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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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Characterization of the MCC. (A) Estimation of MAD2 content in MCC. Quantitative immunoblot of purified MCC with known amounts of recombinant MAD2. Recombinant (His) 6-tagged MAD2 migrated slower than native MAD2. (B) Comparison of APC/C inhibitory activity between the recombinant oligomeric MAD2 and purified MCC. Increasing amounts of oligomeric MAD2 and purified MCC were added to the APC/C assay to compare inhibitory activities. Phosphorimage of the APC/C reactions (upper panel) was quantified to determine the amount of recombinant MAD2 and MCC required to achieve equivalent levels of inhibition (lower panel). (C) MCC subunits exist in near equal stoichiometry. HeLa cells were labeled in 35S-Trans label for 6 h and mitotic and interphase cells were separated and then incubated with nonimmune IgG or hBUBR1 antibodies. Phosphorimage of the immunoprecipitate obtained from mitotic cells shows the radiolabeled hBUBR1, hBUB3, MAD2, and CDC20. The counts from each subunit were normalized to their cysteine and methionine contents (without initiating Met) to estimate their stoichiometry.
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fig5: Characterization of the MCC. (A) Estimation of MAD2 content in MCC. Quantitative immunoblot of purified MCC with known amounts of recombinant MAD2. Recombinant (His) 6-tagged MAD2 migrated slower than native MAD2. (B) Comparison of APC/C inhibitory activity between the recombinant oligomeric MAD2 and purified MCC. Increasing amounts of oligomeric MAD2 and purified MCC were added to the APC/C assay to compare inhibitory activities. Phosphorimage of the APC/C reactions (upper panel) was quantified to determine the amount of recombinant MAD2 and MCC required to achieve equivalent levels of inhibition (lower panel). (C) MCC subunits exist in near equal stoichiometry. HeLa cells were labeled in 35S-Trans label for 6 h and mitotic and interphase cells were separated and then incubated with nonimmune IgG or hBUBR1 antibodies. Phosphorimage of the immunoprecipitate obtained from mitotic cells shows the radiolabeled hBUBR1, hBUB3, MAD2, and CDC20. The counts from each subunit were normalized to their cysteine and methionine contents (without initiating Met) to estimate their stoichiometry.

Mentions: Recombinant MAD2 purified from bacteria can exist as monomers and tetramers, but only tetrameric MAD2 was found to inhibit APC/C in vitro (Fang et al., 1998). Gel filtration of mitotic HeLa extracts cells showed that >90% of MAD2 is not associated with MCC or APC/C (Fig. 3 A) and these fractions did not inhibit APC/C (unpublished data). To understand why recombinant MAD2 can inhibit APC/C whereas MAD2 in HeLa cells cannot, we compared the inhibitory activities between MCC and recombinant MAD2. Optimally, this comparison should be made based on protein concentration. However, we could not estimate the amount of MCC as our preparations were not purified to homogeneity. Therefore, we standardized the inhibitory activities using MAD2 levels. First, we estimated the amount of MAD2 that was present in MCC by quantitative immunoblots using known amounts of recombinant MAD2 (Fig. 5 A). We then compared the inhibitory activities obtained with different amounts of MCC or recombinant tetrameric MAD2 and determined the amount of each inhibitor that gave comparable activities (Fig. 5 B). Based on this analysis, we estimated that the inhibitory activity of the MCC is >3,000-fold greater than recombinant MAD2 oligomers.


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)

Characterization of the MCC. (A) Estimation of MAD2 content in MCC. Quantitative immunoblot of purified MCC with known amounts of recombinant MAD2. Recombinant (His) 6-tagged MAD2 migrated slower than native MAD2. (B) Comparison of APC/C inhibitory activity between the recombinant oligomeric MAD2 and purified MCC. Increasing amounts of oligomeric MAD2 and purified MCC were added to the APC/C assay to compare inhibitory activities. Phosphorimage of the APC/C reactions (upper panel) was quantified to determine the amount of recombinant MAD2 and MCC required to achieve equivalent levels of inhibition (lower panel). (C) MCC subunits exist in near equal stoichiometry. HeLa cells were labeled in 35S-Trans label for 6 h and mitotic and interphase cells were separated and then incubated with nonimmune IgG or hBUBR1 antibodies. Phosphorimage of the immunoprecipitate obtained from mitotic cells shows the radiolabeled hBUBR1, hBUB3, MAD2, and CDC20. The counts from each subunit were normalized to their cysteine and methionine contents (without initiating Met) to estimate their stoichiometry.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196190&req=5

fig5: Characterization of the MCC. (A) Estimation of MAD2 content in MCC. Quantitative immunoblot of purified MCC with known amounts of recombinant MAD2. Recombinant (His) 6-tagged MAD2 migrated slower than native MAD2. (B) Comparison of APC/C inhibitory activity between the recombinant oligomeric MAD2 and purified MCC. Increasing amounts of oligomeric MAD2 and purified MCC were added to the APC/C assay to compare inhibitory activities. Phosphorimage of the APC/C reactions (upper panel) was quantified to determine the amount of recombinant MAD2 and MCC required to achieve equivalent levels of inhibition (lower panel). (C) MCC subunits exist in near equal stoichiometry. HeLa cells were labeled in 35S-Trans label for 6 h and mitotic and interphase cells were separated and then incubated with nonimmune IgG or hBUBR1 antibodies. Phosphorimage of the immunoprecipitate obtained from mitotic cells shows the radiolabeled hBUBR1, hBUB3, MAD2, and CDC20. The counts from each subunit were normalized to their cysteine and methionine contents (without initiating Met) to estimate their stoichiometry.
Mentions: Recombinant MAD2 purified from bacteria can exist as monomers and tetramers, but only tetrameric MAD2 was found to inhibit APC/C in vitro (Fang et al., 1998). Gel filtration of mitotic HeLa extracts cells showed that >90% of MAD2 is not associated with MCC or APC/C (Fig. 3 A) and these fractions did not inhibit APC/C (unpublished data). To understand why recombinant MAD2 can inhibit APC/C whereas MAD2 in HeLa cells cannot, we compared the inhibitory activities between MCC and recombinant MAD2. Optimally, this comparison should be made based on protein concentration. However, we could not estimate the amount of MCC as our preparations were not purified to homogeneity. Therefore, we standardized the inhibitory activities using MAD2 levels. First, we estimated the amount of MAD2 that was present in MCC by quantitative immunoblots using known amounts of recombinant MAD2 (Fig. 5 A). We then compared the inhibitory activities obtained with different amounts of MCC or recombinant tetrameric MAD2 and determined the amount of each inhibitor that gave comparable activities (Fig. 5 B). Based on this analysis, we estimated that the inhibitory activity of the MCC is >3,000-fold greater than recombinant MAD2 oligomers.

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