Figure 1.

Identification of Cdc7/Dbf4 phosphorylation sites in human MCM2. (A) Cdc7wt/Dbf4 (wild type) or Cdc7kd/Dbf4 (kinase dead) complex was produced in Sf9 cells using a baculovirus-expression system and purified by anti-FLAG antibody affinity chromatography followed by gel filtration chromatography. Silver staining of purified Cdc7wt/Dbf4 and Cdc7kd/Dbf4 on an SDS/polyacrylamide gel is shown. (B) (a) Schematic representation of bacterially expressed His-MCM2 protein (wt) and its deletion mutants used in in vitro Cdc7/Dbf4 kinase assay. (b) Bacterially expressed purified His-MCM2 and its deletion mutant proteins described in (a) were incubated with baculovirus-expressed purified Cdc7/Dbf4 in the presence of [γ-³²P]ATP. Proteins were resolved by SDS/PAGE and visualized by autoradiography (right) or Coomassie blue staining (left). (C) ³²P-labeled MCM2 in B and ³²P-labeled endogenous MCM2 immunoprecipitated by α-MCM2M1 were eluted from SDS/polyacrylamide gels and digested with trypsin. The resulting phosphopeptides were separated by electrophoresis (pH 1.9 buffer) in the horizontal dimension (anode on the left) and chromatography (phospho-chromatography buffer) in the vertical dimension. Two-dimensional (2-D) tryptic phosphopeptide maps of His-MCM2 (a), His-MCM2N (unpublished data), His-MCM2M1 (unpublished data), and His-MCM2M5 (b) were very similar to that of endogenous MCM2 (c). A schematic map is shown in d. Note: the phosphopeptides (1–6) and the corresponding phosphorylation sites (Ser27, Ser41, and Ser139) are labeled. Multiple phosphopeptides represent partial tryptic digestion products of a single phosphorylation site on the maps. For details, see text, Materials and Methods, and Supplementary Figure S2. (D) HeLa cells were transfected with control luciferase siRNA (siLuc), Cdc7 siRNA (siCdc7), or Cdk2 siRNA (siCdk2) using the Oligofectamine method. Three days after transfection, cells were lysed with RIPA lysis buffer, and cell lysates were subjected to SDS/PAGE, transferred to the PVDF membrane, and immunoblotted with α-Cdc7 (a), α-Cdk2 (b), α-MCM2M1 (c), α-Cyclin A (d), α-Cyclin E (e), or anti-α-tubulin antibody (f). Same cell lysates used in a–f were immunoprecipitated with α-Cdk2, and immunoprecipitates were incubated with 1 μg of histone H1 in the presence of [γ-³²P]ATP. After incubation, reactions were resolved by SDS/PAGE and visualized by autoradiography (g). HeLa cells were transfected with siLuc, siCdc7, or siCdk2 as in a–g. Three days after transfection, cells were metabolically labeled with ³²P-orthophosphate for 6 h and ³²P-labeled endogenous MCM2 from siLuc-, siCdc7-, or siCdk2-transfected cells was immunoprecipitated by α-MCM2M1. After washing, the immunoprecipitates were subjected to SDS/PAGE and visualized by autoradiography (h). ³²P-labeled MCM2 proteins in panel h were eluted from SDS/polyacrylamide gel and digested with trypsin. The resulting phosphopeptides were separated by electrophoresis (pH 1.9 buffer) in the horizontal dimension (anode on the left) and chromatography (phospho-chromatography buffer) in the vertical dimension. Shown are 2-D tryptic phosphopeptide maps of ³²P-labeled endogenous MCM2 from cells transfected with siLuc (i, 2000 cpm), ³²P-labeled endogenous MCM2 from cells transfected siCdc7 (j, 2000 cpm), and ³²P-labeled endogenous MCM2 from cells transfected siCdk2 (k, 2000 cpm). Note: the intensities of phosphopeptides 1–6 (corresponding to Ser27, Ser41, and Ser139 phosphorylation in panel j) were greatly diminished when compared with those in i and k. The asterisks represent nonspecific phosphopeptides, which show similar intensities in i–k.