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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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Regulation of the MCC. (A) MCC is present and active in interphase HeLa cells. hBUBR1 complex was purified from HeLa cells that were arrested at the G1/S boundary by a double thymidine block. The hBUBR1 and APC/C inhibitor profiles (tested by APC/C partially purified on gel filtration column) from the final Superose 6 column are shown. (B) Only APC/C from mitotic cells is sensitive to inhibition by MCC. APC/C purified from mitotic (M) and interphase (I) cells were incubated in the absence and presence of the MCC that was purified from interphase HeLa cells. Relative APC/C activity denotes the ratio of the ubiquitin ligase activity in reactions performed in the presence and absence of MCC. (C) hBUBR1 binds preferentially APC/C that contains hyperphosphorylated CDC27. Mitotic HeLa lysates were fractionated through a Superose 6 column as described and the portion containing intact APC was immunoprecipitated with hBUBR1 antibodies. The immunoprecipitate (IP) and remaining supernatant (Sup) were separated on SDS-PAGE and probed with hBUBR1, CDC27, CDC16, and APC7 antibodies.
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fig6: Regulation of the MCC. (A) MCC is present and active in interphase HeLa cells. hBUBR1 complex was purified from HeLa cells that were arrested at the G1/S boundary by a double thymidine block. The hBUBR1 and APC/C inhibitor profiles (tested by APC/C partially purified on gel filtration column) from the final Superose 6 column are shown. (B) Only APC/C from mitotic cells is sensitive to inhibition by MCC. APC/C purified from mitotic (M) and interphase (I) cells were incubated in the absence and presence of the MCC that was purified from interphase HeLa cells. Relative APC/C activity denotes the ratio of the ubiquitin ligase activity in reactions performed in the presence and absence of MCC. (C) hBUBR1 binds preferentially APC/C that contains hyperphosphorylated CDC27. Mitotic HeLa lysates were fractionated through a Superose 6 column as described and the portion containing intact APC was immunoprecipitated with hBUBR1 antibodies. The immunoprecipitate (IP) and remaining supernatant (Sup) were separated on SDS-PAGE and probed with hBUBR1, CDC27, CDC16, and APC7 antibodies.

Mentions: We showed previously that the 400-kD hBUBR1 complex was present in both interphase and mitotic cells (Chan et al., 1999). Given that hBUBR1 was found to associate with the APC/C only in mitosis (Chan et al., 1999), we expected that only the mitotic form of the MCC would inhibit the APC/C. Surprisingly, hBUBR1 complex isolated from interphase HeLa cells (synchronized in the G1/S boundary) inhibited APC/C activity and contained the same subunits found in mitotic MCC (unpublished data). Furthermore, both interphase and mitotic forms of MCC exhibited similar activities when equivalent amounts of hBUBR1 were tested (Fig. 6 A). Although this MCC preparation was obtained from cells arrested at the G1/S boundary, analysis of synchronized cells obtained by centrifugal elutriation showed that MCC is present in all stages of the cell cycle (unpublished data).


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)

Regulation of the MCC. (A) MCC is present and active in interphase HeLa cells. hBUBR1 complex was purified from HeLa cells that were arrested at the G1/S boundary by a double thymidine block. The hBUBR1 and APC/C inhibitor profiles (tested by APC/C partially purified on gel filtration column) from the final Superose 6 column are shown. (B) Only APC/C from mitotic cells is sensitive to inhibition by MCC. APC/C purified from mitotic (M) and interphase (I) cells were incubated in the absence and presence of the MCC that was purified from interphase HeLa cells. Relative APC/C activity denotes the ratio of the ubiquitin ligase activity in reactions performed in the presence and absence of MCC. (C) hBUBR1 binds preferentially APC/C that contains hyperphosphorylated CDC27. Mitotic HeLa lysates were fractionated through a Superose 6 column as described and the portion containing intact APC was immunoprecipitated with hBUBR1 antibodies. The immunoprecipitate (IP) and remaining supernatant (Sup) were separated on SDS-PAGE and probed with hBUBR1, CDC27, CDC16, and APC7 antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196190&req=5

fig6: Regulation of the MCC. (A) MCC is present and active in interphase HeLa cells. hBUBR1 complex was purified from HeLa cells that were arrested at the G1/S boundary by a double thymidine block. The hBUBR1 and APC/C inhibitor profiles (tested by APC/C partially purified on gel filtration column) from the final Superose 6 column are shown. (B) Only APC/C from mitotic cells is sensitive to inhibition by MCC. APC/C purified from mitotic (M) and interphase (I) cells were incubated in the absence and presence of the MCC that was purified from interphase HeLa cells. Relative APC/C activity denotes the ratio of the ubiquitin ligase activity in reactions performed in the presence and absence of MCC. (C) hBUBR1 binds preferentially APC/C that contains hyperphosphorylated CDC27. Mitotic HeLa lysates were fractionated through a Superose 6 column as described and the portion containing intact APC was immunoprecipitated with hBUBR1 antibodies. The immunoprecipitate (IP) and remaining supernatant (Sup) were separated on SDS-PAGE and probed with hBUBR1, CDC27, CDC16, and APC7 antibodies.
Mentions: We showed previously that the 400-kD hBUBR1 complex was present in both interphase and mitotic cells (Chan et al., 1999). Given that hBUBR1 was found to associate with the APC/C only in mitosis (Chan et al., 1999), we expected that only the mitotic form of the MCC would inhibit the APC/C. Surprisingly, hBUBR1 complex isolated from interphase HeLa cells (synchronized in the G1/S boundary) inhibited APC/C activity and contained the same subunits found in mitotic MCC (unpublished data). Furthermore, both interphase and mitotic forms of MCC exhibited similar activities when equivalent amounts of hBUBR1 were tested (Fig. 6 A). Although this MCC preparation was obtained from cells arrested at the G1/S boundary, analysis of synchronized cells obtained by centrifugal elutriation showed that MCC is present in all stages of the cell cycle (unpublished data).

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