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.
3.1. Regulation of spindle checkpoint signaling by UbcH10/UbcX
3.1.1. The spindle assembly checkpoint can be inactivated by UbcX in Xenopus egg extract
In mitosis, the presence of improperly attached kinetochores leads to the inactivation of Cdc20 by the checkpoint proteins Mad2, BubR1 and Bub3 by the formation of the inhibitory MCC–Cdc20 complex, therefore the APC/C is inactive and anaphase is delayed (Musacchio and Salmon, 2007). The crystal structure of Mad2 bound to a Cdc20 mimicking peptide argues for a tight interaction between the two proteins (Sironi et al., 2002) and it implies that the Mad2-‐Cdc20 complex has to be actively dissociated. As described previously, contradictory mechanism have been proposed in human cells to inactivate the mitotic checkpoint complex (MCC) and to provide free Cdc20 for APC/C activation, including APC/C dependent proteolysis of Cdc20 (“Cdc20 inactivation model”,(Nilsson et al., 2008) or the polyubiquitylation, but not degradation, of Cdc20 to dissociate the MCC-‐Cdc20-‐APC/C complex (“Cdc20-‐
activation model”;(Reddy et al., 2007).
In support of the Cdc20-‐activation model, UbcH10 has been shown to be responsible for the dissociation of the MCC-‐Cdc20 complex by ubiquitylating Cdc20 and thereby releasing the cell from the mitotic arrest (Reddy et al., 2007). However, a major criticism on this Cdc20-‐activation model concerns the ability of UbcH10 to override SAC arrest under physiological conditions.
Indeed, the overexpression of UbcH10 has been linked to cancer, as it was shown that UbcH10 levels are increased in several cancer cell lines and tumors
UbcX dissociates XErp1 from the APC/C Discussion
(Berlingieri et al., 2007; Okamoto et al., 2003; Pallante et al., 2005; Wagner et al., 2004) and therefore UbcH10 overexpression might inactivate the spindle checkpoint in these cells. The physiological significance of UbcH10 mediated spindle checkpoint inactivation has been put into question, since the amounts used in the original studies by Reddy et al. were suggested to be hundred times the endogenous levels and thus do not represent physiological conditions that could be observed in cancer cells (Walker et al., 2008). Accordingly, overexpression of UbcH10 to three times the endogenous levels did not significantly induce APC/C activation and exit from mitosis in cells arrested by the spindle checkpoint (Walker et al., 2008).
In both studies, overexpression of UbcH10 was induced by transient transfection of cells with a plasmid encoding UbcH10 (Reddy et al., 2007;
Walker et al., 2008). Therefore, the transfection efficiency and thus the amount of overexpression might vary from cell to cell and the amount of overexpression relative to the endogenous protein can only be estimated.
Instead, cell free Xenopus egg extracts allow the addition of recombinant protein, therefore the increase in protein levels is exactly measureable and the same in every condition. To estimate more accurately the levels of UbcX required for SAC inactivation, we determined the amount of endogenous UbcX in extracts by quantitative westernblotting (Figure 2.5.). When eight times the endogenous levels of UbcX were added to SAC arrested extracts, we observed APC/C activation and SAC override (Figure 2.1.). Since this increase in UbcX levels is only modest and can be observed in cancer cells (van Ree et al., 2010), we conclude that increased UbcX or UbcH10 levels can compromise the spindle checkpoint under physiological conditions.
The Xenopus extract system is well established in SAC research and has been a valuable tool for the identification and characterization of proteins important for spindle checkpoint signaling in vertebrate cells (Chen et al., 1998; Chen et al., 1996; Minshull et al., 1994), hence results obtained using Xenopus egg
UbcX dissociates XErp1 from the APC/C Discussion
41 these studies, we contribute to the knowledge of spindle checkpoint signaling, specifically, in its inactivation, by confirming that elevated UbcX activity causes SAC inactivation. We could show that this mechanism is conserved between human and Xenopus. However, we did not test in detail whether Cdc20 destabilization contributes to spindle checkpoint arrest in Xenopus egg extract.
Since we only confirmed one of the two models, the Cdc20-‐activation model, we cannot exclude that the Cdc20-‐inactivation pathway could operate in parallel to regulate SAC mediated APC/C inhibition.
