Cdc20 is required for the post-anaphase, KEN-dependent degradation of centromere protein F

Abstract

Progression through mitosis and cytokinesis requires the sequential proteolysis of several cell-cycle regulators. This proteolysis is mediated by the ubiquitin-proteasome system, with the E3 ligase being the anaphase-promoting complex, also known as the cyclosome (APC/C). The APC/C is regulated by two activators, namely Cdc20 and Cdh1. The current view is that prior to anaphase, the APC/C is activated by Cdc20, but that following anaphase, APC/C switches to Cdh1-dependent activation. However, here we present an analysis of the kinetochore protein Cenp-F that is inconsistent with this notion. Although it has long been appreciated that Cenp-F is degraded sometime during or after mitosis, exactly when and how has not been clear. Here we show that degradation of Cenp-F initiates about six minutes after anaphase, and that this is dependent on a C-terminal KEN-box. Although these two observations are consistent with Cenp-F being a substrate of Cdh1-activated APC/C, Cenp-F is degraded normally in Cdh1-null cells. By contrast, RNAi-mediated repression of APC/C subunits or Cdc20 does inhibit Cenp-F degradation. These findings therefore suggest that the APC/C does not simply ‘switch’ upon anaphase onset; rather, our observations indicate that Cdc20 also contributes to post-anaphase activation of the APC/C. We also show that the post-anaphase, KEN-box-dependent degradation of Cenp-F requires it to be farnesylated, a post-translational modification usually linked to membrane association. Because so many of the behaviours of Cenp-F are farnesylation-dependent, we suggest that this modification plays a more global role in Cenp-F function.

Anaphase-Promoting Complex/Cyclosome Participates in the Acute Response to Protein-Damaging Stress

“…Furthermore, HSF2 was also found to coprecipitate with Cdh1. The finding that HSF2 associates with both Cdc20 and Cdh1 is not unique, since other substrates have also been shown to be regulated by both coactivators (12,92) and some redundancy in substrate recognition occurs in the absence of either Cdc20 or Cdh1 (26,29,33,55). Moreover, HSF2 exhibited a longer half-life and less heat shock-induced ubiquitination when APC/C coactivators or subunits were silenced by specific siRNAs.…”

Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction

“…These observations are potentially in line with a role for increasing Cdc20 activity in anaphase that we and others reported previously. 16 Alternatively, the APC/C activator Cdh1 becomes partially Figure 2. A lysine mutant of cyclin B1 that is degraded slightly less efficiently can halt mitotic exit after the metaphase-to-anaphase transition.…”

“…These findings suggest that, when cyclin B1 is not yet degraded and the APC/C activator Cdh1 should still be repressed, APC/C Cdc20 can be activated by two processes: inactivation of the spindle checkpoint and progression into anaphase. 16 Such a mechanism, which would need to be investigated further, could for instance help to prevent spindle checkpoint re-activation in accidental cases when residual cyclin B1 remains during anaphase.…”

“…Indeed, Cdc20 appears to play a role in anaphase activity of the APC/C. 16 Alternatively, Aurora B or Mps1 kinase inhibitors release some activity of the other APC/C activator, Cdh1, even though cyclin B1-Cdk1 is still active. The latter possibility would imply that APC/C Cdh1 recognizes poor APC/C substrates (such as cyclin B1 with a point-mutation in its D-box, an APC/C substrate which furthermore has no obvious D-box or KEN box) better than APC/C Cdc20 does.…”

BubR1 blocks substrate recruitment to the APC/C in a KEN-box-dependent manner

“…Transgenes were induced with 1 mg/ml tetracycline then imaged by time-lapse microscopy 48 hours later (Morrow et al, 2005). To monitor degradation kinetics, cells co-transfected with securin-DsRed (Holland and Taylor, 2006) or cyclin-B1-Venus (Garnett et al, 2009) were imaged as described (Gurden et al, 2010). To repress Blinkin, cells were transfected with siRNAs (Dharmacon) (supplementary material Table S2) using Interferin (PolyPlus) and analysed 24 hours later.…”