Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

Abstract

The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) from targeting key proteins for ubiquitin-mediated proteolysis. We have examined the mechanism by which the checkpoint inhibits the APC/C by purifying an APC/C inhibitory factor from HeLa cells. We call this factor the mitotic checkpoint complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 checkpoint proteins in near equal stoichiometry. MCC inhibitory activity is 3,000-fold greater than that of recombinant MAD2, which has also been shown to inhibit APC/C in vitro. Surprisingly, MCC is not generated from kinetochores, as it is also present and active in interphase cells. However, only APC/C isolated from mitotic cells was sensitive to inhibition by MCC. We found that the majority of the APC/C in mitotic lysates is associated with the MCC, and this likely contributes to the lag in ubiquitin ligase activity. Importantly, chromosomes can suppress the reactivation of APC/C. Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20. We propose that the preformed interphase pool of MCC allows for rapid inhibition of APC/C when cells enter mitosis. Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

Inducing precocious anaphase in cultured mammalian cells

“…In support of this hypothesis, BubR1 has recently been shown to bind Cdc20 and inhibit the APC/C from ubiqutinating cyclin B, independent of Mad2 [Fang, 2002;Tang et al, 2001]. There is some in vitro biochemical evidence, however, that Mad2 and BubR1 act synergistically to inhibit Cdc20 [Fang, 2002;Sudakin et al, 2001;Tang et al, 2001]. Our studies show that either Mad2⌬C or GST-Mad1F10 can overcome the checkpoint in nocodazole-treated PtK1 cells where kinetochores lack both tension as well as attached microtubules.…”

Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation

“…The spindle assembly checkpoint plays an essential role in maintaining genome integrity by Aurora-A induces mammary tumor formation X Wang et al holding cells with unaligned chromosomes at metaphase (Amon, 1999;Yoon et al, 2002;Andreassen et al, 2003). Mad2 selectively binds to unattached kinetochores and is capable of inhibiting anaphase-promoting complex (APC) activity (Sudakin et al, 2001;Tang et al, 2001;Fang, 2002). It was recently shown that overexpression of Aurora-A abrogates function of Mad2 in inhibiting APC, leading to the failure of the spindle assembly checkpoint (Anand et al, 2003).…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…For example, Bub1p binds to Bub3p throughout the cell cycle [reviewed in [Howell et al, 2000[Howell et al, , 2004Shah et al, 2004]. The association of SAC proteins with kinetochores is important for SAC function [Kadura et al, 2005;Taylor et al, 1998;Vanoosthuyse et al, 2004] perhaps because it alters the binding interactions between SAC proteins and influences the formation of the mitotic checkpoint complex (MCC) [Sudakin et al, 2001], which imposes a cell cycle arrest by binding to the Anaphase Promoting Complex (APC). The APC/C is a multi-protein ubiquitin ligase that targets important cell cycle proteins for degradation by the 26S proteasome.…”