in addition to the regulation of formation of the MCM2-7 complex.

FIGURE 1.

MCM3 interacts with cyclin E/Cdk2. A, co-immunoprecipitation assay. 293T cells were transfected Myc-tagged cyclin E and Cdk2 or Myc-tagged cyclin D and Cdk4 with pcDNA3.1 or FLAG-tagged MCM3. Cell lysates were harvested for immunoprecipitation with FLAG antibody followed by immunoblotting with Myc antibody. B, 293T cells were transfected with Myc-tagged cyclin E and Myc-tagged Cdk2 plasmids. The cell lysates were harvested and subject to GST pulldown with GST or GST-MCM3 fusion proteins and then immunoblotted (IB) with Myc antibody. C, GST protein or GST-MCM3 immobilized on glutathione beads was incubated with purified His-Cdk2. The associated protein was eluted with SDS loading buffer and immunoblotted with His antibody. D, 293T cells were co-transfected with Myc-tagged cyclin E and Myc-tagged Cdk2 with pcDNA3.1 or FLAG-tagged MCM3 (1–193 aa), MCM3 (194–490 aa), MCM3 (1–490 aa), MCM3 (490–807 aa), and full-length MCM3. Cell lysates were subjected to immunoprecipitation with FLAG antibody followed by immunoblotting with Myc antibody. Arrows indicate the bands of FLAG-MCM3 and its truncated forms.

FIGURE 2.

MCM3 is phosphorylated by cyclin E/Cdk2 at Thr-722. A, in vitro kinase assay. 1 μg of GST, GST-MCM3, GST-MCM3 S112A, GST-MCM3 T464A, GST-MCM3 S611A, and GST-MCM3 T722A were incubated with GST-cyclin E and GST-Cdk2 in the presence of [γ-³²P]ATP. Phosphorylation of MCM3 was assessed by SDS-PAGE followed by autoradiography of the gel. B, comparison of amino acid sequences of human and other species on MCM3 peptide around Thr-722. The number at the end of each sequence represented the position of threonine in boldface. C, MCM3 is phosphorylated at Thr-722 in vivo. Tet-on inducible cell lines (vector, MCM3, or MCM3 T722A) were treated with tetracycline for 24 h. Cell lysates were immunoprecipitated with FLAG antibody, followed by immunoblotting (IB) with phospho-threonine-proline antibody. D, GST-MCM3 (419–529 aa), GST-MCM3 (566–656 aa), GST-MCM3 (677–747 aa), and their corresponding phosphorylation site mutations (either Ser or Thr to Ala substitutions) were prepared and subject to in vitro kinase assay as described in B. The arrows indicated the band of phosphorylated MCM3, and the asterisk pointed out the phosphorylated cyclin E (left panel). The purified GST-MCM3 in Coomassie-stained gel is indicated by arrows as well (right panel).

FIGURE 3.

Phosphorylation of MCM3 affects its loading onto chromatin. A, 293T cells were transfected with indicated plasmids. Total cell lysates and chromatin-bound fraction were prepared and subjected to immunoblot with indicated antibodies. β-Actin and lamin B were used as the loading controls. B, quantitcation of chromatin-bound MCM3 and its mutants. The wide-type MCM3 was arbitrarily set to 100. Data are represented as mean ± S.D. (n = 3).

FIGURE 4.

Overexpression of MCM3 blocks S phase entry. A, Tet-on inducible HeLa cells (MCM3 and MCM3 T722A) were treated with or without tetracycline for 24 h. Cell extracts were prepared and subjected to immunoblot with anti-FLAG antibody. B, Tet-on inducible HeLa cells for control, FLAG-MCM3, and FLAG-MCM3 T722A were treated with tetracycline. The chromatin-bound fraction and total cell lysates were prepared for immunoblotting with indicated antibodies. C, Tet-on inducible HeLa cells for control, FLAG-MCM3, and FLAG-MCM3 T722A were treated with tetracycline and subject to flow cytometry analysis. D, schematic view of experimental design. Tet-on inducible cells were treated with 2 mm thymidine for 16 h and released in fresh medium for 8 h and then treated again with 2 mm thymidine and tetracycline for 16 h. Cells then were released for 3 and 6 h and subjected to flow cytometry analysis. E, cell cycle profiles were analyzed by flow cytometry analysis.

FIGURE 5.

Overexpression of MCM3 does not affect M phase exit. A, schematic view of experimental design. Tet-on inducible cells (control, MCM3, and MCM3 T722A) were arrested at G₁/S phase by thymidine treatment for 16 h and then released for 6 h followed by treatment with nocodazole for 6 h. Cells were released for 3 h and subjected to flow cytometry analysis. B, cell cycle profiles at 0 and 3 h after release from nocodazole treatment were analyzed by flow cytometry.

FIGURE 6.

Knockdown of MCM3 does not affect S phase entry. A, schematic view of experimental design. HeLa cells were treated with thymidine and transfected with two rounds of siRNA against MCM3 at the time indicated. B, HeLa cell were treated as described in A. Cells were harvested at 0 h (before final release into S phase) after two rounds of siRNA 1 transfection, and cell lysates were subjected to immunoblot with MCM3 antibody. β-Actin was used as a loading control. C, S phase dynamics after MCM3 depletion. HeLa cells released at 0, 3, and 6 h after double thymine treatment and MCM3 depletion were harvested and subjected to flow cytometry analysis.

FIGURE 7.

Overexpression of MCM3 up-regulates the phosphorylation of Chk1 Ser-345 (A) and Cdk2 Thr-14 (B). Tet-on inducible cells (control, MCM3, and MCM3 T722A) were treated with tetracycline and synchronized at the G₁/S phase. Total cell lysates were harvested and subjected to immunoblot with the indicated antibodies.