4. RESULTS
4.1 P HYSICAL AND FUNCTIONAL INTERACTION OF THE HECT U BIQUITIN L IGASES E6AP
4.1.1 Identification of Herc2 as a new interaction partner of E6AP
Besides the fact that loss of maternal E6AP expression results in the severe neurological phenotype of Angelman Syndrome patients, the physiological background leading to these symptoms is hardly understood. Identification of new E6AP interacting proteins is one approach to gain better insights into E6AP function.
Characterization of these interactions might help to enlighten pathways which involve E6AP and which are deregulated upon loss of E6AP function in Angelman Syndrome patients.
Herc2 was identified as an E6AP binding protein in a Yeast-2-Hybrid screen (Kogel, 2006). In the beginning, characterization of this newly identified interaction was rather limited by the fact that Herc2 is a giant protein with a molecular mass of
∼530kDa; therefore experiments with a full-length protein could not be performed.
During the course of this study we succeded in cloning a full-length Herc2 cDNA and expressing it in cellulo. However, we have not been able to obtain recombinant full-length Herc2 in amounts that are sufficient for in-vitro studies; therefore in-vitro experiments are still limited to the work with Herc2 fragments. Nonetheless, detailed characterization of the E6AP/ Herc2 interaction in in-vitro experiments and in-cellulo analysis of the full-length proteins revealed that these two proteins are physically and functionally interacting with each other.
4.1.2 Endogenous E6AP interacts with endogenous Herc2
As above mentioned, Herc2 was found to interact with E6AP in a Yeast-2-Hybrid screen using a human fetal brain library. To show that the full-length versions of both proteins are indeed forming a complex in cells, we performed co-precipitation experiments using mouse embryonic fibroblast (MEFs) that were isolated from either wild type mice or Ube3a null littermates (Jiang et al., 1998). Immunoprecipitation of E6AP from Ube3a+/+ MEFs clearly showed co-precipitation of Herc2, indicating that Herc2 binds to E6AP in cells (Figure 6 A). Although the Ube3a-/- MEFs were supposed to act as a negative control, a small amount of E6AP was precipitated from these cells markedly lower than from Ube3a+/+ cells and in addition the amount of Herc2
co-Results 47
precipitated from Ube3a-/- mice is significantly lower than from Ube3a+/+ MEFs. For the preparation of E6AP null MEFs, MEFs extracted from different embryos were pooled. Because Ube3a-/- mice are impaired in reproduction, heterozygous animals were crossed. The derived embryos were genotyped before pooling, however mistyping of embryos might have occurred, resulting in a slight contamination of E6AP null MEFs, which may explain the faint E6AP band upon Immunoprecipitation in Ube3a-/- MEFs. This indicates that the amount of Herc2 co-precipitated with anti-E6AP antibody correlates strongly with the amount of E6AP purified, leading to the suggestion that this co-purification is rather specific and Herc2 interacts with E6AP in cells.
To further confirm the interaction of E6AP and Herc2, an additional approach was performed using size-exclusion chromatography. Cell lysates of H1299 cells were fractionated on a Sephacryl S-300 column and a subsequent Western Blot analysis revealed the migration behavior of Herc2 and E6AP (Figure 6 B, upper panel). Herc2 and E6AP show a clear co-fractionation pattern in high molecular mass fractions, while lower molecular mass fractions show monomeric E6AP or E6AP in complex with other
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Results 48
yet unknown proteins. To obtain evidence that the appearance of E6AP in Herc2-containing fractions is dependent on the presence of Herc2, the experiment was repeated using cells that lack either E6AP (Figure 6 B, middle panel) or Herc2 expression (Figure 6 B, lower panel). Knockdown of E6AP and Herc2 expression was respectively achieved by stable expression of shRNA constructs in H1299 cells. Single cell clones were obtained by limited dilution and clones that showed a knockdown of more than 90% were used for further experiments (H1299-K3 for E6AP knockdown, H1299-NA10-2A for Herc2 knockdown). Cell-lysates of stable Herc2 and E6AP knockdown cells were again applied to size-exclusion chromatography and the fractionation behavior of Herc2 and E6AP was analyzed. Upon knockdown of Herc2 the fractionation behavior of E6AP was dramatically changed, i.e. E6AP could not be detected in high molecular mass fractions anymore as in wild type cells. Knockdown of E6AP had no visible effect on the fractionation pattern of Herc2. The obtained results taken together strongly indicate that E6AP is present in Herc2-containing high molecular mass complexes and that Herc2 and E6AP form a complex in cells.
