The HPV E7 oncoprotein is unstable and is a target of the 26S proteasome. E7 functions as a transcription regulator and induces S-phase synthesis in HPV-transformed epithelial cells (reviewed in references 23
, and 58
). The function of E7 is critical for both induction and maintenance of HPV-associated cancer. Thus, it is important to understand the molecular mechanisms that govern the cellular stability of this oncoprotein. In this study, we described the E2 and E3 ubiquitin ligases involved in the ubiquitination of E7. Using in vitro ubiquitination assays, we showed that UbcH7 is the specific ubiquitin-conjugating enzyme involved in E7 ubiquitination. Furthermore, we provided evidence that E7 interacted with and was ubiquitinated by the Cul1- and Skp2-containing ubiquitin ligase complex. Immunofluorescence and cell fractionation studies demonstrated that multiubiquitinated E7 accumulates predominantly in discrete nuclear foci upon inhibition of proteasome-dependent degradation.
Dissection of the enzymatic components involved in the ubiquitination of E7 revealed a role for the E2-conjugating enzyme UbcH7. The E2-conjugating enzymes are a closely related family of proteins (25
). Several E2 enzymes have been characterized (25
). Often, a common E2 enzyme carries out the ubiquitination of multiple substrates. Our results showed that the other E2 enzymes, including the closest homologues UbcH6 and UbcH5a, are significantly less active in E7 ubiquitination (42
). All of the recombinant E2 enzymes used in this study are functionally active, as they can form active thiol esters with ubiquitin (data not shown). Furthermore, UbcH7 is fractionated in FrI of HeLa cell S-100, which was essential for the ubiquitination of E7 in vitro. UbcH7 can function in conjugation with different types of E3 ubiquitin ligases, including a hect-E3, such as E6AP, or a ring-E3, such as c-Cbl (24
). The results presented in this study show that for the ubiquitination of E7, UbcH7 cooperates with the Cul1-Skp2 (SCF) complex, which belongs to the ring-E3 family of ligases (29
The SCF complexes are well-studied ubiquitin ligases that mediate the ubiquitination of diverse cell cycle regulatory and signaling proteins (reviewed in reference 12
). Our results showed that E7 could interact with the Cul1-Skp2 complex both in vitro and in vivo. Moreover, E7 could be ubiquitinated by purified UbcH7 and the Cul1 complex. Finally, we showed that the half-life of E7 was increased from less than 30 min in wild-type MEFs to more than 1 h in Skp2−/−
MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a major role in the ubiquitination and proteolysis of E7. Interestingly, we observed delayed but not complete blockage of E7 proteolysis in Skp2−/−
cells, suggesting that E7 can also be degraded by an Skp2-independent pathway. Similar Skp2-independent proteolysis has been reported for other Skp2 targets, including p27 and E2F-1 (20
). Furthermore, many cellular proteins, including p53, p21, and p27, are targeted by multiple ubiquitin ligases (12
). Among them, p21 is degraded by the proteasome through both ubiquitination-dependent and ubiquitination-independent pathways (49
). The ubiquitination-independent proteolysis of p21 involves its association with the C8 subunit of the proteasome (52
). Previous results showed that E7 could bind to the S4 subunit of the 26S proteasome (2
). The role of the E7-S4 interaction in E7 proteolysis is not clear. Future investigations will provide insight into other mechanisms of E7 proteolysis.
SCF-dependent ubiquitination often requires specific phosphorylation of the substrate (13
). Both p27Kip1
and E2F-1 are phosphorylated by cyclin-dependent kinases prior to ubiquitination by SCF-Skp2 (37
). E7 is phosphorylated by casein kinase II at serine 30 or 31 and by an unknown kinase at serine 71 (33
). E7 also associates with cyclin E/Cdk2 kinase and cyclin A/Cdk2 kinase, but it is not known whether E7 is phosphorylated by these cyclin-dependent kinases (1
). Although our result show that recombinant His-E7 can be efficiently ubiquitinated in vitro, they does not rule out the possibility that the ubiquitination of E7 is regulated by specific phosphorylation. HeLa cell FrIIA used for E7 ubiquitination contained a kinase(s) for E7 phosphorylation, and it is possible that E7 is phosphorylated during in vitro ubiquitination. We observed a doublet corresponding to E7 in extracts of Caski cells after treatment with the proteasome inhibitor MG132 (Fig. ). Further studies will be required to determine whether phosphorylation regulates the ubiquitination of E7 in vivo.
The results presented in this study suggest that E7 proteolysis occurs in the nucleus. There are conflicting reports about the subcellular localization of the HPV E7 oncoprotein (19
). E7 has been reported to be predominantly nuclear in transiently transfected cells (18
). Others have reported the cytoplasmic localization of E7 in CV1 cells (60
) and in normal oral keratinocyte cells (46
). Using biochemical fractionation experiments, we found that a major portion of the E7 protein in Caski cells is cytoplasmic. This is a surprising observation, because multiple nuclear functions of E7, including the transactivation of E2F-regulated genes, binding with the Rb family of transcription regulators, and interactions with the transcription factors Fos, E2F-1, and others, predict the nuclear localization of E7. It has been convincingly demonstrated that these nuclear interactions of E7 are critical for its transformation function. However, interactions of E7 with cytoplasmic proteins, such as actin, M2 pyruvate kinase, and acid α-glucosidase, have also been reported (46
). Interestingly, we recovered a significant level of E7 in the nuclear fraction of Caski cells treated with the proteasome inhibitor MG132. This result suggests that E7 in the nuclear compartment is more labile than cytoplasmic E7. It is also possible that MG132 triggers some modification of E7 that induces its nuclear localization. Immunofluorescence with the E7 monoclonal antibody further confirmed the cell fractionation results. E7 immunofluorescence was readily detected in the cytosolic and perinuclear regions of asynchronously growing Caski cells (Fig. ). These cells also showed some E7 staining in discrete foci in the nuclei.
A previous study showed that the inhibition of proteasome activity causes the deposition of protein aggregates as cytoplasmic inclusion bodies known as aggresomes (27
). Aggresomes generally contain ubiquitin-rich cytoplasmic proteins and are linked to the pathogenesis of many diseases (30
). Following proteasomal inhibition, E7 immunostaining was observed specifically in discrete nuclear foci, not in cytosolic aggresome-like structures, suggesting that E7 proteolysis occurs in the nucleus. In support of this hypothesis, we recovered most of the high-molecular-weight polyubiquitinated E7 in the detergent-insoluble nuclear pellet of MG132-treated Caski cells. The E7 immunostaining was found to partially overlap the PML immunostaining in MG132-treated Caski cells. This result suggests that E7 proteolysis occurs in defined nuclear bodies (Fig. ).
Many questions remain unanswered. (i) What triggers the localization of E7 to the nucleus? (ii) Is E7 ubiquitination regulated by specific phosphorylation? (iii) Are nuclear E7 and cytoplasmic E7 differentially modified? E7 strongly activates the transcription of E2F- and AP1-dependent genes. The major known biochemical function of E7 is the activation of E2F to stimulate DNA replication and cell division. It is intriguing to speculate that in cervical cancer cells, E7 is sequestered in the cytoplasm in a transcriptionally inactive form. Ubiquitin-dependent proteolysis actively regulates the level of E7 in the nuclei of HPV-containing tumor cells and may be linked to the survival mechanism of the tumor cells.