The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase

Abstract

Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2 ؊/؊ mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1-and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.Epidemiological studies have established that the high-risk types of human papillomavirus (HPV) are the main etiological factors for cervical cancer (reviewed in references 23, 35, 50, and 58). Significant percentages (20 to 30%) of premalignant and malignant oral and head and neck cancer lesions have also been documented to contain these high-risk HPVs (41). Cervical cancer alone accounts for almost 12% of all cancers in women (58). Therefore, elucidation of viral functions that contribute to malignant conversion is of major importance.HPVs infect the proliferating epidermal or mucosal epithelial cells. Following persistent infections and after a long latency period, a small percentage of viral lesions progress to carcinoma in situ and squamous cell carcinoma. During this progression to malignancies, the viral genome often integrates into the host chromosome. All HPV-transformed cancer tissues express two HPV-encoded oncoproteins, E6 and E7. Both E6 and E7 possess transformation activity, and they cooperate to transform primary human keratinocytes, fibroblasts, and epithelial cells (reviewed in references 23, 35, 41, 50, and 58). Moreover, continued expression of the E7 protein is necessary for both maintenance of the transformed phenotype and a productive virus life cycle (15,50,51). A recent study showed that a reduction in the expression of E7 by RNA interference induces apoptosis...

Direct characterization of E2-dependent target specificity and processivity using an artificial p27-linker-E2 ubiquitination system

“…The acidic loop that is present in Cdc34, but not in hHR6b and UbcH5b, has been reported to be critical for efficient ubiquitination via the K48 linkage, and the loss of this loop results in a non-K48 ubiquitin linkage (19). However, our data indicate that the presence of residues 184-196 is also critical for the highly processive ubiquitination by Cdc34.…”

“…Moreover, an unstructured protein, like p27, generally interacts with many other cellular proteins, and thus its in vivo ubiquitination of unstructured proteins is likely to be more complicate. Nevertheless, E2 specificity for a target protein could provide an additional point of regulation during the RING-E3 mediated ubiquitination and could explain (i) the reason why some E2s bind RING-E3 but are not able to ubiquitinate a target protein (19,20) and (ii) the presence of many E2 and UEV (ubiquitin E2 variant) proteins.…”

E3 ubiquitin ligases and human papillomavirus-induced carcinogenesis

“…SOCS1 induces ubiquitination and degradation of E7 in a SOCS-box-dependent manner, causing a concomitant increase in pRb levels. 51 These two E7 degradation pathways seem to be complementary, as the first appears to be predominant in the nucleus 50 whereas the second appears to be predominant in the cytoplasm. 51 Interestingly, the stability of E7 is also regulated by its interaction with the deubiquitinating enzyme ubiquitin-specific peptidase 11 (USP11).…”

“…49 Two independent pathways for E7 ubiquitination have been clarified (Table 1): one involves the SCF ubiquitin ligase complex; 50 the second involves the interferon-g-inducible suppressor of cytokine signalling-1 (SOCS1), a cytokine signalling suppressor functioning as an antioncogene against various haematopoietic oncogenic proteins. SOCS1 induces ubiquitination and degradation of E7 in a SOCS-box-dependent manner, causing a concomitant increase in pRb levels.…”

The Emerging Role of the COP9 Signalosome in Cancer

“…[10][11][12]. This function has been proposed to be mediated by the catalytic subunit CSN5 but requires the assembly of the entire CSN holocomplex (10)(11)(12)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). The deneddylation of Cullins is required for Cullin-mediated degradation of E3 substrates.…”