4.1.3 All three E6AP isoforms are able to bind to endogenous Herc2
E6AP is expressed in three different isoforms that occur through alternative splicing.
The three splicing variants only differ in their very N-termini, leading to elongated versions of E6AP in isoform 2 and isoform 3. The fact that all isoforms of E6AP are expressed in cells suggests that they might fulfill different functions and/or are localized to different compartments and/or bind different proteins or substrates. To test if the E6AP isoforms differ in their ability to bind to Herc2, HA-tagged variants of all three isoforms were expressed in H1299 K3 cells and co-immunoprecipitation of E6AP variants and endogenous Herc2 was performed using an anti-HA antibody (Figure 7). All three isoforms of E6AP were able to co-precipitate Herc2 from cell lysates, indicating that they show no difference in their binding behavior with respect to Herc2.
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4.1.4 The Rcc1b domain of Herc2 mediates binding an N-terminal region of E6AP
While strongly indicating that Herc2 and E6AP are interacting and present in a common complex in cells, our results do not distinguish between the possibilities that this complex is composed of Herc2 and E6AP alone or that the complex contains additional proteins. Thus, we cannot conclude that E6AP and Herc2 interact directly with each other and do not need additional proteins for binding. In order to exclude this possibility, several binding studies of Herc2 and E6AP were performed in-vitro.
Additionally, the binding sites on both proteins were identified.
To map the sites on E6AP necessary for binding to Herc2, different E6AP variants, including the N terminus and the HECT domain of E6AP and an E6AP deletion variant that lacks amino acids 150-200 were used (Figure 8 A). The respective HA-tagged expression constructs were transfected into H1299 K3 cells and the E6AP variants precipitated using an anti-HA antibody. Western Blot analysis for Herc2 revealed that
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tagged E6AP constructs used for determination of the Herc2 binding site in co-‐precipitation experiments, lower panel: a 50 amino acid (aa) N-‐terminal region of E6AP is involved in binding to E6AP. HA-‐tagged E6AP
Results 50
the binding site to Herc2 is localized to the N terminus of E6AP, as no interaction was observed for the HECT domain, while the first 500 N-terminal amino acids of E6AP were sufficient to co-precipitate Herc2. Furthermore, the binding site could be further restricted to 50 amino acids in the N-terminal region of E6AP, as a full-length E6AP variant lacking amino acids 150-200 (E6AP Δ150-200) lost its ability to bind to endogenous Herc2. These results suggest that binding of E6AP to Herc2 occurs via the N terminus of E6AP and more precisely via amino acids 150-200.
In the above mentioned Yeast-2-Hybrid screen, which first identified Herc2 as a potential new interacting partner of E6AP, several fragments of Herc2 were isolated. A region common to all these clones spanned the Rcc1b domain of Herc2, indicating that this domain represents the binding site for E6AP. To test this hypothesis, in vitro pulldown experiments were performed using a bacterially expressed GST-tagged version of the Rcc1b domain of Herc2 and in-vitro-translated, S35-radiolabeled E6AP variants (E6AP wild type or E6AP Δ150-200). Co-precipitation of in-vitro translated wild type E6AP with GST-Rcc1b, but not with the GST negative control, was detected, indicating that the Rcc1b domain indeed reflects the binding site on Herc2 for E6AP (Figure 8 B). Furthermore, no binding to the Rcc1b domain was detectable for the deletion mutant E6AP Δ150-200, indicating that the Rcc1b domain binds to the region of E6AP that we already identified as the binding site for Herc2. However, E6AP Δ150-200 was not impaired in its binding to the HPV16E6 oncoprotein compared to wild type E6AP, suggesting that the loss of these 50 amino acid residues does not affect other E6AP functions, e.g., the ability to mediate HPV16E6 induced p53 degradation in cells (data not shown). This experiment clearly identified the Rcc1b domain of Herc2 as the region responsible for binding to a 50 amino acids spanning N-terminal region ofE6AP.
