Regulated degradation of the APC coactivator Cdc20

Abstract

BackgroundCdc20 is a highly conserved activator of the anaphase-promoting complex (APC), promoting cell-cycle-regulated ubiquitination and proteolysis of a number of critical cell-cycle-regulatory targets including securin and mitotic cyclins. APC-Cdc20 activity is tightly regulated, and this regulation is likely important for accurate cell cycle control. One significant component of Cdc20 regulation is thought to be Cdc20 proteolysis. However, published literature suggests different mechanisms and requirements for Cdc20 proteolysis. The degree to which Cdc20 proteolysis is cell-cycle regulated, the dependence of Cdc20 proteolysis on Cdc20 destruction boxes (recognition sequences for APC-mediated ubiqutination, either by Cdc20 or by the related Cdh1 APC activator), and the need for APC itself for Cdc20 proteolysis all have been disputed to varying extents. In animals, Cdc20 proteolysis is thought to be mediated by Cdh1, contributing an intrinsic order of APC activation by Cdc20 and then by Cdh1. One report suggests a Cdh1 requirement for Cdc20 proteolysis in budding yeast; this idea has not been tested further.ResultsWe characterized Cdc20 proteolysis using Cdc20 expressed from its endogenous locus; previous studies generally employed strongly overexpressed Cdc20, which can cause significant artifacts. We analyzed Cdc20 proteolysis with or without mutations in previously identified destruction box sequences, using varying methods of cell cycle synchronization, and in the presence or absence of Cdh1. Cdc20 instability is only partially dependent on destruction boxes. A much stronger dependence on Cdh1 for Cdc20 proteolysis was observed, but Cdh1-independent proteolysis was also clearly observed. Cdc20 proteolysis independent of both destruction boxes and Cdh1 was especially detectable around the G1/S transition; Cdh1-dependent proteolysis was most notable in late mitosis and G1.ConclusionsCdc20 proteolysis is under complex control, with different systems operating at different points in the cell cycle. This complexity is likely to explain apparent conflicts in previously published literature on this subject. A major mode of control of Cdc20 proteolysis occurs in late mitosis/early G1 and is Cdh1-dependent, as in animal cells; this mode may contribute to the known sequential activation of the APC by Cdc20 followed by Cdh1. An independent mode of Cdc20 proteolysis, independent of destruction boxes and Cdh1, occurs at G1/S; we do not know the mechanism or function of this mode of proteolysis, but speculate that it may contribute to sharpening and restricting activation of APC-Cdc20 to early mitosis.

The kinetochore-dependent and -independent formation of the CDC20-MAD2 complex and its functions in HeLa cells

“…Substantial amounts of the complex are still present in anaphase cells when the SAC is supposed to be turned off. This probably should not be too surprising since the MAD2 level remains constant throughout the cell cycle and CDC20 is only degraded by the APC/C Cdh1 in late mitosis and G1303132 with substantial amounts of CDC20 still detectable in anaphase B HeLa cells (Fig. 2a).…”

Suppressor of cytokine signaling 1 blocks mitosis in human melanoma cells

“…NEK2 degradation might also interfere with mitosis progression in BLM-SOCS1 cells, since recent reports suggest a role for NEK2 in abolishing MAD2-Cdc20 control of the metaphase checkpoint [63]. Finally, destruction of targets late in mitosis, including Aurora A and Cdc20 itself, again depends on Cdh1 [31,40]; Cdh1 degradation and metaphase blockade nonetheless prevent BLM-SOCS1 cells from reaching this stage. The combination of markers indicates that Cdh1 is the main SOCS1 target in mitosis and explains why SOCS1 overexpression causes accumulation of cells in metaphase.…”

“…Hence, SOCS1 expression lowers Cdh1 but not Cdc20 levels in BLM cells. Since Cdc20 is a target for the ubiquitin ligase activity of APC/C-Cdh1 complexes [31], SOCS1-mediated Cdh1 degradation might account for Cdc20 accumulation. In accordance with a role for SOCS1 in Cdh1 degradation, cells from mice lacking Cdh1 show proliferative defects and mitotic block reminiscent of BLM-SOCS1 cells, as well as increased Cdc20 levels [59].…”

“…The APC/C is a large ubiquitin E3 ligase complex coordinated by two homologous mitotic coactivators, Cdc20 and Cdh1 [30]. Substrate selection by Cdc20 and Cdh1 results in the sequential degradation of individual target proteins in each step of mitosis; whereas APC/C-Cdc20 targets the anaphase inhibitor securin and initiates chromosome separation, APC/C-Cdh1 promotes degradation of spindle proteins such as Aurora kinases and Cdc20 itself [31]. Because of its role in the metaphaseanaphase transition, APC/C-Cdc20 activity is controlled through the spindle assembly checkpoint [32].…”

Insights into the cellular mechanism of the yeast ubiquitin ligase APC/C-Cdh1 from the analysis of in vivo degrons

“…, 1998; Shirayama et al. , 1998; Robbins and Cross, 2010). To learn more about the degradation sequences of these proteins, we asked whether their N-terminal regions might also be sufficient for cell cycle–regulated proteolysis.…”