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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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APC/C inhibitor is a complex of mitotic checkpoint proteins. (A) MAD2 comigrates with the hBUBR1 kinase complex. Mitotic HeLa extracts were separated through a Superose 6 column and the column fractions were probed for MAD2, hBUBR1, and the APC7. Fractions exhibiting APC/C inhibitory activity are denoted as MCC. (B) MCC consists of hBUBR1, hBUB3, MAD2, and CDC20. Fractions eluting from the 300–400-kD region of the Superose 6 column in A that exhibited APC/C inhibitory activity were pooled and immunoprecipitated with nonimmune IgG, anti-hBUBR1, and anti-MAD2 antibodies, washed, and probed with for hBUBR1, hBUB3, MAD2, and CDC20. (C) MCC can exist independently of APC/C. Fractions 19–28 from the Superose 6 column shown in A were combined and rechromatographed through a MonoQ anion exchange column by FPLC. Fractions were assayed for APC/C activity (solid line) and probed for hBUBR1, hBUB3, CDC20, MAD2, and CDC27 by Western blots. (D) Separation of active and inactive APC/C. Fractions 40–42 from the MonoQ column shown in C that exhibited APC/C activity and MCC were immunodepleted successively with anti-hBUBR1 and anti-CDC20 antibodies. The ubiquitin ligase activity was tested in the supernatants and immunoprecipitates. (Lane 1) Input APC/C activity; (lanes 2 and 3, respectively) APC/C activity in the supernatants after depletion of hBUBR1 and CDC20; (lanes 4 and 5, respectively) APC/C activity associated with corresponding hBUBR1 and CDC20 immunoprecipitates; and (lane 6) immunoprecipitate performed with nonimmune IgG. hBUBR1, CDC20, and nonimmune IgG immunoprecipitates were probed for CDC27 to estimate relative amount of APC/C (bottom panel). Asterisk denotes the contaminant iodinated protein.
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fig3: APC/C inhibitor is a complex of mitotic checkpoint proteins. (A) MAD2 comigrates with the hBUBR1 kinase complex. Mitotic HeLa extracts were separated through a Superose 6 column and the column fractions were probed for MAD2, hBUBR1, and the APC7. Fractions exhibiting APC/C inhibitory activity are denoted as MCC. (B) MCC consists of hBUBR1, hBUB3, MAD2, and CDC20. Fractions eluting from the 300–400-kD region of the Superose 6 column in A that exhibited APC/C inhibitory activity were pooled and immunoprecipitated with nonimmune IgG, anti-hBUBR1, and anti-MAD2 antibodies, washed, and probed with for hBUBR1, hBUB3, MAD2, and CDC20. (C) MCC can exist independently of APC/C. Fractions 19–28 from the Superose 6 column shown in A were combined and rechromatographed through a MonoQ anion exchange column by FPLC. Fractions were assayed for APC/C activity (solid line) and probed for hBUBR1, hBUB3, CDC20, MAD2, and CDC27 by Western blots. (D) Separation of active and inactive APC/C. Fractions 40–42 from the MonoQ column shown in C that exhibited APC/C activity and MCC were immunodepleted successively with anti-hBUBR1 and anti-CDC20 antibodies. The ubiquitin ligase activity was tested in the supernatants and immunoprecipitates. (Lane 1) Input APC/C activity; (lanes 2 and 3, respectively) APC/C activity in the supernatants after depletion of hBUBR1 and CDC20; (lanes 4 and 5, respectively) APC/C activity associated with corresponding hBUBR1 and CDC20 immunoprecipitates; and (lane 6) immunoprecipitate performed with nonimmune IgG. hBUBR1, CDC20, and nonimmune IgG immunoprecipitates were probed for CDC27 to estimate relative amount of APC/C (bottom panel). Asterisk denotes the contaminant iodinated protein.

Mentions: To further characterize the composition of MCC, we probed the complex for MAD2. Fractionation of crude mitotic lysates through a Superose 6 column showed that the vast majority of MAD2 was found as monomers. However, some MAD2 (<5% of total) comigrated with the MCC and the APC/C (Fig. 3 A). MAD2 was determined to be part of MCC, as it was detected in hBUBR1 immunoprecipitates obtained from fractions containing MCC (Fig. 3 B). Furthermore, MCC was found to contain CDC20, an activator of the APC/C that has been shown to target MAD2 to the APC/C (Fang et al., 1998; Wassmann and Benezra, 1998). Consistent with the previous data that showed that hBUB3 was associated with hBUBR1 (Chan et al., 1999, Taylor et al., 1998), hBUB3 was also present in the MCC (Fig. 3 B). The presence of MAD2 in MCC was verified in immunoprecipitation experiments using anti-MAD2 antibodies. The MAD2 immunoprecipitates contained all four MCC subunits (Fig. 3 B). Importantly, we did not detect hBUB1 or MAD1 in the MCC (unpublished data) despite the fact that they have been shown to interact with some of the MCC subunits (hBUB1 with hBUB3, and MAD1with MAD2). Our recently published data indicate that MAD1 binds MAD2 but not CDC20 (Campbell et al., 2001). This suggests that hBUB1 and MAD1 form complexes distinct from the MCC.


