Ubiquitin-like protein 4A 1 2 Ubiquitin
3 26S Proteasome Non-ATPase regulatory subunit 4
1 Proteasome subunit alpha type 2
However, th e m ost interesting feature of th is m ass spectrom etry analysis w as th e identification of another ubiquitin ligase, EDD, in addition to E6AP. M arked in blue and shown in Table 3, EDD was pulled down in both complexes. EDD, a 300kD nuclear protein, is a m am m alian ortholog of the Drosophila melanogaster “hyperplastic
discs” tum or suppressor gene (hyd) (Callaghan et a l., 1998) and it has critical roles in control of cell proliferation and tum origenesis (Mansfield
et al., 1994). Studies have shown th a t EDD (-/-) mice die a t m id
gestation due to failed yolk sac angiogenesis and defective placental development, leading to general failure of embryonic cell proliferation and widespread apoptosis (Saunders et a l., 2004). EDD has been found to be overexpressed in b reast and ovarian cancers (Clancy et al., 2003); while truncating m utations of the protein are commonly found in gastric cancers w ith m icro-satellite instability (Mori et al., 2002). It is also involved in DNA dam age signaling, and, intriguingly, functions as an E3 HECT ubiquitin ligase (Callaghan et al., 1998; Munoz et al., 2007).
HPV-18 E6 B in d s to EDD in vitro
Since EDD is a ubiquitin ligase, we were particularly interested in investigating fu rth er its potential interactions w ith E6 and how this m ight affect and/or contribute to E6 functions. We first analyzed the potential of HPV-18 E6 to interact with EDD in vitro. EDD was in vitro translated, 35S radiolabelled and then incubated w ith GST-18 E6, GST- Dlg, GST-18 E6* or GST alone for comparison. The resu lt of the assay in Figure 14A shows th a t HPV-18 E6 binds to EDD more strongly th a n either Dig or the GST control. Interestingly, th e binding between E6* and EDD is also very weak, suggesting th a t m ost of the interaction m otif lies w ithin th e carboxy term inal h a lf of HPV-18 E6.
We were also in terested in determ ining if EDD binding is specific for HPV-18 E6. Therefore we used GST fused HPV-16 E6 and HPV-11 E6 as comparisons for th e ability to bind EDD. EDD was in vitro tran slated , 35S radiolabelled and then incubated w ith GST-18 E6, GST-
inputs
B
EDD
GST
inputs
Figure 14. HPV-18 E6 protein binds to EDD in vitro. A. In-vitro-
translated EDD was incubated with GST, GST-DIg, GST-18 E6, and GST-18 E6*. Bound proteins were assessed by autoradiography and the input GST fusion proteins were visualized with Coomassie staining (lower panel). Input of EDD (10%) is also shown. Phospholmager quantitation of EDD bound to G S T -18 E6 is 37%. B. /n-wfro-translated EDD was incubated with GST, GST-18E6, GST-16E6, and GST-11E6. Bound proteins were assessed by autoradiography and the input GST fusion proteins were visualized with Coomassie staining (lower panel).
Input of EDD (10%) is also shown.
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16 E6 GST-11 E6 or GST alone for control. The resu lt in Figure 14B shows th a t HPV-18 E6 binds to EDD more strongly th a n both HPV-16 E6 and HPV-11 E6. Interestingly, both HPV-16 E6 and HPV-11 E6 bind EDD a t a sim ilar level, dem onstrating th a t th e ability of E6 to bind EDD is not restricted to th e high risk types.
HPV-18 E6 B in d s to EDD in vivo
We then proceeded to confirm th a t th e interaction between E6 and EDD occurrs in vivo. To do so, we performed pull-down assays in cells where EDD was overexpressed alone, together w ith HA-tagged 18 E6 or in combination w ith HA-tagged 18 E6 and HA-tagged MAGI-2. The results in Figure 15 confirm th e interaction between EDD and 18 E6 in
vivo. In both experim ents (panels A and B) EDD w as co-
im m unoprecipitated when overexpressed w ith E6. When EDD was overexpressed by itself or in combination w ith MAGI-2, binding to th e HA-resin was very w eak or absent. Interestingly, EDD was able to bind to E6 when those two proteins were expressed together, and also when both were expressed in combination w ith MAGI-2, suggesting th a t these three proteins m ight in teract in a triple complex. The fact th a t E6 could interact w ith MAGI-2 and EDD in a complex also suggests th a t th e binding between EDD and E6 does not adversely effect Ed’s ability to bind MAGI-2, fu rth er suggesting th a t th e EDD-binding region on E6 is probably not located in the C-term inal PDZ binding domain of E6.
