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Proc Natl Acad Sci U S A. Aug 9, 2005; 102(32): 11492–11497.
Published online Aug 1, 2005. doi:  10.1073/pnas.0505337102
PMCID: PMC1182135
Microbiology

Association of the human papillomavirus type 16 E7 oncoprotein with the 600-kDa retinoblastoma protein-associated factor, p600

Abstract

The human papillomavirus type 16 (HPV-16) E7 gene encodes a multifunctional oncoprotein that can subvert multiple cellular regulatory pathways. The best-known cellular targets of the HPV-16 E7 oncoprotein are the retinoblastoma tumor suppressor protein pRB and the related pocket proteins p107 and p130. However, there is ample evidence that E7 has additional cellular targets that contribute to its transforming potential. We isolated HPV-16 E7 associated cellular protein complexes by tandem affinity purification and mass spectrometry and identified the 600-kDa retinoblastoma protein associated factor, p600, as a cellular target of E7. Association of E7 with p600 is independent of the pocket proteins and is mediated through the N terminal E7 domain, which is related to conserved region 1 of the adenovirus E1A protein and importantly contributes to cellular transformation independent of pRB binding. Depletion of p600 protein levels by RNA interference substantially decreased anchorage-independent growth in HPV-positive and -negative human cancer cells. Therefore, p600 is a cellular target of E7 that regulates cellular pathways that contribute to anchorage-independent growth and cellular transformation.

Keywords: apoptosis, cervical carcinogensis, retinoblastoma tumor suppressor

Human papillomaviruses (HPVs) are small DNA viruses with double-stranded circular genomes that exhibit a tropism for epithelial cells. Of the >200 HPVs that have been identified, a subgroup of ≈30 HPVs specifically infects mucosal tissues. These HPVs are classified as “low-risk” or “high-risk”, depending on the clinical prognosis of the lesions they cause. More than 99% of cervical carcinomas and ≈20% of oral cancers are associated with high-risk HPV infections (reviewed in ref. 1). The HPV E6 and E7 proteins are consistently expressed in HPV-associated cervical cancers, and persistent expression of these viral oncoproteins is necessary for maintenance of the transformed phenotype. High-risk HPV E7 proteins are sufficient to transform NIH 3T3 cells, can cooperate with the ras oncogene to transform primary baby rat kidney cells, extend the life span of primary human epithelial cells, and in combination with E6 facilitate their immortalization (reviewed in ref. 2). Furthermore, high-risk HPV E7 proteins importantly contribute to malignant progression through induction of centrosome abnormalities that cause genomic instability, thereby fostering development of aneuploidy (reviewed in ref. 3).

HPV-16 E7 encodes a small, 98-aa multifunctional protein. The N-terminal portion of HPV-16 E7 shares amino acid sequence similarity to a portion of conserved regions (CR) 1 and 2 of the adenovirus E1A protein and related sequences in polyomavirus large tumor antigens (4, 5). High-risk HPV E7 proteins exert their biological activities chiefly by interacting with host cellular protein complexes, thereby dysregulating their normal physiological functions. The best-defined cellular target of HPV E7 proteins is the retinoblastoma tumor suppressor protein pRB and the related “pocket proteins” p107 and p130 (reviewed in ref. 6). High-risk HPV E7 proteins associate with the pocket proteins and induce their proteasomal degradation (7, 8). The LXCXE motif within the CR2 homology domain of E7 is sufficient for pocket protein binding (9, 10), but additional sequences located in the immediate N-terminal CR1 homology domain of E7 are required for pocket protein degradation (8, 11), and these sequences are also necessary for the transforming activities of E7 (12, 13). Experiments with tissue culture and animal model systems have shown that some of the transforming activities of HPV-16 E7 are at least in part independent of pocket protein inactivation (14, 15). In addition, the ability of HPV-16 E7 to induce centrosome-associated mitotic abnormalities is also independent of pocket protein inactivation (16). To determine the biochemical basis for the diverse biological activities of high-risk HPV E7 oncoproteins, we isolated HPV-16 E7 oncoprotein-associated cellular protein complexes from cervical carcinoma cells by tandem affinity purification (TAP) and identified individual protein components by mass spectrometry. Here we report the isolation of p600 as a cellular target of E7 and provide evidence that p600 may be involved in regulating cellular pathways that are necessary for cellular transformation.

