MCM3 is phosphorylated at Ser-112, Ser-611, and Thr-719 by CDK1. Tryptic peptide maps of MCM3-WT or mutant MCM3 phosphorylated by CDK1 are shown.

MCM3 is a CDK2 and CDK1 substrate in vitro. (A) Murine MCM3 or a MCM3 mutant devoid of all CDK consensus sites was incubated with active CDK1/2 and [γ-³²P]ATP. MCM3 phosphorylation was assessed by SDS/PAGE followed by autoradiography. In all kinase reactions, negative controls included incubation of MCM3-WT with mock-purified CDK complexes (−). (B) Same as A except that a double-SP, S112A/S611A, and a double-TP, T464A/T719A, MCM3 mutant were analyzed. (C) (Upper) MCM3 phosphorylation was assessed with cyclin A–CDK2 or with cyclin A–CDK2–p21 complexes. (Lower) Before adding MCM3, cyclin B–CDK1 complexes were incubated with roscovitine.

MCM3 phosphorylation is required for continued cell cycle progression. (A) NIH 3T3 cells were released from a nocodazole block into medium containing cycloheximide, and chromatin loading of MCM2–7 was assessed. (B) Nocodazole-arrested NIH 3T3 cells expressing FLAG-MCM3 were released into medium containing cycloheximide, and binding between MCM3 and MCM5 in whole extracts was assessed 3 h after release. (C and D) U20S cells were infected with human-specific MCM3 shRNAs and subsequently transfected with vector, murine MCM3-WT, or S112A where indicated. Efficiency of MCM3 knockdown and MCM2/5/7 levels was assessed by direct Western blotting. (E) Propidium iodide histograms of cells expressing human-specific MCM3 shRNAs with concurrent expression of murine MCM3-WT or S112A.

Regulation of MCM3 phosphorylation in vivo. (A) NIH 3T3 cells released from G₀ were labeled with ³²P_i 2 h before harvest. Endogenous [³²P]MCM3 was precipitated and detected by SDS/PAGE followed by autoradiography. (B) (Top) Asynchronous NIH 3T3 cells expressing FLAG-MCM3 were labeled with ³²P_i and concurrently treated with roscovitine. MCM3 was precipitated (IP), and ³²P-labeled MCM3 was detected as in A. B (Middle and Bottom) Asynchronous NIH 3T3 cells were treated with roscovitine, and endogenous MCM3 precipitates were probed with a phospho-TP/SP antibody (Middle) or with MCM3 (Bottom). Empty vector and normal rabbit serum precipitates served as negative controls for ectopic and endogenous MCM3, respectively (−). (C) MCM3-WT or S112A expressed in NIH3T3 cells was precipitated and probed with a phospho-TP/SP antibody (Upper) or with αMCM3 (Lower). (D) MCM3-WT and S112A were expressed in U20S cells, precipitated, and probed with a phospho-Ser-112-specific antibody (Upper) or with MCM3 (Lower). (E) MCM3 was precipitated from NIH 3T3 cells released from G₀ and probed for phospho-Ser-112 (Upper) or total MCM3 (Lower).

MCM3 phosphorylation regulates MCM2–7 complex assembly. (A) FLAG-MCM3 was precipitated (IP) from lysates prepared from asynchronous or nocodazole-arrested NIH 3T3 cells expressing either MCM3-WT or the MCM3 quadruple mutant (Q4A) and probed for MCM5 and MCM3. (B) (Upper and Middle) Western blot analysis of MCM3 precipitates prepared from asynchronous NIH 3T3 cells. (Bottom) A direct Western blot of MCM5 is shown as an input control. (C) Same as in B, except that MCM3 precipitates were probed for MCM2 (Left) and MCM7 (Right). (D) NIH 3T3 cells expressing empty vector, MCM3-WT, or S112A were fractionated into chromatin-bound and soluble extracts. Fractions were as indicated. Endogenous MCM5 was detected by direct Western blot analysis of the respective fractions as were FBX4 and CREB to confirm purity. Ectopic FLAG-MCM3 or FLAG-MCM3-S112A was first precipitated with the M2 monoclonal antibody before immunoblotting with an MCM3 polyclonal antibody to avoid detection of endogenous MCM3. (E) Same as in D except binding of S112A to MCM5 in chromatin fractions was assessed by loading six times the amount of S112A. The arrow in the figure indicates the specific band for MCM5, and * indicates nonspecific reacting protein. (F) NIH 3T3 cells were released from a nocodazole block, and binding of MCM3-WT and S112A to chromatin was assessed as in D.