To show that these 50 amino acids are sufficient to bind to the Rcc1b domain of Herc2, a GST-tagged construct was cloned consisting only of the above-mentioned region (GST-E6AP 150-200). Pulldown analysis with bacterially expressed GST-E6AP 150-200 and in-vitro translated, S35 radio labeled Rcc1b was performed (Figure 8 C) showing that the isolated 50 amino acids of E6AP are indeed able to bind to the isolated Rcc1b domain. It should additionally be mentioned that Herc2 contains three Rcc1-like domains in total (see Figure 4). Binding of E6AP to the Rcc1a and Rcc1c domain of Herc2 has been tested previously and it could be shown that only the Rcc1b domain of Herc2 and none of the other Rcc1-like domains is able to mediate binding to E6AP (Kogel, 2006) .Taken together, our results suggest that Herc2 and E6AP interact via the Rcc1b domain of Herc2 and 50 amino acids in the N-terminus of E6AP.
Additionally this interaction is very likely a direct interaction, although it cannot be excluded that proteins in the reticulocyte lysate are involved in the interaction.
However, pulldown experiments using bacterially expressed GST-Rcc1b and a purified version of E6AP expressed in the baculo-system, showed binding of recombinant E6AP
Results 51
to Rcc1b (data not shown), minimizing the possibility that other proteins mediate the interaction of E6AP and Herc2. Therefore, we propose that Herc2 and E6AP are direct interaction partners.
4.1.5 Herc2 is not a substrate of E6AP
Being E3 ubiquitin ligases, an obvious possibibility is that Herc2 and E6AP might be substrates of one another. In vitro experiments with Herc2 fragments revealed that a Herc2 fragment (“Herc2 Rcc1b-End”) could indeed be ubiquitinated by recombinant E6AP (data not shown). However experiments in cells did not provide evidence that one of the ligases might be the substrate of the other. Neither overexpression nor knockdown of either ligase showed an effect on protein levels of the other (Figure 9).
This indicates that Herc2 and E6AP do not affect ech other’s half-life by ubiquitination and proteasomal degradation. However ubiquitination mechanisms other than destabilization by proteasomal degradation cannot be excluded.
4.1.6 The isolated Rcc1b domain stimulates E6AP ligase activity in-vitro in a trans-ubiquitination reaction
As Herc2 and E6AP do not appear to serve as substrates for one another, other possibilities were taken into account. It is well established that E6AP auto-ubiquitinates itself (Nuber et al., 1998) and that E6AP auto-ubiquitination can be influenced by interaction with the E6 oncoprotein of high-risk human papillomaviruses.
Binding of HPV16E6 stimulates E6AP auto-ubiquitination probably by switching E6AP auto-ubiquitination from a trans-mechanism to a cis-mechanism (Kao et al., 2000).
Due to this observation, we tested if the isolated Rcc1b domain of Herc2 might also influence E6AP auto-ubiquitination. Therefore, in-vitro auto-ubiquitination kinetics of E6AP were performed. In these assays, in-vitro translated, S35 radiolabeled E6AP was used as a substrate for recombinant baculo E6AP (Figure 10 A). Upon addition of
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Figure 9 Knockdown of E6AP or Herc2 does not affect the protein level of the interaction partner.
Comparison of Herc2 and E6AP protein levels in stable knockdown cellsof either protein (H1299 K3 for E6AP knockdown and H1299-‐NA10 for Herc2 knockdown). An α-‐Tubulin Blot serves as loading control (10% of the sample loaded in the upper panel).
