1 Institute of Parasitology, MacDonald Campus, McGill University, Montreal, Canada, H9X 3V9
2 Lady Davis Institute for Medical Research, McGill University, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Canada, H3T 1E2
3 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy
4 Department of Microbiology and Immunology, McGill University, 3775 University Street, Room 511, Montreal, Canada, H3A 2B4
Correspondence
Greg Matlashewski
greg.matlashewski{at}mcgill.ca
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ABSTRACT |
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Shirin Kazemi and Suiyang Li contributed equally to this work.
Present address: Department of Biochemistry, McIntyre Medical Building, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada, H3G 1Y6.
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MAIN TEXT |
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In order to detect the HPV E6 protein in the cell, we initially constructed plasmids expressing fusion proteins linking a FLAG epitope tag to the N termini of HPV-11 and -18 E6. The FLAG-E6 expression vectors were created by cloning the CMV-FLAG polylinker region of the pFLAG-CMV-2 vector (Sigma) into the multiple cloning region of pCDNA3.1/Zeo (Invitrogen) and the E6 sequences were then cloned between the EcoRI and EcoRV restriction sites of the newly generated vector. To determine whether the N-terminal FLAG-tagged HPV-18 E6 protein was detectable and retained activity, we first generated a stable cell line expressing the HPV-18 E6 fusion protein. Human HT1080 cells were used because they express wild-type p53 protein. The FLAG-tagged HPV-18 E6 expressing and control HT1080 cells were exposed to 1 µM adriamycin to induce p53 expression and the level of p53 was determined by Western blot analysis. As shown in Fig. 1(A), high levels of p53 protein accumulated in HT1080 control cells after 4 h of adriamycin treatment, whereas little p53 was detected in cells expressing FLAG-18E6. Thus, the N-terminal FLAG-tagged 18E6 protein was functional in these cells with respect to mediating p53 degradation. It is noteworthy that although a C-terminal AU epitope-tagged HPV-18 E6 was also able to mediate p53 degradation, it was not able to mediate the degradation of the PDZ domain-containing MAGI-1 protein. In contrast, the N-terminal tagged HPV-18 E6 protein was able to mediate the degradation of both p53 and MAGI-1 (data not shown). Since PDZ domain-containing proteins such as MAGI-1 and DLG are major targets of E6 from oncogenic HPVs (Glausinger et al., 2000
; Gardiol et al., 1999
), we performed subsequent experiments using N-terminal tagged E6 proteins.
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It was of interest to then compare MG-132-mediated stabilization of HPV-11 E6 to HPV-18 E6, and to determine whether HPV-18 E6 mutants unable to interact with E6AP or to mediate the degradation of p53 and DLG were targeted for proteasome degradation. A series of previously characterized HPV-18 E6 mutants were tagged with the FLAG epitope as detailed above and examined for their susceptibility to proteasome-mediated degradation. Following transfection of p53-null 10(1) cells with FLAG-tagged HPV-11 E6, HPV-18 E6, and various mutant HPV-18 E6 expression vectors, cells were incubated with and without MG-132, cell lysates were prepared 4 h following treatment, and Western blotting was performed using the anti-FLAG antibody as detailed above. As shown in Fig. 1(C), both FLAG-11E6 and FLAG-18E6 protein levels increased dramatically in the presence of the proteasome inhibitor. Likewise, all of the FLAG-18E6 mutants were stabilized by proteasome inhibition. Mutants unable to mediate p53 degradation included M2 (R10S, P11G) and
M (delta 2831), while mutations inhibiting DLG degradation lie in the C-terminal half of 18E6 and included mutants
E (delta 101204),
F (delta 113117) and
G (delta126130). Recent studies have shown
M to have a reduced ability to bind the ubiquitin ligase E6AP (Pim & Banks, 1999
). Notably however, these mutants retained the 8 lysines of HPV-18 E6, with the exception of the
F mutant, which does not contain the 7th lysine of the E6 protein. Taken together, these data argue that both the high-risk and low-risk HPV E6 proteins were susceptible to proteasome degradation. Moreover, binding to E6AP or degradation of p53 and DLG was not necessary for proteasome-mediated degradation of HPV-18 E6.