Materials and Methods

Cell Lines. HeLa S3 suspension cells (obtained from Y.N.) were maintained in DMEM containing 10% FBS. Large-scale HeLa S3 suspension cultures were grown in Joklik-modified minimum essential medium (GIBCO Invitrogen), containing 10% calf serum, at 37°C with constant stirring. U2OS, IMR90, IMR90-E7 (17), and Phoenix cells were maintained in DMEM/10% FBS. CaSki cells were grown in RPMI medium 1640 (GIBCO Invitrogen)/10% FBS, and NIH 3T3 cells were grown in DMEM/10% calf serum.

Generation of Plasmids and HPV E7-Expressing HeLa Cell Lines. HPV E7 sequences were inserted into the Xho and NotI sites of pOZ-C or pOZ-N retroviral vectors in frame with the N- or the C-terminal FLAG/hemaglutinin (HA) epitope tags (18). To construct pLXSN CE7 and pLXSN NE7, each coding sequence was PCR amplified from pOZ-CE7 and pOZ-NE7, respectively.

HeLa cells expressing E7 proteins were generated by retroviral infection followed by three consecutive rounds of selection using IL-2R antibody conjugated to magnetic beads (18). Expression of HPV E7 proteins was verified by immunoblotting and/or immunoprecipitation and immunofluorescence.

Immunological Methods. The following antibodies were used: HPV-16 E7 (8C9, Zymed, South San Francisco, CA; ED17, Santa Cruz Biotechnology), pRB (M153, Santa Cruz Biotechnology), and HA (Y11, Santa Cruz Biotechnology). The p600-specific polyclonal antibody was generated by Y.N. M2 FLAG antibody resin was from Sigma, and HA-antibody resin (HA probe F7) was from Santa Cruz Biotechnology. HA (HA.11) and 3× FLAG peptides were from Babco (Richmond, CA) and Sigma, respectively. IL-2R antibody (clone 7G7/B6, Upstate Biotechnology, Lake Placid, NY) was conjugated to Dynalbeads (M-450, Dynal, Great Neck, NY).

For immunoprecipitations, cells were lysed by two freeze–thaw cycles (liquid N2/37°C) in three volumes of 0.3B buffer (20 mM Tris·HCl, pH 8.0/0.3 M KCl/5 mM MgCl2/10% glycerol/0.1% Tween-20/10 mM 2-mercaptoethanol/0.2 mM PMSF) followed by incubation at 4°C for 30 min and centrifugation at 16,000 × g at 4°C, for 30 min. The supernatant was recentrifuged at 16,000 × g at 4°C for 10 min followed by preclearing with protein G PLUS agarose (Santa Cruz Biotechnology). Immunoprecipitations with primary antibodies were performed for 3–4 h at 4°C. Immune complexes were purified by using protein G PLUS agarose and washed three times with 1 ml of 0.3B buffer.

For localization of E7 and p600 by confocal fluorescence microscopy, CaSki cells grown on coverslips were fixed in 4% paraformaldehyde, 0.025% glutaraldehyde in BRB 80 (80 mM Pipes, pH 6.8/1 mM MgCl2/1 mM EGTA) for 15–20 min at 37°C, and rinsed three times with BRB 80 and two times with antibody dilution solution (0.1% Triton X-100/2% BSA in BRB 80). Fixed cells were permeabilized in BRB 80 containing 0.1% Triton X-100 for 10 min at room temperature and incubated with antibody dilution solution for 30 min at 20°C. Cells were incubated with rabbit polyclonal p600 antibody (1:1,000) and monoclonal E7 antibody (1:10) for 2 days at 4°C. After washing with antibody dilution solution and BRB 80 for 30 min each, cells were incubated with FITC-conjugated anti-mouse (1:500) and rhodamine-conjugated anti-rabbit (1:2,000) antibodies for 3 h at 20°C. After rinsing with antibody dilution solution and BRB 80, cells were mounted and analyzed.