Results 52
bacterially expressed GST-Rcc1b, the reaction was about two-fold faster (Figure 10 A, compare 10 and 20min reactions). However, the effect was rather mild, probably because E6AP auto-ubiquitination in-vitro is a processive reaction. To obtain more convincing evidence that Rcc1b stimulates E6AP auto-ubiquitination, additional conditions were tested, e.g. instead of wild type ubiquitin a hydrophobic patch mutant of ubiquitin termed ubiquitin LIA (Leu8 and Ile44 of ubiquitin are replaced by Ala) was used. This mutant is impaired in its ability to interact with ubiquitin-binding proteins and is less efficiently activated by the E1 enzyme(Beal et al., 1996; Dikic et al., 2009).
An E6AP auto-ubiquitination assay with ubiquitin LIA showed that ubiquitination of E6AP is strongly impaired, indicating that E6AP is not able to use ubiquitin LIA as efficiently as wild type ubiquitin (Figure 10 B left panel). However, upon addition of Rcc1b, ubiquitination of E6AP was significantly stimulated, leading to the conclusion that the isolated Rcc1b domain of Herc2 can stimulate E6AP auto-ubiquitination,
!"#$$%&" !"#$$%&" for baculo virus expressed E6AP. The auto-‐ubiquitination assay was performed using a hydrophobic patch mutant of ubiquitin (LIA) (L8A, I44A) in the absence and presence of bacterially expressed GST-‐Rcc1b. Time course analysis of the reaction was performed. Asterisk (∗) indicates ubiquitinated E6AP species.
Results 53
thereby rescueing the impaired reaction in the presence of ubiquitin LIA. This provides a first hint that the interaction of Herc2 and E6AP has functional consequences.
Although we showed that the Rcc1b domain can bind to E6AP, it remains unclear if binding of Rcc1b to E6AP is indeed necessary for the stimulating effect. Therefore we tested if an E6AP mutant that lacks the ability to bind to the Rcc1b domain can still be stimulated by addition of the Rcc1b domain. For this purpose, we used in-vitro translated, radiolabeled E6APΔN199 or E6APΔ150-200 as a substrate for recombinant wild type E6AP. Ubiquitination of none of the binding deficient E6AP mutants was stimulated by addition of Rcc1b (Figure 10 D, E), although E6APΔN199 still represents a target for trans-auto-ubiquitination as can be seen in the experiment with wild type ubiquitin (Figure10 C). Furthermore, the stimulating property of Rcc1b is restricted to this domain as the Rcc1c domain of Herc2, which is supposed to have a very similar structure but is not able to bind to E6AP (data not shown and (Kogel, 2006)), does not stimulate E6AP auto-ubiquitination (Figure 11). In conclusion, specific binding of the Rcc1b domain to E6AP is necessary to achieve the stimulation of auto-ubiquitination.
4.1.7 Binding of Rcc1b on both substrate and ligase site of E6AP is required for stimulation
As mentioned, E6AP auto-ubiquitination is a trans-reaction. This means that one E6AP molecule serves as substrate for a second E6AP molecule. The obtained results so far, demonstrated that binding of Rcc1b to substrate-E6AP is required to achieve stimulation of E6AP autoubiquitination. However, it is not clear if binding of Rcc1b to the ligase-E6AP is required for the stimulation effect. Therefore experiments with E6AP mutants that lack the ability to bind to the Rcc1b domain should not only be restricted
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Figure 11 The Rcc1b domain of HERC2 exclusively stimulates E6AP ligase activity. In-‐vitro translated, radiolabeled E6AP (wild type E6AP) was used in an in-‐vitro auto-‐ubiquitination assay as a substrate for baculo virus expressed E6AP in the absence and presence of bacterially expressed GST-‐Rcc1c . The assay was performed using an ubiquitin LIA mutant (L8A, I44A). Time-‐course analysis of the reaction was performed. Asterisk (∗) indicates ubiquitinated E6AP species.
