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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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Chromosomes inhibit APC/C. (A) Chromosomes prolong the inhibition of the APC/C activity in mitotic lysates. APC/C activity in lysates prepared from interphase (○) and mitotic (⋄) cells were monitored for up to 60 min. The same lysates were assayed for ACP/C activity in the presence of chromosomes (• and ♦). (B) Chromosomes inhibit APC/C fraction directly. Partially purified mitotic APC/C (gel filtration form of the APC/C was prepared as described above), MCC and in vitro–translated CDC20 were preincubated, respectively, in the presence or absence of chromosomes at 300°C. Upon completion of the preincubation, the APC/C inhibitory assays were assembled as described below. After 30 min, E1, E2-C, and radio- labeled substrate were added to initiate the ubiquitin ligase reaction and APC/C activity was determined over the course of 30 min. (1) APC. APC/C was preincubated alone; (2) APC+CR. APC/C was preincubated with chromosomes; (3) APC+CR SUP. Chromosomes were preincubated, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (4) APC+CDC20/CR. In vitro–translated CDC20 was preincubated with chromosomes, the mix was centrifuged for 10 min at 14.000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (5) APC+MCC. MCC was preincubated alone; (6) APC+MCC/CR. MCC was preincubated with chromosomes, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity. All reactions were supplemented with CDC20 to maintain the same concentration with the samples where preincubation of CDC20 with chromosomes has been tested. (C) Chromosomes inhibit highly purified APC/C. Gel filtration fraction of the APC/C was further purified on MonoQ anion exchange FPLC column as described above. The APC/C was preincubated with or without chromosomes and the ubiquitination reaction was initiated upon addition of all necessary ingredients as described above. 0, 5, 10, and 30 min time points were taken to determine the APC/C activity. In all experiments presented in the figure the Ub–substrate conjugates were visualized and quantified as described above.
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fig7: Chromosomes inhibit APC/C. (A) Chromosomes prolong the inhibition of the APC/C activity in mitotic lysates. APC/C activity in lysates prepared from interphase (○) and mitotic (⋄) cells were monitored for up to 60 min. The same lysates were assayed for ACP/C activity in the presence of chromosomes (• and ♦). (B) Chromosomes inhibit APC/C fraction directly. Partially purified mitotic APC/C (gel filtration form of the APC/C was prepared as described above), MCC and in vitro–translated CDC20 were preincubated, respectively, in the presence or absence of chromosomes at 300°C. Upon completion of the preincubation, the APC/C inhibitory assays were assembled as described below. After 30 min, E1, E2-C, and radio- labeled substrate were added to initiate the ubiquitin ligase reaction and APC/C activity was determined over the course of 30 min. (1) APC. APC/C was preincubated alone; (2) APC+CR. APC/C was preincubated with chromosomes; (3) APC+CR SUP. Chromosomes were preincubated, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (4) APC+CDC20/CR. In vitro–translated CDC20 was preincubated with chromosomes, the mix was centrifuged for 10 min at 14.000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (5) APC+MCC. MCC was preincubated alone; (6) APC+MCC/CR. MCC was preincubated with chromosomes, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity. All reactions were supplemented with CDC20 to maintain the same concentration with the samples where preincubation of CDC20 with chromosomes has been tested. (C) Chromosomes inhibit highly purified APC/C. Gel filtration fraction of the APC/C was further purified on MonoQ anion exchange FPLC column as described above. The APC/C was preincubated with or without chromosomes and the ubiquitination reaction was initiated upon addition of all necessary ingredients as described above. 0, 5, 10, and 30 min time points were taken to determine the APC/C activity. In all experiments presented in the figure the Ub–substrate conjugates were visualized and quantified as described above.

Mentions: The ability of MCC from interphase cells to block APC/C shows that its activity does not require the presence of unattached kinetochores, as mature kinetochores are not found until mitosis. To clarify the interactions between MCC, APC/C, and kinetochores, we set out to reconstitute the checkpoint inhibition of the APC/C in lysates prepared from HeLa cells. Kinetic studies showed that the APC/C activity in crude mitotic lysates exhibited a reproducible lag of ∼15 min, compared with the APC/C activity in lysates prepared from asynchronous cells that were >90% interphase (Fig. 7 A, compare ⋄ and ○). The initial lag seen in the mitotic lysates may have been due to checkpoint inhibition, as lysates were prepared from mitotically arrested cells whose APC/C is associated with MCC. In the absence of chromosomes, the checkpoint inhibition cannot be sustained and the APC/C eventually recovers its activity. This was confirmed when addition of purified chromosomes to the mitotic lysate suppressed the reactivation of ACP/C (Fig. 7 A, ♦). Chromosomes do not permanently inactivate the APC/C, as APC/C was reactivated when chromosomes were removed from during the incubation (unpublished data). Chromosomes were only effective in mitotic lysates as they modestly reduced APC/C activity in asynchronous lysates (Fig. 7 A, •). This modest reduction can be attributed to the small fraction of mitotic cells that were present in the asynchronous population.


