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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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Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.
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fig4: Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.

Mentions: Next, we sought evidence to directly support our finding that hBUBR1 is part of the inhibitory complex that blocks APC/C activity. Initial attempts to purify MCC with the hBUBR1 antibodies were unsuccessful because the harsh conditions required to elute hBUBR1 from the antibody-linked beads destroyed the complex. An alternative approach was to express a glutathione S-transferase (GST)-tagged hBUBR1 in human embryonic kidney cell line HEK293T and then use glutathione beads to affinity purify it along with the appropriate subunits from extracts of transfected cells (Fig. 4, A and B) . Western blot analysis confirmed that the affinity-purified GST–hBUBR1 formed the MCC, as it contained endogenous hBUBR1, hBUB3, CDC20, and MAD2. As shown previously, only a small fraction of the total pool of MAD2 was associated with GST–hBUBR1:MCC (Fig. 4 A, lane 3). All of these associations are specific to hBUBR1, as these proteins did not associate with GST alone (Fig. 4 A, lane 1). Purified GST–hBUBR1:MCC was found to inhibit ubiquitin ligase activity of the APC/C (Fig. 4 B, lane 5) as was seen with conventionally purified MCC (Fig. 4 B, lane 4). GST purified from transfected cells did not exhibit significant inhibitory activity (Fig. 4 B, lane 3).


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)

Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.
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Related In: Results  -  Collection

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fig4: Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.
Mentions: Next, we sought evidence to directly support our finding that hBUBR1 is part of the inhibitory complex that blocks APC/C activity. Initial attempts to purify MCC with the hBUBR1 antibodies were unsuccessful because the harsh conditions required to elute hBUBR1 from the antibody-linked beads destroyed the complex. An alternative approach was to express a glutathione S-transferase (GST)-tagged hBUBR1 in human embryonic kidney cell line HEK293T and then use glutathione beads to affinity purify it along with the appropriate subunits from extracts of transfected cells (Fig. 4, A and B) . Western blot analysis confirmed that the affinity-purified GST–hBUBR1 formed the MCC, as it contained endogenous hBUBR1, hBUB3, CDC20, and MAD2. As shown previously, only a small fraction of the total pool of MAD2 was associated with GST–hBUBR1:MCC (Fig. 4 A, lane 3). All of these associations are specific to hBUBR1, as these proteins did not associate with GST alone (Fig. 4 A, lane 1). Purified GST–hBUBR1:MCC was found to inhibit ubiquitin ligase activity of the APC/C (Fig. 4 B, lane 5) as was seen with conventionally purified MCC (Fig. 4 B, lane 4). GST purified from transfected cells did not exhibit significant inhibitory activity (Fig. 4 B, lane 3).

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