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)

APC/C inhibitor is a complex of mitotic checkpoint proteins. (A) MAD2 comigrates with the hBUBR1 kinase complex. Mitotic HeLa extracts were separated through a Superose 6 column and the column fractions were probed for MAD2, hBUBR1, and the APC7. Fractions exhibiting APC/C inhibitory activity are denoted as MCC. (B) MCC consists of hBUBR1, hBUB3, MAD2, and CDC20. Fractions eluting from the 300–400-kD region of the Superose 6 column in A that exhibited APC/C inhibitory activity were pooled and immunoprecipitated with nonimmune IgG, anti-hBUBR1, and anti-MAD2 antibodies, washed, and probed with for hBUBR1, hBUB3, MAD2, and CDC20. (C) MCC can exist independently of APC/C. Fractions 19–28 from the Superose 6 column shown in A were combined and rechromatographed through a MonoQ anion exchange column by FPLC. Fractions were assayed for APC/C activity (solid line) and probed for hBUBR1, hBUB3, CDC20, MAD2, and CDC27 by Western blots. (D) Separation of active and inactive APC/C. Fractions 40–42 from the MonoQ column shown in C that exhibited APC/C activity and MCC were immunodepleted successively with anti-hBUBR1 and anti-CDC20 antibodies. The ubiquitin ligase activity was tested in the supernatants and immunoprecipitates. (Lane 1) Input APC/C activity; (lanes 2 and 3, respectively) APC/C activity in the supernatants after depletion of hBUBR1 and CDC20; (lanes 4 and 5, respectively) APC/C activity associated with corresponding hBUBR1 and CDC20 immunoprecipitates; and (lane 6) immunoprecipitate performed with nonimmune IgG. hBUBR1, CDC20, and nonimmune IgG immunoprecipitates were probed for CDC27 to estimate relative amount of APC/C (bottom panel). Asterisk denotes the contaminant iodinated protein.
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fig3: APC/C inhibitor is a complex of mitotic checkpoint proteins. (A) MAD2 comigrates with the hBUBR1 kinase complex. Mitotic HeLa extracts were separated through a Superose 6 column and the column fractions were probed for MAD2, hBUBR1, and the APC7. Fractions exhibiting APC/C inhibitory activity are denoted as MCC. (B) MCC consists of hBUBR1, hBUB3, MAD2, and CDC20. Fractions eluting from the 300–400-kD region of the Superose 6 column in A that exhibited APC/C inhibitory activity were pooled and immunoprecipitated with nonimmune IgG, anti-hBUBR1, and anti-MAD2 antibodies, washed, and probed with for hBUBR1, hBUB3, MAD2, and CDC20. (C) MCC can exist independently of APC/C. Fractions 19–28 from the Superose 6 column shown in A were combined and rechromatographed through a MonoQ anion exchange column by FPLC. Fractions were assayed for APC/C activity (solid line) and probed for hBUBR1, hBUB3, CDC20, MAD2, and CDC27 by Western blots. (D) Separation of active and inactive APC/C. Fractions 40–42 from the MonoQ column shown in C that exhibited APC/C activity and MCC were immunodepleted successively with anti-hBUBR1 and anti-CDC20 antibodies. The ubiquitin ligase activity was tested in the supernatants and immunoprecipitates. (Lane 1) Input APC/C activity; (lanes 2 and 3, respectively) APC/C activity in the supernatants after depletion of hBUBR1 and CDC20; (lanes 4 and 5, respectively) APC/C activity associated with corresponding hBUBR1 and CDC20 immunoprecipitates; and (lane 6) immunoprecipitate performed with nonimmune IgG. hBUBR1, CDC20, and nonimmune IgG immunoprecipitates were probed for CDC27 to estimate relative amount of APC/C (bottom panel). Asterisk denotes the contaminant iodinated protein.
Mentions: To further characterize the composition of MCC, we probed the complex for MAD2. Fractionation of crude mitotic lysates through a Superose 6 column showed that the vast majority of MAD2 was found as monomers. However, some MAD2 (<5% of total) comigrated with the MCC and the APC/C (Fig. 3 A). MAD2 was determined to be part of MCC, as it was detected in hBUBR1 immunoprecipitates obtained from fractions containing MCC (Fig. 3 B). Furthermore, MCC was found to contain CDC20, an activator of the APC/C that has been shown to target MAD2 to the APC/C (Fang et al., 1998; Wassmann and Benezra, 1998). Consistent with the previous data that showed that hBUB3 was associated with hBUBR1 (Chan et al., 1999, Taylor et al., 1998), hBUB3 was also present in the MCC (Fig. 3 B). The presence of MAD2 in MCC was verified in immunoprecipitation experiments using anti-MAD2 antibodies. The MAD2 immunoprecipitates contained all four MCC subunits (Fig. 3 B). Importantly, we did not detect hBUB1 or MAD1 in the MCC (unpublished data) despite the fact that they have been shown to interact with some of the MCC subunits (hBUB1 with hBUB3, and MAD1with MAD2). Our recently published data indicate that MAD1 binds MAD2 but not CDC20 (Campbell et al., 2001). This suggests that hBUB1 and MAD1 form complexes distinct from the MCC.

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
Related in: MedlinePlus