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

Sudakin V, Chan GK, Yen TJ - J. Cell Biol. (2001)

Bottom Line: 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.Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20.Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

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.

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MCC inhibition of the APC/C requires hBUBR1. Purified mitotic APC/C was incubated in the presence of buffer (lane 1), MCC (lane 2), MCC that was mock depleted with nonimmune IgG (lane 3) or immunodepleted with hBUBR1 antibodies (lane 4), and then assayed for ubiquitin ligase activity. (A) Ubiquitination assay. Mock- and hBUBR1-immunodepleted supernatants were probed for hBUBR1 (top panel). Asterisk denotes the contaminant iodinated protein. (B) Quantification of the ubiquitination assay on phosphorimager.
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fig2: MCC inhibition of the APC/C requires hBUBR1. Purified mitotic APC/C was incubated in the presence of buffer (lane 1), MCC (lane 2), MCC that was mock depleted with nonimmune IgG (lane 3) or immunodepleted with hBUBR1 antibodies (lane 4), and then assayed for ubiquitin ligase activity. (A) Ubiquitination assay. Mock- and hBUBR1-immunodepleted supernatants were probed for hBUBR1 (top panel). Asterisk denotes the contaminant iodinated protein. (B) Quantification of the ubiquitination assay on phosphorimager.

Mentions: To test whether hBUBR1 is an essential component of MCC, fractions containing high levels of MCC activity were immunodepleted of hBUBR1 and the remaining supernatant was tested for APC/C inhibitory activity. Incubation of MCC with nonimmune IgG did not deplete hBUBR1 or reduce its inhibitory activity relative to untreated MCC (Fig. 2 A, compare lanes 2 and 3). In contrast, MCC fractions that were immunodepleted of hBUBR1 did not block APC/C activity to the same extent as mock-depleted or untreated MCC (Fig. 2 A, compare lane 4 with lanes 2 and 3). Because hBUBR1 was not completely depleted from this MCC fraction (∼25% of the input hBUBR1 was still detected by Western analysis, Fig. 2 A, upper panel), some residual APC/C inhibitory activity was detected when compared with APC/C that was incubated with buffer alone (Fig. 2 A, compare lanes 1 and 4, Fig. 2 B). These results show that hBUBR1 or an associated factor is essential for MCC activity.


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

Sudakin V, Chan GK, Yen TJ - J. Cell Biol. (2001)

MCC inhibition of the APC/C requires hBUBR1. Purified mitotic APC/C was incubated in the presence of buffer (lane 1), MCC (lane 2), MCC that was mock depleted with nonimmune IgG (lane 3) or immunodepleted with hBUBR1 antibodies (lane 4), and then assayed for ubiquitin ligase activity. (A) Ubiquitination assay. Mock- and hBUBR1-immunodepleted supernatants were probed for hBUBR1 (top panel). Asterisk denotes the contaminant iodinated protein. (B) Quantification of the ubiquitination assay on phosphorimager.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196190&req=5

fig2: MCC inhibition of the APC/C requires hBUBR1. Purified mitotic APC/C was incubated in the presence of buffer (lane 1), MCC (lane 2), MCC that was mock depleted with nonimmune IgG (lane 3) or immunodepleted with hBUBR1 antibodies (lane 4), and then assayed for ubiquitin ligase activity. (A) Ubiquitination assay. Mock- and hBUBR1-immunodepleted supernatants were probed for hBUBR1 (top panel). Asterisk denotes the contaminant iodinated protein. (B) Quantification of the ubiquitination assay on phosphorimager.
Mentions: To test whether hBUBR1 is an essential component of MCC, fractions containing high levels of MCC activity were immunodepleted of hBUBR1 and the remaining supernatant was tested for APC/C inhibitory activity. Incubation of MCC with nonimmune IgG did not deplete hBUBR1 or reduce its inhibitory activity relative to untreated MCC (Fig. 2 A, compare lanes 2 and 3). In contrast, MCC fractions that were immunodepleted of hBUBR1 did not block APC/C activity to the same extent as mock-depleted or untreated MCC (Fig. 2 A, compare lane 4 with lanes 2 and 3). Because hBUBR1 was not completely depleted from this MCC fraction (∼25% of the input hBUBR1 was still detected by Western analysis, Fig. 2 A, upper panel), some residual APC/C inhibitory activity was detected when compared with APC/C that was incubated with buffer alone (Fig. 2 A, compare lanes 1 and 4, Fig. 2 B). These results show that hBUBR1 or an associated factor is essential for MCC activity.

Bottom Line: 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.Chromosomes did not affect the inhibitory activity of MCC or the stimulatory activity of CDC20.Unattached kinetochores then target the APC/C for sustained inhibition by the MCC.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

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.

Show MeSH