Figure 1.
Serine 207 and 243 are potential phosphorylated residues and serine 243 mediates the chromosomal association of the HPV-16 E2 protein.
A) Immunopurification and mass spectrometric analysis of phosphorylated serine on the HPV-16 E2 protein. Proteins extracted from 293 cells carrying plasmids expressing EGFP-tagged E2 proteins or the control vector were immunoprecipitated with anti-EGFP antibody in the presence of protease inhibitor (MG132). The purified lysates were resolved by SDS-PAGE followed by staining with Coomassie brilliant blue (upper) or immune-blotting with anti-EGFP antibody (lower). The visible band at size 74 kDa (marked by * in the upper panel) was cut and analyzed by mass spectrometry for the phosphorylated residues. B) HPV-16 E2 proteins are phosphorylated at serine 207 and 243. Amino acids 202 to 286 from the hinge region of HPV-16 E2 are shown and the both potential phosphor-serines are underlined. C) Chromosome associating phenotypes of the E2 proteins with mutations in the hinge at G1/S, metaphase and anaphase. Localization of EGFP-tagged HPV-16 E2 proteins, including with substitutions of serine 207, serine 243 and R37A/I73A, in mitotic COS-7 cells is shown in green and DNA was stained with DAPI (blue) and visualized by confocal microscopy.
Figure 2.
Residue serine 243 is critical for the association of HPV-16 E2 with Brd4.
A) 293 cells were transfected with expression vectors for EGFP-tagged HPV-16 E2 or mutated E2. Cell extracts were immunoprecipitated with anti-Brd4 antibody or anti-IgG as a control in incubation buffer containing EtBr and the immunoblotting was performed with antibodies as indicated. B) C33A cells were cotransfected with HPV-16 LCR reporter and EGFP-tagged E2 or mutants. At 48 h posttransfection, chromatin extracts were immunoprecipitated with antibody against Brd4. The eluted DNA-protein complexes were subjected to PCR analysis (ChIP) or further immunoprecipitation with anti-Brd4 antibody (reChIP). C) The S243-mutated E2 protein (S243A) has a shorter half-life than the wild type. 293 cells were transfected with EGFP-tagged wild type or mutated E2. At 36 h posttransfection, the cells were treated with cycloheximide (50 µg/ml) for up to 8 h. Cell extracts were collected at the times shown and immunoblotted with the antibodies indicated. The graph below presents the quantified intensities of the bands. The expression levels of E2 were normalized using β-actin and the percentage of E2 at each time point relative to that of the control (without CHX treatment, set at 100) are shown. Data shown are means of results from three independent experiments (mean ± standard deviation; n = 3).
Figure 3.
Phosphorylation at serine 243 of HPV-16 E2 mediates binding to Brd4.
A) Phosphorylation analysis and HPV-16 E2 proteins. 293 cells were transfected with expression vectors for wild type or mutated hinge region of HPV-16 E2 fused to EGFP. Equivalent amounts of protein lysate were immunoprecipitated using anti-EGFP antibody and the samples were analyzed by western blotting with antibody against phosphor-serine. B) The graph presents the quantified intensities of the phosphorylated signal. The level of phosphorylated E2 hinge was normalized using total E2 immunoprecipitated. C) Mutational analysis of E2 proteins binding to Brd4. 293 cells were transfected with vectors expressing various E2 proteins containing amino acid substitutions in the hinge region. Cell lysates were collected and immunoprecipitated with anti-Brd4 antibody. The protein complexes were analyzed by SDS-PAGE and subjected to immunoblotting with anti-EGFP antibody. The expression level of E2 proteins was normalized using β-Actin, and the percentage of E2 at each time point relative to that of the control (without CHX treatment, set at 100) is shown (lower panel) Data shown are means of results from three independent experiments. Asterisk, non-specific band. D) Summary of the Brd4-binding activity of the HPV-16 E2 proteins and mutants.
Figure 4.
Chromosome association phenotypes of Brd4 and E2 proteins with amino acid substitutions in the hinge.
A, B) Phosphorylation at residue 243 is critical for the co-localization of HPV-16 E2 and Brd4 and association with mitotic chromosomes. COS-7 cells transfected with plasmids expressing HPV-16 wild type E2, S207-mutated E2 (S207A) or S243-mutated E2 (S243A, S243D, S243N, S243E, S243Q) were visualized by confocal microscopy to determine the location of E2 (green), Brd4 (red) and cellular DNA (blue) during mitosis. A) metaphase and B) anaphase.
Table 1.
E2-expressing mitotic cells showing the focus binding pattern shown in Fig. 4.
Figure 5.
HPV-16 E2 binds to the chromosomes in a Brd4-dependent manner.
A) C33A cells were transfected with the expression plasmids for EGFP-fused HPV-16 wild type, S243A E2, or EGFP proteins. After fixation, the cells were subjected to fluorescence microscopy after counterstaining of the DNA with DAPI. The cells in metaphase (left) and anaphase (right) are shown. B) Inhibition of Brd4 chromatin binding causes E2 to dissociate from the chromosomes during mitosis. COS-7 cells were transfected with EGFP-tagged E2 and treated with DMSO or 0.5 µM JQ1(+). Location of protein complexes was detected by confocal microscopy for E2 (green), Brd4 (red) and DNA (DAPI).