HPV E6 proteins do not inhibit or activate APC/C in Xenopus laevis egg extracts 78

activity of APC/C may not be detectable in U2OS cells. Therefore, we switched the system to Xenopus laevis egg extracts. CSF (cytostatic factor) Xenopus laevis egg extract is arrested in metaphase of meiosis II due to inhibition of APC/C by phosphorylated Xerp1. Addition of calcium ions (Ca²⁺) mimics fertilization and thereby leads to degradation of Xerp1 (Rauh et al., 2005). This results in activation of APC/C and release of the extract into interphase. As these extracts are an “open system” it is possible to analyze the influence of proteins or other components on APC/C activity in a simple manner (Hannak and Heald, 2006).

Before analyzing the effect of bacterially expressed GST-E6 proteins on APC/C activity in this system, we investigated if they also bind to Xenopus laevis APC/C. Pulldown experiments with GST-11E6, GST-16E6 or GST-18E6 meiotic (CSF) extracts showed that all of them were able to bind to APC/C. However, binding to interphase APC/C was only detected for GST-16E6 and GST-18E6 but not for GST-11E6 (figure 31A).

In the next step, we tested if GST-16E6 is active in Xenopus laevis egg extracts and as a consequence is able to induce degradation of in vitro translated p53 added to extracts. For E6-induced degradation of p53 E6AP is required. Thus, we first attempted to determine if E6AP is present in Xenopus laevis egg extracts. In a western blot analysis of Xenopus laevis egg extract E6AP was not detected. However, the anti-E6AP antibody used was prepared against the human E6AP HECT domain and therefore it may not recognize Xenopus laevis E6AP (data not shown). Therefore, for the degradation assay, p53 was in vitro translated in wheat germ extract and not in reticulocyte lysate, as the latter contains E6AP. Subsequent, p53 degradation assays in meiotic and released extracts showed that addition of high risk GST-16E6 protein induced the degradation of p53 within 5 to 10 minutes independent of the cell cycle status. Supplement of baculovirus expressed E6AP to the extracts did not have an additional effect on the degradation of p53 (figure 31B, upper two panels). Moreover, low risk GST-11E6 protein and GST alone did not induce the degradation of p53 as expected (figure 31B, panel 3-6). From these experiments we concluded that Xenopus laevis egg extracts contain active E6AP and that it can form an active E3 ligase complex with GST-16E6 to degrade p53 and that GST-16E6 is functional in Xenopus laevis egg extracts.

Figure 31: GST-E6 proteins are functional in Xenopus laevis egg extracts and bind to Xenopus laevis APC/C

A: Pulldown experiments were performed with bacterially expressed GST, GST-11E6, GST-16E6 and GST-18E6. Coomassie stained gel shows input levels of GST proteins (right panel). GST-fusion proteins were incubated with Xenopus laevis egg extracts (CSF or interphase extract). Binding of APC/C was analyzed by SDS-PAGE with subsequent western blot analysis using anti-Cdc27 antibody (left panel) (Nadine Dreser, under supervision). B: Xenopus laevis egg extract (CSF) was supplemented with in vitro translated p53 and GST-fusion proteins and baculovirus expressed E6AP as indicated. Degradation of p53 was analyzed in CSF extract (-Ca²⁺) and in CSF extracts directly after the addition of Ca²⁺ (+Ca²⁺).

Samples were taken after 0, 5, 10, 20, 40, 60 and 90 minutes and analyzed by SDS-PAGE and subsequent fluorography.

After verifying that GST-16E6 is active in Xenopus laevis egg extracts, we analyzed its effect on APC/C activity. To do so, we supplemented extracts with in vitro translated Securin, a known substrate of APC/C (Zou et al., 1999), to monitor its E3 ligase activity. In meiotic extracts (CSF), which contain inactive APC/C, Securin was stable whereas after addition of Ca²⁺ APC/C

was activated leading to degradation of Securin after 5-10 minutes. Addition of GST-16E6 to extracts did not show any inhibition or activation of APC/C as Securin remained stable in meiotic CSF extract and was also degraded 5-10 minutes after release of extract into interphase (figure 32). In conclusion, E6 proteins do not have an effect on APC/C mediated ubiquitylation of Securin in Xenopus laevis extracts (see 5.5.2).

Figure 32: GST-16E6 does not inhibit or activate APC/C in Xenopus laevis egg extract

Xenopus laevis egg extract (CSF) was supplemented with in vitro translated Securin and bacterially expressed GST or GST-16E6 protein as indicated. Degradation of Securin was analyzed with CSF extract (-Ca²⁺) and with CSF extract directly after the addition of Ca²⁺ (+Ca²⁺). Samples were taken after 0, 5, 10, 20, 40, 60, and 90 minutes and analyzed by SDS-PAGE and subsequent fluorography.

4.5.7 APC/C does not ubiquitylate HPV E6 proteins

E6 proteins are substrates of a so far unknown E3 ligase (Kehmeier et al., 2002; Stewart et al., 2004). As APC/C interacts with the E6 proteins, another potential function of this interaction could be that APC/C is an E3 ligase for E6. To analyze this hypothesis we performed E6 degradation assays in Xenopus laevis egg extracts. We used in vitro translated GFP-E6 fusion proteins for this assay, as E6 proteins themselves only exhibit one or two methionines and therefore cannot be efficiently radiolabeled. For degradation assay, meiotic extracts were supplemented with GFP, GFP-11E6, GFP-16E6 (figure 33) or GFP-18E6 (data not shown) and part of the extracts were released by Ca²⁺ into interphase which results in activation of APC/C. The degradation assay showed that all GFP fusion proteins were stable after the addition of CSF and released extract (figure 33, data not shown). Xerp1 was degraded within 5 minutes upon release of extracts showing that APC/C was activated. Furthermore, 20 minutes after release unphosphorylated newly synthetized Xerp1 was detectable as expected (figure 33). For all GFP-E6 proteins tested a higher migrating band was detected in the degradation assays. However, this band was probably due to the incorporation of free radiolabeled methionine into a newly synthesized protein in the extract. This band was also visible when using GFP in the assay (data not show). From these degradation assays in Xenopus laevis egg extract we conclude that APC/C is not an E3 ligase for E6 ubiquitylation.

Figure 33: In vitro translated GFP-E6 proteins are not degraded by Xenopus laevis egg extracts Xenopus laevis egg extract (CSF) was supplemented with in vitro translated GFP or GFP-E6 proteins as indicated. Degradation of GFP-fusion proteins was analyzed in CSF extract (-Ca²⁺) or directly after the addition of Ca²⁺ to CSF extract (+Ca²⁺). Samples were taken after 0, 5, 10, 20, 40, 60 and 90 minutes and analyzed by SDS-PAGE following fluorography and western blot analysis using anti-Xerp1 and anti-tubulin antibodies. Protein levels of tubulin served as loading control.

4.5.8 Analysis of HPV E1, E2 and E6 protein expression on cell cycle progression