Phosphorylation of Mcm2 by Cdc7 promotes pre-replication complex assembly during cell-cycle re-entry

Abstract

Cyclin E has been shown to have a role in pre-replication complex (Pre-RC) assembly in cells re-entering the cell cycle from quiescence. The assembly of the pre-RC, which involves the loading of six MCM subunits (Mcm2-7), is a prerequisite for DNA replication. We found that cyclin E, through activation of Cdk2, promotes Mcm2 loading onto chromatin. This function is mediated in part by promoting the accumulation of Cdc7 messenger RNA and protein, which then phosphorylates Mcm2. Consistent with this, a phosphomimetic mutant of Mcm2 can bypass the requirement for Cdc7 in terms of Mcm2 loading. Furthermore, ectopic expression of both Cdc6 and Cdc7 can rescue the MCM loading defect associated with expression of dominant-negative Cdk2. These results are consistent with a role for cyclin E-Cdk2 in promoting the accumulation of Cdc6 and Cdc7, which is required for Mcm2 loading when cells re-enter the cell cycle from quiescence.

Figure 1. Phosphorylation of Mcm2 at aminoterminal sites regulates chromatin loading

(a) Serines at the aminoterminus of Mcm2 found to be phosphorylated in the current and other studies (red) and sites found in other studies (blue). (b) Retroviral transduction of HA-Mcm2 leads to at most modest overexpression. Extracts from a population of 293A cells transduced with retroviral empty vector (left lane) and with HAMcm2 wild-type retrovirus (right lane) were separated by SDS-PAGE and then the indicated proteins were detected by western blotting. (c) Phosphorylation site mutations at the Mcm2 aminoterminus affect chromatin loading. Extracts from mimosine-arrested 293A cells stably transduced with retroviruses expressing the indicated phosphorylation site mutations (either S to A or S to D substitutions) were separated into chromatin bound (released by DNAseI digestion) and detergent soluble fractions. Chromatin (upper panel) and soluble (lower panel) fractions were then separated by SDS-PAGE and the indicated proteins detected by western blotting. The graph shows the relative levels of wild-type and phopshorylation site mutant HA-Mcm2 normalized to endogenous Mcm7. The chromatin and soluble levels of wild-type Mcm2 have been arbitrarily set to 1.0. (d) Aminoterminal phosphorylation site mutations of Mcm2 do not affect MCM complex formation. The same retrovirally transduced populations expressing wild-type and mutant Mcm2 as in (c) were separated into chromatin-bound and detergent soluble fractions. Immunoprecipitations using anti-HA antibody were then carried out on each fraction (right panels). Immunoprecipitates were separated by SDS-PAGE and the indicated MCM proteins were detected by western blotting. Panels on the left correspond to 10% of amount of the respective fractions used for the immunoprecipitations.

Figure 2. Cdc7-Dbf4 phosphorylates residues in the Mcm2 Ser4,5,7 cluster in vitro

(a) Phosphorylation in vitro of recombinant Mcm2 by recombinant cyclin E1-Cdk2, cyclin A-Cdk2 and Dbf4-Cdc7. All proteins were expressed in baculovirus-transduced insect cells and purified using NiTA chromatography. Increasing amounts of Mcm2- S13/27A or Mcm2-S4/5/7/13/27A were added to reaction mixtures containing γ[³²P]-ATP. Reactions were then separated by SDS-PAGE. Gels were then silver stained and then analyzed using a Phosphorimager. Upper panel, autoradiograph, low exposure. Middle panel, autoradiograph, high exposure. Lower panel, silver stain of the same gel. Numbers below the middle panel correspond to the specific radioactivities of Mcm2 bands, where the Mcm2-S13/27A signal is set to 100%. (b) Serine 5 of Mcm2 is the primary in vitro aminoterminal target of Dbf4-Cdc7. GST fusions purified from E. coli of amino acids 2–24 of wild-type Mcm2, S5A and S4/5/7A derivatives were incubated with recombinant insect-cell-expressed Dbf4-Cdc7 and γ[³²P]-ATP. Reactions were then separated by SDS-PAGE, which were silver stained and then analyzed using a Phosphorimager. Upper panel, autoradiograph. Lower panel, Coomassie blue stain of same gel. Numbers below upper panel correspond to the specific radioactivities of GST and GST-fusion bands, where the wild-type signal is set to 100% and the background (GST) is set to 0%.