TAP and Mass Spectrometry. Cellular protein complexes associated with E7 were isolated from 5–10 liters of stable HeLa suspension cell lines (18). After a first round of affinity purification on M2 FLAG antibody resin (Sigma), proteins were eluted with 0.5 mg/ml 3× FLAG peptide. For subsequent purification on HA antibody resin, samples were incubated with HA antibody resin (HA-probe F-7, Santa Cruz Biotechnology) for 3 h at 4°C with rotation. Beads were washed three times with 0.1B buffer (20 mM Tris·HCl, pH 8.0/0.1 M KCl/5 mM MgCl2/10% glycerol/0.1% Tween-20/10 mM 2-mercaptoethanol/0.2 mM PMSF). Protein complexes were eluted with HA peptide (0.5 mg/ml) for 30 min at 20°C and separated on SDS/4–12% Bis-Tris polyacrylamide gradient gels (Invitrogen). Individual bands were visualized by colloidal blue (Bio-Rad) staining, excised, and analyzed by mass spectrometry at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School).

Knockdown Experiments. The human p600-specific small hairpin RNA (shRNA) expression plasmid was generated by using the sequence GCAGTACGAGCCATTCTAC expressed from pRetro/Super (19). A reverse orientation shRNA expression vector CATCTTACCGAGCATGACG was used as a control. The pRetro/Superbased mouse p600-specific shRNA expression vector was generated by Y.N. Recombinant p600 shRNA expressing retroviruses were generated by transfecting Phoenix cells using FuGENE 6 (Roche Diagnostics). NIH 3T3 cells were infected with p600 shRNA or control shRNA expressing retrovirus and selected in 2 μg/ml puromycin. To generate HPV-16 E6 and/or E7 expressing populations, selected stable p600 or reverse orientation control shRNA expression vector transduced lines were infected with pLXSN, pLXSN E7, or pLXSN E6/E7 retroviral supernatants followed by selection in 500 μg/ml G418 for ≈2 weeks. The stable NIH 3T3 cell lines generated were maintained in puromycin (2 μg/ml) and G418 (500 μg/ml). Stable p600 and control knockdown U2OS and CaSki cell lines were similarly established after selection with 2 μg/ml puromycin.

Anchorage-Independent Growth Assays. Cells (2,500 per well of a six-well plate) were suspended in 0.3% Agar Noble (Difco) dissolved in tissue culture medium and layered onto dishes coated with 0.5% Agar Noble. Colony formation was evaluated in duplicate wells after 2–3 weeks by phase-contrast microscopy.

Results

Construction and Characterization of HA/FLAG Epitope-Tagged HPV-16 E7 Proteins. To isolate and identify cellular protein complexes associated with the HPV-16 E7 protein in human cervical cancer cell lines by TAP, HPV-16 E7 coding sequences were cloned into pOZ-N and pOZ-C to generate FLAG/HA tagged HPV-16 E7 at the N (N-E7) or C (C-E7) terminus, respectively. In each case, the epitope tags are separated from the E7 coding sequence by a linker and the FLAG and HA tags are also separated by linkers (Fig. 1A). Because some N-terminally tagged HPV-16 E7 proteins are functionally defective (20), we performed transformation assays in NIH 3T3 cells to assess functionality. Consistent with our previous findings, the N-terminally double-tagged HPV-16 E7 protein (N-E7) was impaired for NIH 3T3 cell transformation, whereas the C-terminally double tagged E7 protein (C-E7) was competent for transformation at a level similar to the untagged HPV-16 E7 protein (Fig. 1B).

Fig. 1.
Construction and characterization of epitope-tagged HPV-16 E7 proteins. (A) Schematic representation of the FLAG/HA double tagged HPV-16 E7 fusion proteins constructed for this study. C-E7 and N-E7 contain FLAG/HA tags fused to their C and N termini, ...