EDD In h ib its H P V 18 E6 D eg ra d a tio n o f D ig in vitro
As already mentioned, HPV E6 is not able to degrade targ ets by itself, it needs to form a complex w ith a cellular ubiqutin ligase, such as E6AP, and w ith such targ et proteins as p53, Bak, or various PDZ domain- containing proteins for proteasom al degradation to occur (H anda et al., 2007; Kuballa et a l., 2007; Scheffner et a l., 1993; Thomas and Banks, 1998; Thomas and Banks, 1999). Since EDD is a ubiquitin ligase we were obviously interested in investigating the potential role of EDD in E6’s degradatory activities. To do this E6, Dig and EDD were tran sla te d in vitro and co-incubated a t 30°C for 1 and 2 hrs. The level of Dig protein rem aining was th en ascertained by SDS-PAGE and autoradiography. The results in Figure 16 show the levels of in vitro tran sla te d Dig a t tim e-points 0, 60 and 120 m inutes. As expected, Dig rem ained relatively stable over th is tim e period. After th e addition of E6 th e levels of Dig were dram atically decreased a t th e 60 m inute time- point, while a t 120 m inutes tim e-point Dig was alm ost completely degraded. Interestingly, in th e presence of EDD, Dig protein degradation was greatly reduced a t both th e 60 and 120 m inute time- points. This observation is opposite to the outcome th a t one m ight expect if EDD was acting as an E6-dependent ubiquitin ligase to degrade its targets. The resu lt from Figure 16 suggests th a t EDD somehow interferes w ith E6 degradatory activity.
ED D In terferes w ith H PV 18 E6 D egrad ation o f D ig, MAGI-2 and p53 in vivo
To investigate these effects of EDD further, we analyzed w hether EDD could rescue Dig protein levels in the presence of E6 in vivo in hum an
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O Ni Q CQ LU O EDD full lengthIP: HA18E6, probed with IP: HA18E6 and HAMAGI-2, anti EDD antibody 293 probed with anti EDD
cells antibody 293 cells
Figure 15. HPV-18 E6 protein binds to EDD1 in
vivo. 293 cells were transfected with 10 pg of HA
tagged HPV18 E6, 5 pg EDD1, or 5 pg HA-tagged MAGI-2, alone or in combination. After 24 h cells were incubated for 3h with proteasome inhibitor (CBZ) before harvesting. Soluble fractions were incubated with HA-antibody conjugated agarose beads for 2h at 4°C. Immunoprecipitated EDD1 protein was assessed by Western blot analysis using anti-EDD1 antibody (A and B). EDD1 protein was only detected in lanes which included co-transfected HA-18 E6 or combination of HA-18 E6 and HA-MAGI-2.
Inputs
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Figure 16. EDD down-regulates HPV-18 E6 degradation of
Dig in vitro. EDD, HPV-18 E6 and Dig were translated, and
co-incubated at 30°C for the times indicated. Residual Dig was then detected by immunoprecipitation, SDS-PAGE and autoradiography. Inputs are also shown (lower panel). WPL refers to water prime lysate.
293 cells which were transfected w ith plasm ids expressing Dig alone or in combinations w ith HPV E6 and EDD (Figure 17A). In addition, we introduced MAGI-2 into th e same experim ent (Figure 17B), to determ ine w hether th e observations from Figure 16 were Dlg-specific, or w hether EDD could have th e sam e effects on other E6 PDZ domain- containing targets. Finally, we also included p53 (Figure 17C) in th is experim ent to investigate if EDD could have th e sam e effect on rescuing p53 from E6-mediated degradation. 24 h post-transfection, cells were harvested in SDS lysis buffer and Dig, MAGI-2 and p53 levels were assessed by W estern blot analysis. The resu lts in Figure 17 show degradation of Dig, MAGI-2 and p53 in th e presence of E6 as expected. Interestingly, and sim ilar to w hat was seen in th e Figure 16, after EDD was co-transfected w ith both Dig and MAGI-2 protein levels were restored back to control levels. Interestingly, p53 protein levels were also rescued in th e sam e way. Furtherm ore, w hen EDD and MAGI-2, Dig or p53 were combined in the absence of E6 there were no m ajor changes in the protein levels of these targets. These results suggest th a t th e effects of EDD in rescuing MAGI-2, Dig and p53 from Ed- induced degradation are m ost likely associated w ith a direct effect on E6 function, rath e r th a n an effect on th e targ e t proteins per se, or on indirect effects involving general components of th e proteasom e pathw ay.