Figure 3. Mcm2 phosphorylation site mutants affect chromatin loading of other MCM subunits

Overexpression of Mcm2 wild-type, as well as 4/5/7A and 4/5/7D derivatives was achieved by transient transfection in 293A cells. Extracts were separated into chromatin-bound and detergent-soluble fractions, separated by SDS-PAGE and the indicated proteins (or phosphoepitope) were detected by western blotting. V indicates the empty vector control. Graphs to the right represent quantitation of the indicated signal normalized to the actin signal.

Figure 4. Aminoterminal phosphorylation of Mcm2 regulates chromatin loading in cells re-entering the cell cycle from quiescence

(a) Time course analysis of chromatin loading and expression of cell cycle regulatory proteins as IME cells re-enter the cell cycle. IME cells were synchronized in G0 by contact inhibition and serum/growth factor removal and then stimulated to re-enter the cell cycle by replating in complete medium. Left panel, chromatin bound proteins. Right panel, soluble proteins. Right lane for each panel corresponds to 28-hour time point where the Cdk inhibitor, roscovitine was added at the time of replating (0-hour). Left lane for each panel corresponds to whole cell extract (WCE) from asynchronous IME cells. (b) Time course analysis of cyclin E-Cdk2 kinase activity as IME cells re-enter the cell cycle. Cyclin E1 immunoprecipitations were carried on extracts prepared from IME cells synchronized as in (a). Immune complexes were assayed for histone H1 kinase activity. (c) Expression of HA-Mcm2 wild-type by retroviral transduction in IME cells leads to only modest overexpression of Mcm2. Extracts from asynchronous populations of IME cells transduced with vector and HA-Mcm2 retrovirus were analyzed for the indicated proteins by SDS-PAGE and western blotting. (d) Aminoterminal phosphorylation site mutants affect chromatin loading of Mcm2 as IME cells re-enter the cell cycle. Extracts from IME cells stably transduced with retroviruses expressing the indicated phosphorylation site mutations (either S to A or S to D substitutions) synchronized as in (a) and released from G0 block for 15 hours were separated into chromatin bound (released by DNAseI digestion) and detergent soluble fractions. Chromatin (left panel) and soluble (right panel) fractions were then separated by SDS-PAGE and the indicated proteins detected by western blotting. Asterisk indicates a non-specific background band in the Mcm6 panel. The graph shows the relative levels of wild-type and phosphorylation site mutant HA-Mcm2 normalized to endogenous Mcm6. The chromatin and soluble levels of wild-type Mcm2 have been arbitrarily set to 1.0. (e) Mcm2 phosphorylated on serine 5 loads onto chromatin with the same kinetics as general MCM protein loading. Chromatin fractions from IME cells synchronized as in (a) were analyzed for the indicated proteins and phosphoepitope by SDS-PAGE and western blotting.

Figure 5. Phosphorylation of Mcm2 by Cdc7 is required for chromatin loading upon cell cycle re-entry

(a) Expression of a dominant-negative Cdc7 blocks chromatin loading of Mcm2 and Mcm4. IME cells synchronized as described in Fig. 4a were transduced with a recombinant adenovirus expressing dominant negative Cdc7 (Cdc7-dn) prior to stimulating cell cycle re-entry by replating in complete medium. Cells harvested at 15 hours post-stimulation were separated into soluble and chromatin-bound fractions (sonication) and analyzed by SDS-PAGE and western blotting for the indicated proteins. (b) A phosphomimetic Mcm2 mutant can bypass the requirement for Cdc7 phosphorylation in Mcm2 chromatin loading during cell cycle re-entry. IME cells expressing either HA-tagged wild-type Mcm2 (WT) or an Mcm2 allele containing aspartate substitutions at serines 4, 5, 7, 23, 40, 41, and 53 (7D) introduced by retroviral transduction were synchronized as described in Fig. 4a. Either no adenovirus (−) or two different Cdc7-dn adenovirus doses were used. Cells harvested at 15 hours post-stimulation were separated into soluble and chromatin-bound fractions (released by sonication) and analyzed by SDS-PAGE and western blotting for the indicated proteins. For Mcm2, only the epitope-tagged protein is shown.