Results 54
to the substrate site (see 4.1.6). In order to find out if Rcc1b binding has to occur on the ligase site of the reaction pair (Figure 12 A), experiments were performed with an in-vitro-translated, radio labeled catalytically inactive E6AP variant (E6AP C820A) as a substrate for recombinant E6APΔN199. While the recombinant E6APΔN199 variant was still able to ubiquitinate catalytic inactive E6AP in the presence of wild type ubiquitin (Figure 12, A), addition of Rcc1b did neither stimulate the reaction (Figure 12 B) nor rescue the impaired ubiquitination in the presence of ubiquitin LIA (Figure 12 C). Thus we conclude that binding of E6AP to both the ligase and the substrate site of the trans-ubiquitination reaction is necessary for its stimulating effect.
4.1.8 Rcc1b stimulates E6AP auto-ubiquitination at the E3 ligase level of the ubiquitination cascade
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level of the ubiquitination cascade. However, to exclude that Rcc1b might affect other enzymes of the ubiquitination cascade as well, thioester kinetics were performed and the effect of Rcc1b on thioester-formation was examined at all levels. For this, either wild type ubiquitin or ubiquitin LIA were in-vitro translated and used in an E1 thioester assay. A time-course experiment was performed, after 15min of incubation E2 was added to the reaction and again a time-course was performed over a period of 15min.
In a third step E6AP was added to the reaction and again a 15 min time-course was carried out. Thioester formation on E1 and E2 levels were comparable in reactions with or without addition of Rcc1b (Figure 13 A).
Addition of Rcc1b exclusively affected the rate of ubiquitin conjugation at the E6AP level, which is additionally apparent by the consumption of free ubiquitin. As expected from previous reports the hydrophobic patch mutation of ubiquitin LIA impaired E1 thioester formation. However, this impairment was not affected by addition of Rcc1b to the reaction, but instead conjugation of ubiquitin on E6AP was enhanced again
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Figure 13 Effect of Rcc1b on different levels of the ubiquitination cascade. Time-‐course analysis of thioester formation using S35 labeled ubiquitin wild type (A) or LIA (B) was performed. First, thioester formation of the E1 enzyme level was examined for the indicated time points. After 15min reaction time, E2 was added and thioester formation on the E2 enzyme was visualized. After additional 15min E6AP (E3) was added to the reaction and kinetics performed (30min). Thioester-‐time courses were analysed in the absence and presence of GST-‐Rcc1b. Rcc1b does not affect thioester formation on E1 and E2 levels but increases ubiquitin conjugation on E6AP.
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(Figure 13 B), implying that the effect of Rcc1b on E6AP auto-ubiquitination is restricted to the E3 level of the ubiquitination cascade and that Rcc1b increases the reaction rate of ubiquitin conjugation by stimulating E6AP activity.
4.1.9 Rcc1b stimulates ubiquitination of E6AP substrates.
The physiological relevance of E6AP auto-ubiquitination is not understood to date.
Stimulation of E6AP auto-ubiquitination by the isolated Rcc1b domain of Herc2 might of course have an impact on E6AP half-life and, thus, on E6AP function but the physiological significance is highly ambiguous. Hence we investigated if Rcc1b would also influence E6AP-dependent ubiquitination of substrates. As substrate, we chose the reported E6-independent E6AP substrate Ring1b (Zaaroor-Regev et al., 2010).
Ring1b is a RING E3 ubiquitin ligase and ubiquitinates itself very efficiently, thus to differentiate E6AP-dependent Ring1b ubiquitination from Ring1b auto-ubiquitination,
RING-inactive mutants of Ring1b had to be used in this study (Ring1b I53A). A Ring1b ubiquitination assay was performed using in-vitro translated, S35 labeled Ring1b I53A as substrate for recombinant E6AP. Because Ring1b is very efficiently ubiquitinated by E6AP in vitro, the hydrophobic patch ubiquitin mutant LIA, was used again in order to obtain a better time-dependent resolution of the ubiquitination reaction. Using
RING-inactive mutants of Ring1b had to be used in this study (Ring1b I53A). A Ring1b ubiquitination assay was performed using in-vitro translated, S35 labeled Ring1b I53A as substrate for recombinant E6AP. Because Ring1b is very efficiently ubiquitinated by E6AP in vitro, the hydrophobic patch ubiquitin mutant LIA, was used again in order to obtain a better time-dependent resolution of the ubiquitination reaction. Using