Isolation of Cellular Protein Complexes Associated with HPV-16 E7 in the HeLa Cervical Carcinoma Cell Line. With these results in mind, we next generated populations of the HPV-18-positive cervical carcinoma cell line HeLa with stable expression of the C-E7 or N-E7 proteins. HeLa cells were chosen for these experiments because they are derived from a cervical cancer, contain an inherently intact retinoblastoma tumor suppressor pathway (21, 22), and the ectopic expression of HPV-16 E7 in these cells can rescue the biological and biochemical defects that are caused by loss of expression of endogenous HPV-18 E7 (23, 24). The resulting HeLa cell populations expressed the tagged E7 proteins at low levels; similar to those detected in the SiHa cervical carcinoma cell line where E7 is expressed from integrated low-copy partial HPV-16 genomes (2527) (data not shown). Low-level expression of the tagged E7 protein is desirable for these experiments because it minimizes the possibility of formation of aberrant complexes (18). A total of 5–10 liters of C-E7-expressing or control HeLa S3 cells were grown in spinner flasks, and C-E7-associated protein complexes were purified by consecutive rounds of immuno-affinity purification using HA and FLAG antibody beads. To control for nonspecific protein binding to the antibody columns, we performed the same purification steps with an equal amount of vector transduced HeLa S3 cells. Protein complexes were resolved on SDS-polyacrylamide gels and visualized by Colloidal Blue staining (Fig. 2A). Individual bands were excised, digested with trypsin, and identified by mass spectrometry. These experiments yielded evidence for ≈100 different cellular proteins as potential components of HPV-16 E7 associated host cellular protein complexes. Among the proteins identified are previously described cellular targets of E7 including pRB, p107, p130, several E2F and DP family members, cyclin A, cyclin E, CDC2, and CDK2 (Fig. 2 A), thus clearly supporting the viability of the TAP approach.

Fig. 2.
Identification of p600 as an interactor of HPV-16 E7 and mapping of E7 domains necessary for p600 association. (A) SDS/4–12% PAGE analysis of cellular protein complexes associated with C-E7 in HeLa cells after TAP. C denotes TAP of an equivalent ...

The p600 Protein Associates with HPV-16 E7 N-Terminal Sequences That Are Important for Transformation Independent of pRB Binding. Some of the other proteins may presumably represent additional cellular E7 targets. For this study, we focused on a C-E7 associated protein band that migrates at a molecular size of >188 kDa. In multiple experiments, we obtained ≈100 peptides corresponding to p600, a 5,183-aa protein (GenBank accession number AF348492; Fig. 2 A).

Because p600 has been identified as a component of pRB-associated protein complexes in the HPV-18-positive HeLa cervical carcinoma cells (Y.N., unpublished results), we determined whether association of E7 with p600 may be indirect through pRB and, thus, depends on the integrity of the core pRB-binding site of HPV-16 E7. A series of HeLa-based cell lines with stable expression of various C-terminally HA/FLAG tagged HPV-16 E7 mutants were generated and their ability to associate with p600 and pRB was compared by immunoprecipitation/immunoblot experiments (Fig. 2B). These experiments showed that HPV-16 E7 mutants H2P and Δ6–10 that target the N-terminal E7 CR1 homology domain and retain the ability to associate with pRB are defective for association with p600 (Fig. 2B). In contrast the pRB-binding-deficient HPV-16 E7 Δ21–24 mutant associated with p600 at levels similar to wild-type HPV-16 E7 (Fig. 2B) Similarly, HPV-16 E7 mutants within a C-terminal domain that is important for cellular transformation independent of pRB binding and degradation (14) retained the capacity to associate with p600, albeit at reduced levels (Fig. 2B). The cell lines expressing the different E7 mutants contained similar p600 protein levels (data not shown).

The transformation-defective N-terminally HA/FLAG tagged HPV-16 E7 fusion protein (N-E7), was unable to interact with p600, even though mass spectrometric analyses revealed that N-E7 efficiently interacted with pRB and other pocket proteins (data not shown). Hence, association of HPV-16 E7 with p600 is independent of the ability to interact with pRB.

Low-Risk HPV-6b and HPV-11 E7 Proteins Can Associate with p600. Low-risk HPV-6b and HPV-11 E7 proteins are impaired for cellular transformation, associate with pRB at a decreased efficiency, and are defective for inducing its degradation (reviewed in ref. 6). To determine whether the HPV-6b and HPV-11 E7 proteins can associate with p600, we generated HeLa-based cell lines with stable expression of C-terminally HA/FLAG-tagged HPV-6b or HPV-11 E7 proteins and performed immunoprecipitation/immunoblot experiments. HPV-6b and HPV-11 E7 associated with p600 as efficiently as HPV-16 E7, whereas consistent with earlier reports (9, 28), HPV-6b and -11 E7 proteins interacted with pRB at a decreased efficiency (Fig. 2C).