3.1.2. Is an APC/C inhibitor targeted for ubiquitylation during SAC signaling?
The similarities between UbcX induced SAC-‐ and CSF-‐override presented here suggest that ubiquitylation of APC/C inhibitors could be a general mechanism to regulate the activity of APC/C inhibitory complexes. We could show that an increase in UbcX levels induces the ubiquitylation of XErp1 rather than Cdc20.
Maybe, also during spindle checkpoint signaling, the APC/C inhibitor needs to be ubiquitylated, and not Cdc20, to dissociate the MCC-‐Cdc20 complex. Could this be an explanation for the controversial findings on Cdc20 regulation during SAC signaling?
Under SAC arrest conditions, it has been suggested that APC/CCdc20 bound to the MCC proteins is inactive (Musacchio and Salmon, 2007). Electron microscopy studies of the APC/CCdc20-‐MCC complex favor this idea, as they revealed that substrate engagement to the APC/C is hindered when the MCC proteins are bound (Herzog et al., 2009). Additionally, the non-‐APC/C-‐bound, free pool of Cdc20 is either in an inhibitory complex bound to the MCC proteins (Musacchio and Salmon, 2007) or degraded upon ubiquitylation by the APC/C (Nilsson et al., 2008).
UbcX dissociates XErp1 from the APC/C Discussion
Figure 3.1. Model of the dynamic spindle checkpoint mediated APC/C inhibition regulated by ubiquitylation. During SAC arrest, the MCC proteins bind to Cdc20 to inhibit the APC/C.
Ubiquitylation of the MCC proteins and Cdc20 by the APC/C could result in MCC-‐Cdc20 complex dissociation and free ubiquitylated of Cdc20 is targeted for degradation. During SAC arrest, the dissociation could be counteracted by the deubiquitylating activity of USP44. Once the SAC is satisfied, the existing MCC-‐Cdc20 complexes are rapidly dissociated by ubiquitylation, free Cdc20 activates the APC/C and cells progress to anaphase.
A possible model for the dynamic regulation of APC/C by the SAC (Figure 3.1.) could be that Cdc20 once synthesized, is incorporated in the MCC complex and recruited to the APC/C. The APC/C ubiquitylates Cdc20 and targets it for degradation, perhaps to keep Cdc20 at a constant level despite continuous synthesis. Additionally, the APC/C ubiquitylates another component of the inhibitory MCC to induce the dissociation of the complex. It has been shown previously that BubR1 is ubiquitylated by the APC/C in vitro (Garnett et al., 2009), which was suggested to be important for progression through mitosis (Choi et al., 2009). Therefore it is tempting to speculate that BubR1 could be the target of APC/C mediated ubiquitylation. The stability of the Cdc20-‐MCC complex could be additionally promoted by USP44 to counteract dissociation of ubiquitylated Cdc20 to a certain degree. In this scenario, Cdc20 bound to the MCC complex is stable but inactive, whereas free, ubiquitylated Cdc20 is targeted for degradation. As a result there is no free Cdc20 available for APC/C activation, and at the same time, due to e.g. BubR1 ubiquitylation and deubiquitylation, the Cdc20-‐MCC complex remains dynamic and responsive to changes i.e. fully attached kinetochores. Together, these mechanisms-‐ Cdc20
UbcX dissociates XErp1 from the APC/C Discussion
43 a robust SAC arrest in the presence of unattached kinetochores, but at the same time a fast, switch-‐like inactivation of the SAC once it is satisfied, thus ensuring the faithful segregation of the genetic material into the two descending daughter cells.
In future, it will be interesting to test if also the Cdc20 degradation can be verified in SAC arrest conditions in Xenopus, or if the two models are mutually exclusive and only the Cdc20 activation model applies. Additionally, it could be tested whether BubR1, Mad2 and/or Bub3 are regulated by ubiquitylation similar to XErp1. If this is the case, lysine less mutants of the relevant proteins could be generated which cannot be inactivated by ubiquitylation anymore.
These mutants should be inducing a spindle checkpoint arrest when overexpressed in somatic cells or a delay in anaphase onset despite a satisfied spindle checkpoint, since the APC/C cannot inactivate inhibitory MCCs anymore.
3.2. UbcX mediated ubiquitylation of XErp1 regulates its APC/C inhibitory