Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling

Abstract

The mitotic checkpoint (also known as the spindle assembly checkpoint) prevents premature anaphase onset through generation of an inhibitor of the E3 ubiquitin ligase APC/C, whose ubiquitination of cyclin B and securin targets them for degradation. Combining in vitro reconstitution and cell-based assays, we now identify dual mechanisms through which Bub3 promotes mitotic checkpoint signaling. Bub3 enhances signaling at unattached kinetochores not only by facilitating binding of BubR1 but also by enhancing Cdc20 recruitment to kinetochores mediated by BubR1's internal Cdc20 binding site. Downstream of kinetochore-produced complexes, Bub3 promotes binding of BubR1's conserved, amino terminal Cdc20 binding domain to a site in Cdc20 that becomes exposed by initial Mad2 binding. This latter Bub3-stimulated event generates the final mitotic checkpoint complex of Bub3-BubR1-Cdc20 that selectively inhibits ubiquitination of securin and cyclin B by APC/C(Cdc20). Thus, Bub3 promotes two distinct BubR1-Cdc20 interactions, involving each of the two Cdc20 binding sites of BubR1 and acting at unattached kinetochores or cytoplasmically, respectively, to facilitate production of the mitotic checkpoint inhibitor.

Fig. 1.

Bub3 directly promotes Mad2-dependent BubR1 inhibition of APC/C. (A) Schematic of functional domains of BubR1. (B) E409K mutation in BubR1 disrupts the BubR1–Bub3 interaction, analyzed by GFP immunoprecipitation. (C) Schematic for the protocol used to replace endogenous BubR1 with a GFP-tagged version and to determine duration of nocodazole-induced mitotic arrest in HeLa cells. (D) Time-lapse microscopy was used to determine nocodazole (100 ng/mL)-induced mitotic duration after replacing endogenous BubR1 with GFP–BubR1 variants using siRNA. (E) Coomassie staining of purified checkpoint components. (F) In vitro APC/C activity assay for checkpoint protein-mediated inhibition of APC/C-mediated cyclin B ubiquitination. (G) Effect on APC/C^Cdc20-mediated ubiquitination of cyclin B_1–102 by various combinations of BubR1, Mad2, and Bub3. (H) Quantification from G. Data represent mean ± SEM, n = 3. (I) Time titration for the inhibition of APC/C ubiquitination of cyclin B by Mad2 and BubR1 in the presence or absence of Bub3.

Fig. 2.

Bub3 binding promotes the conserved N-terminal Mad3 homology domain of BubR1-mediated inhibition of APC/C^Cdc20. (A) Schematic for the Mad3 homology domain of BubR1 (BubR1^N, 1–477 aa). (B) Inhibition of APC/C^Cdc20 activity by various combinations of checkpoint proteins. (C) Quantification from B. (D) Coomassie staining of purified wild-type or E409K mutant BubR1^N. (E) Test for requirement of Bub3 binding to BubR1 for Bub3 stimulation of BubR1^N. (F) Quantification from E. Data represent mean ± SEM. ***P < 0.001 from Student t test.

Fig. 3.

Bub3 binding promotes the Mad2-dependent N-terminal Mad3 homology domain of BubR1 binding to Cdc20. (A) In vitro assay of Bub3-dependent effects on binding of APC/C-bound Cdc20 to the N-terminal Cdc20 binding site of BubR1. (B) Quantification from A. (C) In vitro test if Bub3 stimulation of the BubR1–Cdc20 interaction in A required the Bub3 binding domain of BubR1. (D) Assay (using GFP immunoprecipitation of extracts from nocodazole-induced mitotic HeLa cells) to measure E409K GFP–BubR1 mutant association with Bub3, Cdc20, and the APC/C subunit Cdc27. (E) Quantification from D. Data represent mean ± SEM. *P < 0.05 and **P < 0.01 from Student t test, n = 3.

Fig. 4.

Enabling a cytosolic BubR1 to localize dynamically to the kinetochore enhances mitotic checkpoint signaling.(A) Schematic showing three tests for assessing BubR1’s role at kinetochores, including (i) blocking BubR1 binding at kinetochores with the E409K mutation, (ii) tethering BubR1 to kinetochores by fusion to Mis12, and (iii) directly tethering BubR1 to Bub3 to mediate kinetochore association. (B) Levels of accumulation of various GFP–BubR1 variants after expression in HeLa cells. (C) Localization of BubR1^FL or Bub3 or Mis12-tagged cytosolic BubR1^E409K. (D) Schematic of the protocol used in E to determine duration of nocodazole-induced mitotic arrest in cells expressing recombinant BubR1 variants. (E) Test of mitotic checkpoint function of various BubR1 variants. Time-lapse microscopy was used to determine nocodazole-induced mitotic duration after replacing endogenous BubR1 with GFP–BubR1 variants using siRNA. (F) Quantitation of mitotic timing in individual cells assayed as in E. (G) Indirect immunofluorescence to assess kinetochore binding of BubR1^E409K fused to Bub3 with or without an additional R183E mutation in Bub3.

Fig. 5.

Targeting a cytosolic BubR1 variant with only its N-terminal Cdc20 binding site to kinetochores does not enhance mitotic checkpoint signaling. (A) Schematics for (i) blocking BubR1^N (BubR1_1–363) binding to kinetochores by deleting the Bub3 binding site or by driving its kinetochore localization through fusion to (ii) Mis12 or (iii) Bub3. (B) Levels of accumulation of GFP–BubR1 variants after expression in HeLa cells. (C) Intracellular localization of Bub3 or Mis12-tagged cytosolic BubR1_1–363. (D–F) FRAP analysis of GFP–BubR1 variants in a nocodazole-induced mitotic cell. (D) Wild-type GFP–BubR1, (E) GFP–BubR1_1–363–Bub3, and (F) GFP–Mis12–BubR1_1–363. (G) Quantification from D–F. n/a, not available; t_1/2, time (seconds) for 50% recovery of fluorescence. (H) Schematic for the protocol used to determine mitotic timing. (I) Mitotic checkpoint function of BubR1 N terminus variants, analyzed by time-lapse microscopy.

Fig. 6.

Bub3 mediates kinetochore recruitment of Cdc20 via BubR1’s internal Cdc20 binding site to enhance mitotic checkpoint signaling. (A) Effect of kinetochore localization of BubR1 on kinetochore localization of Cdc20 in HeLa cells arrested in mitosis with monastrol (100 μM). (B) Quantification of Cdc20 intensity on kinetochores from A. (C) Indirect immunofluorescence assay of Cdc20 binding to kinetochores in the presence of BubR1 variants with either of its Cdc20 binding sites. (D) Quantification of Cdc20 intensity on kinetochores from C. (E) Duration of mitosis after reducing endogenous BubR1 (with siRNA) and expression of BubR1^FL or BubR1 deleted in its internal Cdc20 binding site. Time-lapse microscopy was used to determine nocodazole-induced duration of mitotic arrest after replacing endogenous BubR1 with a MycGFP–BubR1 in DLD-1 cells. (F) A model for dual modes of BubR1 activation by Bub3 for generating the mitotic checkpoint inhibitor. Mitotic checkpoint signaling is promoted (a) at kinetochores by Bub3-dependent recruitment to those kinetochores of BubR1 and Cdc20 (through binding of the internal Cdc20 binding site of BubR1 to Cdc20) and (b) in the cytosol by Bub3 stimulation of Mad2-dependent BubR1–Cdc20 formation (through binding of the conserved N-terminal Cdc20 binding site of BubR1).