Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2

MCC inhibition of the APC/C requires hBUBR1. Purified mitotic APC/C was incubated in the presence of buffer (lane 1), MCC (lane 2), MCC that was mock depleted with nonimmune IgG (lane 3) or immunodepleted with hBUBR1 antibodies (lane 4), and then assayed for ubiquitin ligase activity. (A) Ubiquitination assay. Mock- and hBUBR1-immunodepleted supernatants were probed for hBUBR1 (top panel). Asterisk denotes the contaminant iodinated protein. (B) Quantification of the ubiquitination assay on phosphorimager.

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.

Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.

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.

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.

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.