Human papillomavirus type 16 E7 oncoprotein associates with the cullin 2 ubiquitin ligase complex, which contributes to degradation of the retinoblastoma tumor suppressor

Abstract

Human papillomavirus type 16 (HPV16) and other high-risk HPVs are etiologically linked to the development of cervical carcinomas and contribute to a number of other tumors of the anogenital tract, as well as oral cancers. The high-risk HPV E6 and E7 oncoproteins are consistently expressed in cervical cancer cells and are necessary for the induction and maintenance of the transformed phenotype. An important aspect of HPV16 E7's oncogenic activities is destabilization of the retinoblastoma tumor suppressor (pRB) through a ubiquitin/proteasome-dependent mechanism, although the exact molecular mechanism is unknown. Here, we report that HPV16 E7 is associated with an enzymatically active cullin 2 ubiquitin ligase complex and that the HPV16 E7/pRB complex contains cullin 2. Depletion of cullin 2 by RNA interference causes increased steady-state levels and stability of pRB in HPV16 E7-expressing cells, and ectopic expression of HPV16 E7 and the cullin 2 complex leads to pRB ubiquitination in vivo. Hence, we propose that the HPV16 E7-associated cullin 2 ubiquitin ligase complex contributes to aberrant degradation of the pRB tumor suppressor in HPV16 E7-expressing cells.

FIG. 1.

HPV16 E7 associates with cullin 2. (A) Tandem-affinity purification of cellular protein complexes associated with C-E7 expressed in HeLa cells. Representative HPV16 E7-associated cellular proteins isolated by this procedure are indicated (31). Vector-transfected HeLa cells (lanes C) were used as a control. (B) Coprecipitation of cullin 2 with E7 antibodies in HPV16 E7-expressing RKO colon carcinoma cells. Parental RKO cells (lanes C) were used as a control. (C) E7 preferentially associates with the slower-migrating form of cullin 2 (top). The blot was stripped and reprobed with NEDD8-specific antibody to demonstrate that this band corresponds to NEDD8-modified cullin 2 (middle). A blot demonstrating immunoprecipitation of E7 is also shown (bottom). Lysates of HeLa cells with stable expression of C-E7 were immunoprecipitated with FLAG antibody, followed by Western blotting with the corresponding antibodies. Vector-transfected HeLa cells (lanes C) were used as a control. (D) Relative levels of NEDD8-conjugated cullin 2 are increased in HPV16 E7-expressing RKO cells (lane E7) compared to parental RKO cells (lane C). (E) Low-risk HPV11 and HPV6b and high-risk HPV18 E7 do not form a detectable complex with cullin 2. Lysates of HeLa cells with stable expression of the corresponding FLAG/HA-tagged E7 proteins were analyzed by immunoprecipitation with FLAG antibody, followed by Western blotting with the indicated antibodies. Vector-transfected HeLa cells (lane C) were used as a control. (F) HPV16 E7 binds to cullin 2, but not to cullin 1, 4, or 5. Lysates of HeLa cells with stable expression of C-E7 were immunoprecipitated with FLAG antibody, followed by Western blotting with the corresponding cullin antibodies. Vector-transfected HeLa cells (lanes C) were used as a control.

FIG. 2.

HPV16 E7 associates with an active cullin 2 ubiquitin ligase complex. (A) HPV16 E7 associates with cullin 2, elongin B, elongin C, and Rbx1. Lysates of HeLa cells with stable expression of C-E7 were immunoprecipitated with FLAG antibodies, followed by Western blotting with the corresponding cullin 2 ubiquitin ligase components. Vector-transfected HeLa cells (lanes C) were used as a control. (B) HPV16 E7 interacts with the cullin 2 ubiquitin ligase complex produced in insect cells. Sf9 insect cells were coinfected with recombinant baculoviruses encoding the various components of the cullin 2 ubiquitin ligase complex and C-E7 as indicated. E7 immunoprecipitations were performed with FLAG antibody, and coprecipitated components of the cullin 2 complex were detected by immunoblotting. (C) The HPV16 E7/cullin 2 complex isolated from insect cells (panel B) was subjected to an in vitro ubiquitination reaction including ubiquitin and UbcH5a (E2) with or without E1. Ubiquitin polymerization was analyzed by Western blotting using ubiquitin antibody. (D) HPV16 E7 binds to elongin C in vitro. [³⁵S]methionine/cysteine-labeled cullin 2, Rbx1, elongin C, elongin B, and pRB were synthesized by in vitro transcription/in vitro translation using rabbit reticulocyte lysates. The in vitro-translated proteins were incubated with bacterially produced GST-HPV16 E7 fusion protein (GST-E7) or GST in a buffer containing 0.1% NP-40, followed by affinity chromatography on glutathione-Sepharose.