In order to confirm the previous observation that proteasome inhibition stabilized E6, it was necessary to use an epitope tag other than FLAG in which we could determine whether the epitope tag itself was stabilized by proteasome inhibitor. Hence, constructs expressing E6 fusion proteins linking GFP to the N termini of HPV-11 and -18 E6 were generated. Briefly, E6 cDNA sequences were first amplified by PCR, followed by cloning into the pEGFP-C3 vector (Clontech) at the BglII and EcoRI restriction sites present within the oligonucleotide primers. The generation of in-frame fusions was confirmed by DNA sequencing. To determine whether the constructs expressed fusion proteins of the expected size, p53-null 10(1) cells were transfected with equal amounts of GFP, GFP-11E6, GFP-18E6 or p53-GFP expression plasmids and a lacZ expression vector. Total cell lysates were prepared by sonication and -galactosidase activity was determined to confirm equal transfection efficiency. Equal amounts of protein were subjected to Western blot analysis with an anti-GFP antibody (Clontech). As shown in Fig. 2
(A), native GFP and p53-GFP had the expected molecular masses of 27 kDa and 80 kDa respectively. Both GFP-11E6 and GFP-18E6 fusion proteins had the predicted molecular masses of 4446 kDa. These data confirm the expression and detection of intact GFP-E6 fusion proteins of the predicted size in the transfected cells.
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To address whether the increase in the steady-state levels of E6 upon proteasome inhibition was independent of the epitope tag, p53-null 10(1) cells transiently expressing GFP-E6 fusion proteins were treated with the proteasome inhibitor MG-132 at a concentration of 40 µM for 4 h and transfection efficiencies were determined as before. Cell lysates, prepared by sonication, were subjected to Western blot analysis as detailed above. As shown in Fig. 2(C), a significant increase in the steady-state levels of both GFP-11E6 and GFP-18E6 was observed following MG-132 treatment. In contrast, MG-132 treatment did not increase the steady-state level of GFP alone. These results support the previous data obtained with the FLAG-11E6 and FLAG-18E6 proteins in showing that proteasome inhibition results in the stabilization of E6 and this was independent of the epitope tag used to detect E6. Furthermore, lactacystin, another specific inhibitor of the proteasome, also mediated an increase in both GFP-11E6 and GFP-18E6 in transfected 10(1) cells (data not shown), further supporting the involvement of the proteasome in the degradation of HPV E6.
In order to ensure that the observed stabilization of GFP-11E6 and GFP-18E6 proteins in the presence of the proteasome inhibitor was due to the direct effect of inhibiting degradation by the proteasome, and not to secondary influences on E6 transcription or translation, we investigated the turn-over rate of the GFP-E6 proteins in the presence and absence of proteasome inhibitor. The protein half-life analysis for the GFP-E6 fusion proteins was carried out as described previously for GFP fusion proteins, by determining GFP fluorescence at various times in cells treated with the protein synthesis inhibitor cycloheximide (CHX) (Li et al., 1998). H1299 cells, null for p53, were transfected with GFP, GFP-11E6 or GFP-18E6 expression vectors, and cells were split into 60 mm dishes 9 h post-transfection in order to equalize transfection efficiencies. Twenty-four hours following transfection, cells were treated with either 100 µg CHX (Sigma) ml1, or 100 µg CHX ml1 with 50 µM MG-132 to inhibit proteasome function. Control cells were incubated in the presence of equal volumes of solvent. Cells were collected at 0, 2, 5 and 9 h following treatment, washed and resuspended in PBS with 1 % FBS, and fluorescence intensity was measured on a FACScan (Becton Dickinson). An arbitrary fluorescence of 100 % was attributed to cells collected at the 0 h time-point. As shown in Fig. 3
(A), control GFP-expressing cells in the absence of the CHX showed an increase in fluorescence over time, while CHX-treated cells showed a minor reduction in fluorescence levels. Cells treated with CHX and MG-132 showed fluorescence levels similar to CHX alone. These results are consistent with GFP not being targeted for proteasome-mediated degradation, resulting in the relatively long half-life of GFP. In comparison to native GFP, fusing the HPV-11 and -18 E6 proteins to GFP markedly reduced fluorescence in a time-dependent manner, which was clearly most evident in the presence of CHX where the estimated half-lives were approximately 7 h for both GFP-11E6 and GFP-18E6 proteins (Fig. 3B, C
). However, inhibition of the proteasome with MG-132 in the CHX-treated cells restored the half-lives of GFP-11E6 and GFP-18E6 to levels similar to the control cells, confirming that proteasome inhibition increased the half-lives of the GFP-E6 fusion proteins (Fig. 3B, C
). Thus, proteasome inhibition significantly reduced the degradation rates of both GFP-11E6 and GFP-18E6, to the same extent.