Association of HPV-16 E7 with p600 in the HPV-16-Positive CaSki Cervical Cancer Line. To evaluate association of untagged HPV-16 E7 with endogenous p600, we performed coimmunoprecipitation experiments in the HPV-16 positive CaSki cervical carcinoma cell line. The HPV-negative C33A cervical cancer cell line was used as a negative control. Immunoblot experiments revealed that CaSki and C33A cells expressed similar amounts of p600. Immunoprecipitation experiments were performed by using HPV-16 E7-specific monoclonal antibodies, and p600 was coprecipitated in the HPV-16-positive CaSki cells but not in HPV-negative C33A cells (Fig. 3A). Therefore, association of HPV-16 E7 with p600 is detected in a cervical carcinoma cell line under conditions of endogenous p600 and HPV-16 E7 expression.

Fig. 3.
Association and colocalization of HPV-16 E7 with p600 in cervical cancer cells. (A) HPV-16 E7 associates with p600 in the HPV-16 positive CaSki human cervical carcinoma cell line. Immunoprecipitation was performed with E7-specific antibodies followed ...

To further document HPV-16 E7/p600 association in vivo, we also performed confocal immunofluorescence experiments in CaSki cells. HPV-16 E7 and p600 staining was observed in cytoplasmic as well as nuclear structures with partial colocalization of the HPV-16 E7 and p600 signals (Fig. 3B).

Association of HPV-16 E7 with p600 Does Not Contribute to E7-Mediated pRB Degradation. A computer search of the conserved domain database (29) revealed that the p600 protein contains a RING finger-like structure in its N-terminal domain (amino acid residues 1,660–1,717). RING fingers are cysteine/histidine-containing zinc-binding structures that are also detected in some ubiquitin ligases (reviewed in ref. 30). The RING finger in p600 is most closely related to the RING finger in N-recognin (31, 32), a specificity factor and ubiquitin ligase in the N-end rule pathway (reviewed in ref. 33). Therefore, p600 may also be associated with E3 ubiquitin ligase activity. Because our mapping studies revealed that the association with p600 requires N-terminal HPV-16 E7 sequences (Fig. 2B) that are necessary for E7-mediated pRB degradation (11), we next determined whether the interaction of E7 with p600 may be involved in HPV-16 E7-mediated pRB destabilization. We used small interfering RNA (siRNA) technology to deplete p600 in HPV-16 E7-expressing cells. If p600 was a rate-limiting factor in E7-mediated pRB degradation, it may be expected that pRB levels be elevated in p600-depleted, E7-expressing cells. However, experiments in E7-expressing IMR90 human diploid lung fibroblasts (17) and HPV-16-positive CaSki human cervical cancer cells by stable expression of a p600-specific shRNA expression vector provided no evidence for elevated pRB levels upon p600 depletion (Fig. 3C). The HPV-16 E7 protein has a short half-life and is degraded through a proteasome-dependent pathway involving conjugation of ubiquitin to the N-terminal residue (34). Depletion of p600 had no effect on HPV-16 E7 steady state levels (data not shown) and, hence, it is unlikely that p600 plays a rate-limiting role in E7-mediated pRB destabilization or HPV-16 E7 turnover.

