(A) Expression and phosphorylation of HsCdc6 during the cell cycle. U2OS cells were synchronized by a “thymidine/nocodazole” block at G₂/M phase and then released for different times (h) as indicated. Fifty micrograms of cell lysates from the indicated time point were subjected to SDS/PAGE, transferred to Immobilon-P membrane, and then immunoblotted with affinity-purified anti-HsCdc6 antibodies (Upper). Cell cycle position of U2OS cells used in the immunoblotting analysis at indicated time points (Upper) was determined by flow cytometry (Lower). (B) U2OS cells were synchronized at various stages of the cell cycle as indicated and in vivo labeled with [³²P]orthophosphate for a period of 4 h. HsCdc6 was immunoprecipitated by using anti-HsCdc6 antibodies. After washing, the immunoprecipitates were subjected to SDS/PAGE and visualized by autoradiography (Middle). The bands containing ³²P-labeled HsCdc6 proteins were quantified by a PhosphorImager (Bottom). The amounts of immunoprecipitated HsCdc6 protein were determined by using anti-HsCdc6 antibodies for immunoblotting (Top).

Chromatin/nuclear matrix association and subcellular localization of (Myc)₆-HsCdc6wt, (Myc)₆-HsCdc6A1A2A3, and (Myc)₆-HsCdc6E1E2E3. (A) U2OS cells were transiently transfected with DNA constructs expressing (Myc)₆-HsCdc6wt, (Myc)₆-HsCdc6A1A2A3, (Myc)₆-HsCdc6E1E2E3, and control, (Myc)₆. Lysates from indicated transfected cells were subjected to SDS/PAGE and then analyzed by immunoblotting with the 9E10 mAb. (B) Cells expressing indicated proteins in A were subjected to chromatin/nuclear matrix fractionation. The soluble fraction from the first extraction (I), the soluble fraction from the third extraction (II), and the chromatin/nuclear matrix fraction (III) from the indicated cells were subjected to SDS/PAGE and then analyzed by immunoblotting with either the 9E10 mAb or anti-histone H1 mAb. (C) U2OS cells were grown on glass coverslips and transiently transfected with DNA constructs expressing indicated proteins as in A. After fixation, samples were incubated with the 9E10 mAb. Immunofluorescence staining was performed with Texas red-conjugated goat anti-mouse antibodies, and nuclei were visualized with Hoechst dye 33258. Fluorescent images were obtained by using a fluorescence microscope. (D) Subcellular localization of endogenous HsCdc6 during the cell cycle. HeLa cells were synchronized by a “double thymidine” block in G₁/S phase and then released into S/G₂ phase in fresh medium for a period of 6 h. Subcellular fractionation was performed as described (42, 43). Equal amounts of cytoplasmic and nuclear proteins extracted from G₁/S and S/G₂ cells were subjected to SDS/PAGE and then analyzed by immunoblotting with anti-HsCdc6 antibodies (Top), anti-IκBα (a cytoplasmic protein) antibodies [Middle (42)], or anti-Prc1 (a nuclear protein) antibodies [Bottom (29)], respectively.

Expression of the HsCdc6A1A2A3 mutant inhibits initiation of DNA replication. HSF8 cells expressing the indicated GFP-HsCdc6 proteins and uninfected control cells were synchronized in G₀ by serum deprivation and then stimulated with 20% FCS in fresh medium in the presence of 10 μM BrdUrd. Cells were fixed and stained with anti-BrdUrd antibody and Hoechst 33258 at the indicated times. (A) Representative photomicrographs of B at 20 and 30 h are shown. (B) Percentages of BrdUrd-positive cells in indicated points are shown. Each value represents a minimum count of 100 cells.

HsCdc6 is a Cdk substrate. (A) Substrate specificity of cyclin-Cdk complexes against GST-HsCdc6 in comparison with a known substrate protein, GST-Rb C terminus (residues 768–928). One to 2 μg GST-HsCdc6 or GST-Rb was incubated with purified baculovirus-expressed cyclin D1-Cdk6 (D1/K6), cyclin E-Cdk2 (E/K2), cyclin A-Cdk2 (A/K2), or cyclin B1-Cdc2 (B/K1) in the presence of [γ-³²P]ATP. Proteins were resolved by SDS/PAGE and visualized by autoradiography (Upper) or Coomassie blue staining (Lower). (B) The GST-HsCdc6wt and its mutant proteins described in Materials and Methods were incubated with cyclin E-Cdk2 in the presence of [γ-³²P]ATP. Proteins were resolved by SDS/PAGE and visualized by autoradiography (Upper) or Coomassie blue staining (Lower). (C) ³²P-labeled immunoprecipitated HsCdc6 protein from cells in S/G₂ described in Fig. 1B (in vivo) and GST-HsCdc6 protein phosphorylated by cyclin E-Cdk2 described in A (in vitro) were eluted from SDS/polyacrylamide gels and then digested with trypsin. The resulting phosphopeptides were separated by electrophoresis (pH 1.9 buffer) in the horizontal dimension (anode on the left) and chromatography (isobutyric acid buffer) in the vertical dimension. Shown are 2D tryptic phosphopeptide maps of: in vivo, HsCdc6 from in vivo ³²P-labeled G₂/M cells (2,000 cpm); in vitro, GST-HsCdc6 phosphorylated by cyclin E-Cdk2 in vitro (2,000 cpm); mix, mix of in vivo (1,000 cpm) and in vitro (1,000 cpm) and the schematic map. The major phosphopeptides, 1–6, are labeled. PAA of in vivo ³²P-labeled HsCdc6 and GST-HsCdc6 phosphorylated by cyclin E-Cdk2 in vitro were performed, and radioactive phosphoamino acids were visualized by autoradiography (Insets).

Subcellular localization of GFP-HsCdc6 and its mutants upon LMB treatment. (A) GFP, GFP-HsCdc6wt, GFP-HsCdc6A1A2A3, and GFP-HsCdc6E1E2E3 were transiently expressed in HSF8 cells by retrovirus-mediated transduction for 24 h. After fixation, GFP fluorescent images were obtained by using a fluorescence microscope. (B) HSF8 cells expressing the indicated GFP-HsCdc6 proteins were treated with or without cycloheximide (CHX, 50 μg/ml) and LMB (20 nM) for 4 h. After fixation, GFP fluorescent images were obtained by using a fluorescence microscope. (C) Summary of quantitative results from B, in which ≈100 cells expressing the indicated GFP-HsCdc6 proteins treated with (+) or without (−) CHX+LMB were counted. Solid bars represent the percentage of GFP-HsCdc6 proteins in the nucleus, and open bars represent the percentage of GFP-HsCdc6 proteins in the cytoplasm.

Model for regulation of DNA replication by Cdk phosphorylation of HsCdc6 in human cells (for details, see text).