FIG. 3.

HPV16 E7 sequences in CR1 and the carboxyl-terminal region contribute to association with the cullin 2 complex. (A) Schematic representation of HPV16 E7 functional regions. (B) Association of the cullin2 ubiquitin ligase complex with different HPV16 E7 mutants. Lysates of HeLa cells with stable expression of the corresponding C-E7 proteins were immunoprecipitated with FLAG antibody, followed by Western blotting with the corresponding antibodies; pRB and E7 blots (using HA antibody) are shown as controls.

FIG. 4.

Cullin 2 is a component of the HPV16 E7/pRB complex. (A) Cosedimentation of the HPV16 E7-associated pRB, p107, p130, and cullin 2 complexes on a glycerol gradient. HPV16 E7-associated protein complexes isolated from HeLa cells expressing C-E7 by immunoprecipitation with FLAG antibody followed by elution with FLAG peptide were resolved on a 10 to 40% glycerol gradient. Representative fractions were analyzed by silver staining (left) and Western blotting with the indicated antibodies (right). (B) The HPV16 E7/pRB complex contains cullin 2. HPV16 E7-associated proteins were isolated from HeLa cells expressing C-E7 by immunoprecipitation (IP) with FLAG antibody, followed by elution with FLAG peptide. The eluted protein complexes were reprecipitated with pRB antibody. The presence of cullin 2 and E7 in pRB-associated protein complexes was assessed by Western blotting. Vector-transfected HeLa cells (lanes C) were used as a control.

FIG. 5.

The cullin 2 ubiquitin ligase contributes to HPV16 E7-mediated pRB degradation. (A) Cullin 2 depletion by transfection of two siRNA oligonucleotide duplex populations results in pRB accumulation in the HPV16-positive CaSki cervical cancer cell line. Transfection of a control siRNA duplex (Ci) was used as a control. The concentration of the siRNA duplexes in the transfection mixture was 47.5 nM. Bar graphs with quantification of pRB (after normalization to GAPDH [glyceraldehyde-3-phosphate dehydrogenase] levels) and RB mRNA levels as determined by real-time PCR analysis from the same cells are shown below the blots. The RB mRNA levels represent averages and standard deviations of a single analysis performed in duplicate. Two additional experiments with higher siRNA concentrations (225 nM) yielded similar results. (B) Inhibition of pRB degradation in HPV16 E7-expressing cells upon cullin 2 depletion by siRNA. Control and HPV16 E7-expressing hTERT-immortalized NOK were transfected with the cullin 2-specific siRNA oligonucleotide duplex no. 1 (Cul2i) or a control siRNA (Ci), followed by treatment with cycloheximide to inhibit new protein synthesis. The cells were harvested at 0 and 3 h after cycloheximide treatment, and pRB levels were assessed by Western blotting. Cullin 2 and GAPDH levels are shown as controls. The concentration of the siRNA duplexes in the transfection mixture was 225 nM. (C) The HPV16 E7/cullin 2 ubiquitin ligase complex enhances pRB ubiquitination. 293 cells were cotransfected with the indicated plasmids. At 24 h after transfection, the ubiquitinated (Ub) proteins were immunoprecipitated with HA antibody beads, and ubiquitinated pRB products were detected by Western blotting with pRB antibody. Immunoglobulin heavy and light chains are denoted by asterisks.

FIG. 6.

A hypothetical model of the HPV16 E7/cullin 2 complex. (A) HPV16 E7 associates with the cullin 2 complex and recruits cellular proteins, including pRB, p130, and potentially additional, unidentified cellular proteins (?) for ubiquitination [(Ub)n]. In addition, cullin 2-associated ubiquitin ligase activity may be increased in HPV16 E7-expressing cells due to increased cullin 2 neddylation. (B) HPV16 E7 contains two BC box-like sequences (BCBL1 and BCBL2) and a cullin 2 box-like amino acid sequence within the carboxyl-terminal domain. The carboxyl-terminal HPV16 E7 sequence (amino acid residues 51 to 98) is shown, with positions of mutations that are necessary for efficient association with the cullin 2 ubiquitin ligase complex underlined in blue. BC box-like sequences (BCBLS1 and -2) are indicated by red boxes, and a sequence related to the cullin 2 box in the VHL tumor suppressor is shown in green. Detailed sequence comparisons are shown underneath, and identical residues are boxed.