Depletion of p600 Decreases Anchorage-Independent Growth. Association of p600 with HPV-16 E7 is mediated through N-terminal E7 sequences that are necessary for cellular transformation, including anchorage-independent growth in NIH 3T3 cells (12). To determine whether p600 depletion in CaSki cells may affect their transformed phenotype, we assessed their potential to form colonies in soft agar-containing growth medium. Compared to CaSki cells that express the reverse orientation shRNA expression vector, p600-depleted CaSki cells displayed a >10-fold decreased capacity to form colonies in soft agar-containing medium (Fig. 4A). Similarly, p600 depletion in HPV-16 E7 or HPV-16 E6/E7 transformed NIH 3T3 cells resulted in a 30–40% decrease in anchorage-independent growth (Fig. 4 B and C). This finding is somewhat less dramatic than in the CaSki cell line, which may reflect genetic differences between these two cell types. To determine whether this effect was specific for HPV E7-transformed cells, we also depleted p600 in U2OS human osteosarcoma cells, which do not contain HPV sequences. Similar to HPV-16-transformed CaSki cells, p600 depletion in U2OS cells resulted in an ≈10-fold decreased capacity to form colonies in soft agar (Fig. 4D). Importantly, p600 depletion did not markedly reduce growth of U2OS cells in monolayer culture, suggesting that p600 depletion does not generally interfere with cellular proliferation (data not shown). Likewise, p600 depletion in parental NIH 3T3 cells caused a decrease in anchorage-independent growth. Hence, p600 depletion in HPV-positive or -negative cells inhibits their capacity to grow in the absence of a substratum, which represents an important hallmark of cellular transformation (reviewed in ref. 35).

Fig. 4.
Depletion of p600 by stable expression of specific shRNA expression plasmids (600i) decreases anchorage independent growth in HPV-16 positive CaSki cervical cancer (A), HPV-16 E7 expressing NIH 3T3 (B), and HPV-16 E6/E7 expressing NIH 3T3 (C) and HPV-negative ...

Discussion

Here we report the isolation of host cellular protein complexes associated with the HPV-16 E7 oncoprotein in the HeLa cervical cancer cell line. In addition to well established cellular target proteins such as pRB and E2F family members, cyclins A and E, cdk2, and cdk1 (reviewed in ref. 2), we discovered additional potential cellular proteins in association with HPV-16 E7 (Fig. 2 A). These include the 600-kDa retinoblastoma protein associated factor, p600. This protein was initially discovered in HeLa cells by TAP as a pRB-associated protein (Y.N., unpublished results). Our results show that the p600/HPV-16 E7 interaction is mediated through an N-terminal E7 domain that is related to a portion of CR1 in Ad E1A (4) (Fig. 2B). Association of p600 with HPV-16 E7 is independent of pRB because the pRB binding-deficient HPV-16 E7 Δ21–24 mutant associates with p600 at similar levels as wild-type HPV-16 E7. In addition, the bovine papillomavirus type 1 (BPV-1) E7 protein, which lacks the LXCXE core pRB-binding domain and is pRB-binding deficient (9), was shown in an independent study to associate with p600 through related N-terminal sequences (36). Additional studies are necessary to determine whether the HPV-16 E7/p600 complex also contains pRB or whether the HPV-16 E7/pRB and HPV-16 E7/p600 complexes represent biochemically and biologically distinct entities. In addition, it remains to be determined whether E7 associates directly with p600 or whether complex formation is mediated through another cellular protein.

However, our experiments strongly suggest that p600 is an important cellular target that mediates some of the transforming activities of HPV-16 E7. For one, p600 binding-deficient HPV-16 E7 mutants within the N-terminal CR1 homology domain of E7 have a reduced transforming potential (12, 13). Similarly, the ability of BPV-1 E7 to cooperate with BPV-1 E6 to induce anchorage-independent growth (37) maps to related N-terminal BPV-1 E7 sequences that are necessary for p600 association (36). Moreover, p600 depletion in HPV-16 E7 and E6/E7-transformed NIH 3T3 mouse fibroblasts and HPV-16-positive CaSki cervical cancer cells interfered with the transformed phenotype, as evidenced by a marked reduction of anchorage independent growth (Fig. 4). However, this effect was not specific to HPV oncogene-transformed cells, and p600 depletion in the HPV-negative U2OS human osteosarcoma cell line resulted in a similar decrease in anchorage-independent growth (Fig. 4). Hence, p600 may be an important regulator of cellular processes that modulate cellular proliferation and/or survival in response to detachment from the matrix.