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)

Chromosomes inhibit APC/C. (A) Chromosomes prolong the inhibition of the APC/C activity in mitotic lysates. APC/C activity in lysates prepared from interphase (○) and mitotic (⋄) cells were monitored for up to 60 min. The same lysates were assayed for ACP/C activity in the presence of chromosomes (• and ♦). (B) Chromosomes inhibit APC/C fraction directly. Partially purified mitotic APC/C (gel filtration form of the APC/C was prepared as described above), MCC and in vitro–translated CDC20 were preincubated, respectively, in the presence or absence of chromosomes at 300°C. Upon completion of the preincubation, the APC/C inhibitory assays were assembled as described below. After 30 min, E1, E2-C, and radio- labeled substrate were added to initiate the ubiquitin ligase reaction and APC/C activity was determined over the course of 30 min. (1) APC. APC/C was preincubated alone; (2) APC+CR. APC/C was preincubated with chromosomes; (3) APC+CR SUP. Chromosomes were preincubated, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (4) APC+CDC20/CR. In vitro–translated CDC20 was preincubated with chromosomes, the mix was centrifuged for 10 min at 14.000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (5) APC+MCC. MCC was preincubated alone; (6) APC+MCC/CR. MCC was preincubated with chromosomes, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity. All reactions were supplemented with CDC20 to maintain the same concentration with the samples where preincubation of CDC20 with chromosomes has been tested. (C) Chromosomes inhibit highly purified APC/C. Gel filtration fraction of the APC/C was further purified on MonoQ anion exchange FPLC column as described above. The APC/C was preincubated with or without chromosomes and the ubiquitination reaction was initiated upon addition of all necessary ingredients as described above. 0, 5, 10, and 30 min time points were taken to determine the APC/C activity. In all experiments presented in the figure the Ub–substrate conjugates were visualized and quantified as described above.
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fig7: Chromosomes inhibit APC/C. (A) Chromosomes prolong the inhibition of the APC/C activity in mitotic lysates. APC/C activity in lysates prepared from interphase (○) and mitotic (⋄) cells were monitored for up to 60 min. The same lysates were assayed for ACP/C activity in the presence of chromosomes (• and ♦). (B) Chromosomes inhibit APC/C fraction directly. Partially purified mitotic APC/C (gel filtration form of the APC/C was prepared as described above), MCC and in vitro–translated CDC20 were preincubated, respectively, in the presence or absence of chromosomes at 300°C. Upon completion of the preincubation, the APC/C inhibitory assays were assembled as described below. After 30 min, E1, E2-C, and radio- labeled substrate were added to initiate the ubiquitin ligase reaction and APC/C activity was determined over the course of 30 min. (1) APC. APC/C was preincubated alone; (2) APC+CR. APC/C was preincubated with chromosomes; (3) APC+CR SUP. Chromosomes were preincubated, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (4) APC+CDC20/CR. In vitro–translated CDC20 was preincubated with chromosomes, the mix was centrifuged for 10 min at 14.000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity; (5) APC+MCC. MCC was preincubated alone; (6) APC+MCC/CR. MCC was preincubated with chromosomes, the mix was centrifuged for 10 min at 14,000 rpm to remove chromosomes, and the supernatant was tested for the APC/C inhibitory activity. All reactions were supplemented with CDC20 to maintain the same concentration with the samples where preincubation of CDC20 with chromosomes has been tested. (C) Chromosomes inhibit highly purified APC/C. Gel filtration fraction of the APC/C was further purified on MonoQ anion exchange FPLC column as described above. The APC/C was preincubated with or without chromosomes and the ubiquitination reaction was initiated upon addition of all necessary ingredients as described above. 0, 5, 10, and 30 min time points were taken to determine the APC/C activity. In all experiments presented in the figure the Ub–substrate conjugates were visualized and quantified as described above.
Mentions: The ability of MCC from interphase cells to block APC/C shows that its activity does not require the presence of unattached kinetochores, as mature kinetochores are not found until mitosis. To clarify the interactions between MCC, APC/C, and kinetochores, we set out to reconstitute the checkpoint inhibition of the APC/C in lysates prepared from HeLa cells. Kinetic studies showed that the APC/C activity in crude mitotic lysates exhibited a reproducible lag of ∼15 min, compared with the APC/C activity in lysates prepared from asynchronous cells that were >90% interphase (Fig. 7 A, compare ⋄ and ○). The initial lag seen in the mitotic lysates may have been due to checkpoint inhibition, as lysates were prepared from mitotically arrested cells whose APC/C is associated with MCC. In the absence of chromosomes, the checkpoint inhibition cannot be sustained and the APC/C eventually recovers its activity. This was confirmed when addition of purified chromosomes to the mitotic lysate suppressed the reactivation of ACP/C (Fig. 7 A, ♦). Chromosomes do not permanently inactivate the APC/C, as APC/C was reactivated when chromosomes were removed from during the incubation (unpublished data). Chromosomes were only effective in mitotic lysates as they modestly reduced APC/C activity in asynchronous lysates (Fig. 7 A, •). This modest reduction can be attributed to the small fraction of mitotic cells that were present in the asynchronous population.

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