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Since the majority of cellular proteins targeted for degradation by the 26S proteasome are first covalently bound to a chain of ubiquitin proteins, it was important to determine whether HPV-11 and -18 E6 are ubiquitinated. To investigate this possibility, HT1080 cells were co-transfected with an expression construct for HA-tagged ubiquitin (Treier et al., 1994) and expression plasmids for FLAG-11E6 and FLAG-18E6, using the vaccinia virus/T7 system as described previously (Li et al., 1999
). Cells were harvested 24 h post-transfection and protein extracts were subjected to immunoprecipitation with anti-FLAG antibody and protein Aagarose (Pharmacia). Immunoprecipitated and non-immunoprecipitated lysate (W.C. 10 % input) were then subjected to Western blot analysis using either anti-FLAG or anti-HA antibody (HRP-conjugated mouse monoclonal IgG; ICN). As shown in Fig. 4
(lane 3, upper panels A and B of the anti-HA Western blots), high molecular mass, polyubiquitinated E6 proteins were only detected in cells co-transfected with the HA-tagged ubiquitin vector and the FLAG-tagged E6 expressing constructs. This revealed that both FLAG-11E6 and FLAG-18E6 proteins were ubiquitinated.
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The higher expression of FLAG-11E6 as compared to FLAG-18E6, however, did permit improved detection of ubiquitinated FLAG-11E6 species using an anti-FLAG antibody, as observed in lane 3 of the anti-FLAG Western blot in the lower panel of Fig. 4(B) (indicated with asterisks). As expected, detection of HA-tagged polyubiquitinated FLAG-11E6 with anti-HA antibody (Fig. 4B
, lane 3, upper panel,) was again more sensitive than with anti-FLAG antibody (lower panel). The increased sensitivity of the anti-HA Western blot explains the increase in the number of polyubiquitinated FLAG-11E6 species detected, as compared to the anti-FLAG Western blots. Taken together, these data demonstrate that HPV-11 and -18 E6 are both susceptible to ubiquitination, thus supporting the previous experiments and arguing that the E6 proteins from low- and high-risk HPV types can be degraded via the ubiquitin-mediated proteasome degradation pathway.
A recent study reported that high-risk HPV-16 and -18 E6, but not the low-risk HPV-6a and -11 E6 proteins, were stabilized by inhibition of the proteasome degradation pathway (Kehmeier et al., 2002). Our results are in agreement with this study with respect to the high-risk HPVs and we further extended these observations by showing that these E6 proteins become ubiqutinated. However, our observations differ with respect to the low-risk HPV types. In the present study, we showed that the HPV-11 E6 protein was stabilized by proteasome inhibition and this was supported by the observation that HPV-11 E6 was polyubiqutinated. Furthermore, fusing HPV-11 and -18 E6 to GFP reduced the half-life of GFP in the presence of CHX and this was reversed upon the addition of proteasome inhibitor.
Differences in the observations regarding the low-risk HPV-11 E6 may be due to the different experimental design in which Kehmeier et al. (2002) used a C-terminal AU epitope-tagged E6, whereas the present study used N-terminal tagged E6 proteins. Despite retaining the ability to target p53 for degradation, we observed that C-terminal AU-tagged E6 was unable to mediate the degradation of PDZ-containing MAGI-1. Since PDZ domain-containing proteins represent a major target for high-risk HPV E6 proteins, we used N-terminal tagged E6, which is functional for targeting MAGI-1, in this study.
It is, however, important to highlight that the present study supports the conclusion of Kehmeier et al. (2002) with respect to high-risk HPV-18 E6 being a target for proteasome degradation. Certainly, this is an important development in defining the regulation of this oncogenic protein in HPV-infected cells, and therefore merits independent confirmation as provided by this study.
In summary, the present study reveals a potentially important common trait, arguably the first identified biochemical process shared between low- and high-risk HPV E6 proteins, with respect to both being targets for ubiquitination and proteasome-mediated degradation. In addition to acting as a target for degradation, it is possible that ubiqutination may enhance biological function(s) of the E6 proteins. Recent reports have shown roles for ubiquitination in cellular trafficking, kinase activation and transcriptional regulation (reviewed by Pickart, 2001), and may be relevant with respect to HPV E6 modification by ubiquitin. However, since both low-risk and high-risk HPV E6 proteins became polyubiquitinated this argues that ubiquitination does not contribute to the oncogenic properties of high-risk HPV E6. Exploring the mechanisms involved in the ubiquitination and proteasome-mediated degradation of HPV E6 will shed light on the significance of these phenomena in the context of the virus life-cycle and the pathology of infection.
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ACKNOWLEDGEMENTS |
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Received 24 September 2003;
accepted 16 February 2004.