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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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Identification of an APC/C inhibitor that copurifies with hBUBR1 kinase. (A) Identification of APC/C inhibitory activity in mitotic lysates. Mitotic lysate (S100) was fractionated through a Superose 6 gel filtration column by FPLC and proteins eluting at approximately the 300–400-kD range were found to inhibit APC/C activity. To maintain some physiological relevance to the APC/C inhibition assays, equal cell equivalents of APC/C and the various column fractions were used. Purified mitotic APC/C was incubated for 30 min with either buffer B alone (lane 2) or fractions from the 300–400-kD range of the Superose 6 column (lane 4) and then assayed for ubiquitin ligase activity. The same column fractions were assayed without addition of APC/C to monitor contaminating APC/C activity (lane 3). A reaction containing only substrate served as a negative control (lane 1). Asterisk denotes an iodinated contaminant bacterial protein that is not a substrate for APC/C and was excluded from the quantitation. (B) Flow chart of the purification of the APC/C inhibitory complex from HeLa cells. (C) Inhibitor of APC/C cofractionates with the hBUBR1 kinase complex. The elution profiles of hBUBR1 (inset) and APC/C inhibitory activity from the final Superose 6 column is shown. Arrows point to thyroglobulin (670 kD), γ-globulin (158 kD), and ovalbumin (44 kD), which served as migration standards.
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fig1: Identification of an APC/C inhibitor that copurifies with hBUBR1 kinase. (A) Identification of APC/C inhibitory activity in mitotic lysates. Mitotic lysate (S100) was fractionated through a Superose 6 gel filtration column by FPLC and proteins eluting at approximately the 300–400-kD range were found to inhibit APC/C activity. To maintain some physiological relevance to the APC/C inhibition assays, equal cell equivalents of APC/C and the various column fractions were used. Purified mitotic APC/C was incubated for 30 min with either buffer B alone (lane 2) or fractions from the 300–400-kD range of the Superose 6 column (lane 4) and then assayed for ubiquitin ligase activity. The same column fractions were assayed without addition of APC/C to monitor contaminating APC/C activity (lane 3). A reaction containing only substrate served as a negative control (lane 1). Asterisk denotes an iodinated contaminant bacterial protein that is not a substrate for APC/C and was excluded from the quantitation. (B) Flow chart of the purification of the APC/C inhibitory complex from HeLa cells. (C) Inhibitor of APC/C cofractionates with the hBUBR1 kinase complex. The elution profiles of hBUBR1 (inset) and APC/C inhibitory activity from the final Superose 6 column is shown. Arrows point to thyroglobulin (670 kD), γ-globulin (158 kD), and ovalbumin (44 kD), which served as migration standards.

Mentions: We undertook a biochemical approach to identify factors in mitotically arrested cells that were responsible for inhibiting the APC/C in vivo. HeLa cells were chosen because they exhibit a robust checkpoint arrest in response to spindle or kinetochore defects. Lysates prepared from mitotically blocked cells were chromatographed through a gel filtration column and fractions were tested for their ability to inhibit APC/C in a standard in vitro assay (Sudakin et al., 1995). Our assay relied on APC/C that was partially purified from mitotic HeLa cells, and was based on a protocol used to purify the cyclosome from clam oocytes (Sudakin et al., 1995). Iodinated substrate consisting of protein A fused to the destruction box of cyclin B1 allowed quantitation of the APC/C activity (Glotzer et al., 1991). We found that fractions eluting from the 400-kD region of the gel filtration column strongly inhibited APC/C activity (Fig. 1 A, lane 4) whereas no inhibitory activity was detectable from other fractions from the column (unpublished data). The inhibitory factor eluted at a position that was very close to that described previously for the mitotic checkpoint kinase hBUBR1 (Chan et al., 1999). We attempted to separate the inhibitor away from hBUBR1 by using additional column chromatography (Fig. 1 B). At each of the three successive chromatographic steps, the peaks of APC/C inhibitory activity and hBUBR1 coincided. The results obtained from the final Superose 6 column show coelution of the APC/C inhibitor (MCC) and hBUBR1 (Fig. 1 C).