In addition, we found that, similar to HPV-16 E7-expressing cells (11), p600-depleted IMR90 human diploid fibroblasts and NIH 3T3 murine fibroblasts are sensitized to cell death under conditions of confluence and growth factor deprivation (K.W.H. and K.M., unpublished observations). These observations suggest that p600 may be a component of the trophic sentinel-signaling pathway, an important intrinsic tumor suppressor pathway that functions to thwart outgrowth of early neoplastic cell clones that have suffered mutations in oncogene pathways (reviewed in ref. 38).

Even though p600 is a very large protein, it does not contain many conserved domains that yield much information regarding its biological function. Of note is a RING finger domain between amino acid residues 1660 and 1717 that is most closely related to a similar motif in N-recognin, a ubiquitin ligase in the N-end rule pathway that directly binds to N-terminal destabilizing amino acid residues (31, 32). Hence, it is tempting to speculate that p600 may also function as a regulator of ubiquitin-mediated proteolysis. Despite the fact that p600 associates with HPV-16 E7 through N-terminal sequences that are necessary for E7 induced pRB destabilization, our siRNA experiments suggest that p600 is not a rate-limiting factor for HPV-16 E7-mediated pRB degradation (Fig. 3C) or proteasome-mediated turnover of HPV-16 E7 (data not shown).

Proteins related to p600 have also been isolated from other organisms. These include the Arabidopsis thaliana BIG protein (39) and the Drosophila melanogaster calossin/pushover protein (40). The Arabidopsis BIG protein mediates polar auxin transport (39) as well as hormone and light responses (41), whereas the Drosophila calossin/pushover protein has been implicated in regulating neuronal excitability and transmitter release processes at neuromuscular junctions as well as glial growth (40, 42). Thus, it is tempting to speculate that p600 may be similarly involved in vesicle trafficking in mammalian cells. Given its large size, p600 is most likely a multifunctional protein; however, it is presently unclear whether and how such potential vesicle trafficking functions may be related to the regulation of anchorage-dependent growth in mammalian cells that is inferred from our studies (Fig. 4). Nevertheless, because HPV-16 E7 colocalizes and associates with cytoplasmic as well as nuclear p600 (Fig. 3B), association of the HPV-16 E7 protein may affect nuclear as well as cytoplasmic p600 activities.

In summary, we have identified p600 as a cellular target of the HPV-16 E7 oncoprotein. Our results indicate that p600 may mediate some of the pRB-independent transforming activities of HPV-16 E7. The simultaneous identification of p600 as a potential transformation target of BPV-1 E7 (36) suggests that this mechanism is conserved with other papillomavirus types. In addition, the E7 sequences that are necessary for p600 association are also conserved within the adenovirus E1A and simian virus 40 T antigen sequences (4, 5), and it will be interesting to determine whether these oncoproteins also target p600. Clearly, p600 association of HPV E7 proteins per se is not sufficient for cellular transformation because the transformation-defective low-risk HPV E7 proteins efficiently associate with p600 (Fig. 2C). This finding is similar to what has been previously reported for E7/pRB complex formation, where cellular transformation has been linked to pRB degradation, and not mere binding (20). Hence, p600 association may be necessary but not sufficient for transformation by papillomavirus E7 proteins. Therefore, it will be important to determine the functional consequences of p600 association of transforming and nontransforming papillomavirus E7 proteins in more detail.

Acknowledgments

We thank Dr. Galloway (Fred Hutchinson Cancer Center, Seattle) for the pLXSN based expression vectors and Dr. Agami (Center for Biomedical Genetics, Utrecht, The Netherlands) for his kind gift of the pRetro/Super plasmid. We also thank Kei-ichiro Ishiguro and Hideaki Tagami, two previous members of the Nakatani laboratory, for their kind help with various aspects of tandem affinity purification. This work was supported by Public Health Service Grants R01-CA081135 (to K.M.) and P01-CA050661 (to P.M.H.). J.D. was supported by American Association Society Fellowship PF-0414701.

Notes

Author contributions: K.-W.H. designed research; K.-W.H. performed research; H.O. and Y.N. contributed new reagents/analytic tools; K.-W.H., J.D., P.M.H., and K.M. analyzed data; and K.-W.H. and K.M. wrote the paper.

Abbreviations: HPV, human papillomavirus; CR, conserved region; TAP, tandem affinity purification; HA, hemagglutinin; shRNA, small hairpin RNA.

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