E n d o g en o u s EDD D ir ec tly R e g u la te s H PV E 6 A c tiv ity in vivo
To fu rth er elucidate these observations and to investigate th e role of EDD in the context of HPV E6 function in cervical tumor-derived cell lines, we performed siRNA EDD silencing experim ents in HPV-18- positive HeLa and HPV-16-positive CaSKi cells. Both H eLa and CaSKi
cells were transfected w ith siRNAs directed against eith er EDD, HPV- 18 or HPV-16 E6, respectively, or Luciferase for comparison, and the levels of both proteins were th en analysed by w estern blotting after 72 hrs. The results obtained are shown in Figure 18. As can be seen, the reduction of E6 levels by siRNA results in a m arked upregulation in the level of p53 expression (Figure 18 A and B), and this is consistent w ith previous observations showing th a t E6 uses E6AP in targ etin g p53 for proteasom e-m ediated degradation (Kao et a l., 2000). Interestingly however, depletion of E6AP expression results in even greater upregulation in p53 protein levels, to levels th a t are even higher th an those obtained using the E6 siRNA. These results reflect our observations in Figure 8, where we show th a t E6 levels are lower in cells treated w ith siRNA E6AP th a n in cells treated w ith siRNA E6 only, highlighting again th a t E6 protein stability is highly dependent on E6AP levels.
However, even more striking results were observed from cells treated w ith siRNA EDD. In both cell lines (Figure 18 A and B), cells treated w ith siRNA EDD show a dram atic reduction in p53 levels in comparison w ith th e control p53 levels, suggesting th a t the presence of EDD is inhibiting E6 degradatory function w ith respect to p53. Interestingly, there were still some residual levels of EDD after siRNA treatm ent, suggesting th a t complete silencing of EDD protein levels would probably resu lt in even g reater p53 degradation. These results show th a t EDD is a crucial component of the E6 degradatory m achinery and th a t it directly or indirectly has a m ajor effect on E6’s ability to direct the degradation of its m ain target, p53.
E ffects o f siR N A EDD D e p le tio n o n H PV E6 and E6AP in H eL a C ells
From the results m entioned above it is clear th a t EDD does not have any m ajor direct effects on E6 targ ets such as the PDZ domain- containing proteins, b u t th a t it ra th e r h a s a direct effect on E6 or on some component linking E6 to th e proteasom e pathway. Therefore, we were interested in investigating th e potential effects of EDD upon th e levels of both E6 and E6AP. Assays were performed as above in HeLa cells, using siRNA Luciferase as a negative control and siRNA against E6AP, siRNA against EDD, and combined siRNAs against E6AP and EDD. 72 hours post transfection cells were harvested and to tal cellular lysates were subjected to W estern blot analysis using the anti-18 E6 antibody, an anti-E6AP antibody, and an anti-Tubulin antibody as a loading control. The results obtained are shown in Figure 19. As shown above, w hen E6AP was silenced in HPV-positive cells the levels of E6 were dram atically reduced, and th a t observation was confirmed in th is experiment. However, more in terestin g results were observed in cells treated w ith siRNA EDD, where upregulation of both E6 and E6AP was observed after EDD was silenced. To exclude th e possibility th a t EDD is th e ubiquitin ligase which tu rn s over E6 w hen E6AP is not present, we also included cells treated w ith siRNA EDD and siRNA E6AP. As can be seen from lane four, there was no significant E6 protein rescue w hen both ubiquitin ligases were silenced. These results dem onstrate th a t EDD h as a potential direct effect on E6AP stability, since EDD silencing leads to E6AP accumulation, which consequently resu lts in E6 upregulation. The results also suggest th a t E6 protein turnover is not EDD-dependent in the absence of E6AP, which suggests several possibilities th a t will be discussed in more detail in the Discussion.
H PV E6, EDD and E6AP ca n In tera ct in vitro
In order to investigate if E6, EDD and E6AP can potentially interact, EDD and E6AP were in vitro tran slated in W heat Germ Extract. The reasoning for using W heat Germ system was because it does not contain E6AP which is present in th e Rabbit Reticulocyte system. The in vitro tran slated proteins were incubated individually or in combination with GST-18 E6, and GST alone for th e negative control. The result of the binding assay in Figure 20A shows th a t both proteins bind to E6. The introduction of either E6AP or EDD to th e m ixture does not increase the binding affinity of the proteins towards E6. Additionally, this result suggests th a t EDD could form a complex w ith E6 independently of E6AP.
F urtherm ore we w anted to investigate if an E6 m u ta n t (I130T), which is significantly reduced in its ability to bind E6AP, would also have any effects on EDD binding. EDD and E6AP were in vitro translated, 35S radiolabelled and th en incubated w ith GST-18 E6, GST-18 E6 (I130T), and GST alone for control. The resu lt of the assay in Figure 20B shows th a t HPV-18 E6 binds to EDD sim ilarly to the HPV-18 E6 (I130T) m u tan t, while there is a difference between wild type HPV-18 E6 binding to E6AP in comparison to the I130T m utant. This again suggests a possibility th a t E6 does not require E6AP for EDD binding and th a t the binding sites for EDD and E6AP are distinct on E6.
To clarify th is even further, and to additionally confirm th a t EDD can bind to E6 w ithout E6AP, we decided to perform pull-down assays in E6AP-null cells. NIH3T3 mouse fibroblasts were used as the positive control since they are positive for E6AP. Prior to harvesting, cells were trea te d w ith the proteasome inhibitor CBZ for 2.5 hrs and then
:Z-