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)

Identification of an APC/C inhibitor that copurifies with hBUBR1 kinase. (A) Identification of APC/C inhibitory activity in mitotic lysates. Mitotic lysate (S100) was fractionated through a Superose 6 gel filtration column by FPLC and proteins eluting at approximately the 300–400-kD range were found to inhibit APC/C activity. To maintain some physiological relevance to the APC/C inhibition assays, equal cell equivalents of APC/C and the various column fractions were used. Purified mitotic APC/C was incubated for 30 min with either buffer B alone (lane 2) or fractions from the 300–400-kD range of the Superose 6 column (lane 4) and then assayed for ubiquitin ligase activity. The same column fractions were assayed without addition of APC/C to monitor contaminating APC/C activity (lane 3). A reaction containing only substrate served as a negative control (lane 1). Asterisk denotes an iodinated contaminant bacterial protein that is not a substrate for APC/C and was excluded from the quantitation. (B) Flow chart of the purification of the APC/C inhibitory complex from HeLa cells. (C) Inhibitor of APC/C cofractionates with the hBUBR1 kinase complex. The elution profiles of hBUBR1 (inset) and APC/C inhibitory activity from the final Superose 6 column is shown. Arrows point to thyroglobulin (670 kD), γ-globulin (158 kD), and ovalbumin (44 kD), which served as migration standards.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196190&req=5

fig1: Identification of an APC/C inhibitor that copurifies with hBUBR1 kinase. (A) Identification of APC/C inhibitory activity in mitotic lysates. Mitotic lysate (S100) was fractionated through a Superose 6 gel filtration column by FPLC and proteins eluting at approximately the 300–400-kD range were found to inhibit APC/C activity. To maintain some physiological relevance to the APC/C inhibition assays, equal cell equivalents of APC/C and the various column fractions were used. Purified mitotic APC/C was incubated for 30 min with either buffer B alone (lane 2) or fractions from the 300–400-kD range of the Superose 6 column (lane 4) and then assayed for ubiquitin ligase activity. The same column fractions were assayed without addition of APC/C to monitor contaminating APC/C activity (lane 3). A reaction containing only substrate served as a negative control (lane 1). Asterisk denotes an iodinated contaminant bacterial protein that is not a substrate for APC/C and was excluded from the quantitation. (B) Flow chart of the purification of the APC/C inhibitory complex from HeLa cells. (C) Inhibitor of APC/C cofractionates with the hBUBR1 kinase complex. The elution profiles of hBUBR1 (inset) and APC/C inhibitory activity from the final Superose 6 column is shown. Arrows point to thyroglobulin (670 kD), γ-globulin (158 kD), and ovalbumin (44 kD), which served as migration standards.
Mentions: We undertook a biochemical approach to identify factors in mitotically arrested cells that were responsible for inhibiting the APC/C in vivo. HeLa cells were chosen because they exhibit a robust checkpoint arrest in response to spindle or kinetochore defects. Lysates prepared from mitotically blocked cells were chromatographed through a gel filtration column and fractions were tested for their ability to inhibit APC/C in a standard in vitro assay (Sudakin et al., 1995). Our assay relied on APC/C that was partially purified from mitotic HeLa cells, and was based on a protocol used to purify the cyclosome from clam oocytes (Sudakin et al., 1995). Iodinated substrate consisting of protein A fused to the destruction box of cyclin B1 allowed quantitation of the APC/C activity (Glotzer et al., 1991). We found that fractions eluting from the 400-kD region of the gel filtration column strongly inhibited APC/C activity (Fig. 1 A, lane 4) whereas no inhibitory activity was detectable from other fractions from the column (unpublished data). The inhibitory factor eluted at a position that was very close to that described previously for the mitotic checkpoint kinase hBUBR1 (Chan et al., 1999). We attempted to separate the inhibitor away from hBUBR1 by using additional column chromatography (Fig. 1 B). At each of the three successive chromatographic steps, the peaks of APC/C inhibitory activity and hBUBR1 coincided. The results obtained from the final Superose 6 column show coelution of the APC/C inhibitor (MCC) and hBUBR1 (Fig. 1 C).

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