Figure 6. Cyclin E-Cdk2 promotes chromatin loading of Mcm2 by regulating expression of Cdc6 and Cdc7

(a) Expression of a dominant-negative allele of Cdk2 (Cdk2-dn) impairs accumulation of Cdc6 and Cdc7 as IME cells re-enter the cell cycle from quiescence. Extracts of IME cells synchronized in G0 by contact inhibition and serum starvation and then transduced with control or Cdk2-dn adenoviruses were stimulated to re-enter the cell cycle by replating in complete medium. Extracts were prepared at the indicated times and analyzed by SDS-PAGE and western blotting. Quantitation of Cdc6 and Cdc7 relative to actin for the 11-hour samples is shown in the graphs below. (b) Cdk2 activity is required for chromatin loading of Mcm2. IME cells were synchronized in G0 and transduced with the indicated recombinant adenoviruses. When cells were not transduced with Cdk2-dn expressing viruses, empty vector virus was substituted. Cells were then restimulated to enter the cell cycle by replating in complete medium and 15 hours later separated into chromatin and soluble fractions. The indicated proteins were analyzed by SDS-PAGE and western blotting. Left panel, chromatin bound proteins (sonication). Right panel, soluble proteins. The Cdc6 allele expressed has phosphomimetic mutations at four serines to prevent proteasomal degradation. The graph below represents quantitation of western blots from two independent experiments where chromatin bound Mcm2 was normalized to chromatin bound cyclin E1. Error bars correspond to the variance of the mean. (c) Cdk2 activity is required to accumulate Cdc6 and Cdc7 mRNA in IME cells re-entering the cell cycle from quiescence. IME cells were synchronized as in (a) and RNA prepared at 11 hours after stimulation to re-enter the cell cycle. The indicated mRNA levels were determined by quantitative real-time PCR, with the values given normalized to actin mRNA. Error bars correspond to the standard deviation from three experiments. (d) Cyclin E is required for expression of Cdc6 and Cdc7 in IME cells re-entering the cell cycle from quiescence. IME cells were synchronized in G0 by contact inhibition and serum starvation, transfected with siRNAs targeting cyclins E1 and E2 or control siRNAs (GFP), and then stimulated to re-enter the cell cycle by replating in complete medium. At 11 hours post stimulation, cells were separated into chromatin-bound and soluble fractions and the indicated proteins were detected by SDS-PAGE followed by western blotting. Part of the blot stained for protein using imido black prior to incubation with antibodies is given as a loading control. The asterisk denotes a non-specific band recognized by the Cdc6 antibody. (e) A phosphomimetic mutant of Mcm2 can load onto chromatin in the absence of Cdk2 and Cdc7 activity if Cdc6 is supplied. IME cells expressing either HA-tagged wild-type Mcm2 (WT) or an Mcm2 allele containing aspartate substitutions at serines 4, 5, 7, 23, 40, 41, and 53 (7D) introduced by retroviral transduction were synchronized in G0. Either no adenovirus (−) or two different Cdc7-dn adenovirus doses were used in conjunction with a constant dose of Cdk2-dn and Cdc6 adenoviruses. Cells harvested at 15 hours post-stimulation were separated into soluble and chromatin-bound fractions (released by sonication) and analyzed by SDS-PAGE and blotting for the indicated proteins. For Mcm2, only the epitope-tagged protein is shown. For Cdc7, faster migrating species corresponds to endogenous Cdc7 whereas more slowly migrating species corresponds to ectopic Myc-Cdc7-dn.

Figure 7. Serine 5-phosphorylated Mcm2 accumulates in the soluble fraction when chromatin loading is prevented

IME cells were synchronized in G0 and transduced with the indicated recombinant adenoviruses. When cells were not transduced with Cdk2-dn expressing viruses, empty vector virus was substituted. Cells were then restimulated to enter the cell cycle by replating in complete medium and 15 hours later separated into chromatin bound (released by sonication) and soluble fractions. The indicated proteins and the Mcm2 phospho-Ser5 epitope were analyzed by SDS-PAGE and western blotting. Left panel, chromatin-bound proteins. Right panel, soluble proteins.