UbcX dissociates XErp1 from the APC/C Results
Figure 2.11. Dissociation of XErp1 from the APC/C upon phosphorylation is independent of ubiquitylation. CSF-‐extracts were treated as indicated and Cdc27 was immunoprecipitated.
The untreated and CIP treated input samples and immunoprecipitates were analyzed by WB for Cdc27 and XErp1. OA, ocadaic acid; CIP, calf intestinal alkaline phosphatase; ci, catalytic inactive;
2.13. Cdc20 degradation is not required for CSF arrest maintenance
In somatic cells arrested by the SAC, Cdc20 is ubiquitylated by the APC/C.
However the model in which this ubiquitylation of Cdc20 liberates it from the mitotic checkpoint complex has been challenged. Instead it has been proposed, that the consequence of Cdc20 ubiquitylation is proteasomal degradation. Thus, while the “activation-‐model” proposes that UbcH10 dependent ubiquitylation of Cdc20 results in its release from the inhibitory SAC-‐complex, i.e. APC/CCdc20 activation (Reddy et al, 2007), the “inactivation-‐
model“ proposes that it induces Cdc20 degradation, i.e. APC/CCdc20 inactivation (Nilsson et al, 2008). If a similar inactivation model were valid during CSF arrest, we would expect that in the presence of XErp1, Cdc20 bound to the APC/C is turned into a substrate inducing its ubiquitylation and degradation.
Therefore we analyzed if Cdc20 is short-‐lived in CSF-‐extract. WB-‐analyses of CSF-‐extract treated with cycloheximide (CHX) to block protein synthesis demonstrated that the levels of endogenous Cdc20 did not significantly decrease during a time period of 120 minutes (Figure 2.12. a, panel 1). To corroborate this finding, we analyzed the levels of Cdc20 when protein destruction was blocked. As shown in figure 2.12. b, panel 1, Cdc20 levels did not detectably increase over a time frame of 120 minutes in extract treated with the potent proteasome inhibitor MG262 compared to DMSO. Calcium
UbcX dissociates XErp1 from the APC/C Results
37 failed to induce degradation of cyclin B in MG262-‐treated extract (Figure 2.12.
b, panel 3) confirming efficient inhibition of the proteasome.
Figure 2.12. Cdc20 is stable in CSF arrest. (a) CSF extract treated with CHX was supplemented with buffer control, UbcXwt or calcium. At the indicated time points samples were taken, treated with phosphatase and analyzed by WB for Cdc20, cyclin B2, XErp1 and α-‐tubulin. (b) CSF extract treated with MG262 was supplemented with buffer control, UbcXwt, or calcium and samples were taken. After phosphatase treatment Cdc20, cyclin B2, XErp1 and α-‐tubulin were analyzed by WB. CHX, cycloheximide; CIP, calf intestinal alkaline phosphatase; wt, wild type;
Thus, Xenopus Cdc20 is a stable protein and has a low turnover in egg extract.
Moreover, when re-‐synthesis is inhibited, the amount of Cdc20 still present in the extract is sufficient to effectively target APC/C substrates for destruction when UbcX or calcium is added to the extract (Figure 2.12. a, panel 2 and 3).
These results together with our observation that exogenous UbcX induces premature CSF-‐release rather than an enhancement of the arrest suggests that a stable CSF-‐state is not aided by the destabilization of Cdc20.
UbcX dissociates XErp1 from the APC/C Discussion
3. DISCUSSION
How the APC/C is regulated in oocytes, as well as in somatic cells, is an important and still elusive question in cell-‐cycle research. The studies presented here identify a novel layer of APC/C activity regulation via XErp1 in Xenopus egg extracts. The CSF component XErp1 arrests oocytes at metaphase of meiosis II by directly inhibiting the APC/C (Ohe et al., 2010; Schmidt et al., 2005). Fertilization of oocytes triggers the SCFβTRCP dependent destruction of XErp1, thereby causing APC/C activation and the irreversible exit from meiosis (Hansen et al., 2006; Liu and Maller, 2005; Rauh et al., 2005). During CSF arrest, transient Cdk1-‐mediated inactivation of XErp1 is important for the compensation of continuous cyclin B synthesis (Wu and Kornbluth, 2008; Wu et al., 2007b). Here, we provide evidence that an autocatalytic mechanism regulating APC/C activity is involved in CSF arrest in Xenopus. First, elevated UbcX activity causes APC/C activation in CSF arrested egg extract. Second, UbcX induces the non-‐proteolytic ubiquitylation of XErp1 and the dissociation of the APC/C–XErp1 complex, suggesting that increased ubiquitylation of XErp1 decreases its affinity for the APC/C. Third, the UbcX induced ubiquitylation of XErp1 is mediated by the APC/C, therefore the APC/C is able to activate itself in an autocatalytic manner in the presence of elevated UbcX activity. Similarly, UbcX can trigger activation of the APC/C in Xenopus egg extracts with an activated spindle checkpoint. This suggests that in vertebrate cells, APC/C dependent ubiquitylation is important to overcome SAC arrest, thus favoring the self-‐activation model, where the APC/C can liberate itself from SAC inhibition by ubiquitylating Cdc20, over the inactivation model of the APC/C, in which SAC arrest is mediated by the APC/C dependent destruction of Cdc20.
In addition, our findings that Cdc20 is stable during CSF arrest suggest that CSF arrest is not mediated via the destabilization of Cdc20.
UbcX dissociates XErp1 from the APC/C Discussion
39 In the following section, the implications of these novel findings on our view of the spindle checkpoint arrest as well as on CSF arrest will be discussed, models for the physiological function of APC/C dependent XErp1 ubiquitylation will be suggested and open questions as well as possible future directions will be proposed.