Results

hBUBR1 Is Required for the Mitotic Checkpoint
Two complementary approaches were used to examine if hBUBR1 function is required for the mitotic checkpoint in Hela cells. Our first strategy to inhibit hBUBR1 activity was to microinject hBUBR1 antibodies into cells and test the response of the injected cells to the microtubule inhibitor nocodazole. The hBUBR1 antibodies used for the injection experiments have been shown not to cross-react with hBUB1 as determined by Western blots and immunoprecipitations (Chan et al. 1998; Jablonski et al. 1998). Hela cells synchronized at the G1/S boundary were microinjected with hBUBR1 or nonimmune antibodies shortly after they were released from the G1/S boundary. Approximately 2 h before the synchronized cells were expected to enter mitosis, nocodazole was added to the medium and the injected cells were sampled at various times during the ensuing 8 h (Fig. 1a and Fig. b). Mitotic cells were scored by the presence of condensed chromosomes which aggregated near the center of the cell when the spindle is abolished by nocodazole (Fig. 1 A, rows 1 and 2, left). Approximately 12 h after release from the G1/S boundary, most cells have entered mitosis. At this time, there was no significant difference between the mitotic indices of the cells that were injected with hBUBR1 or the nonimmune antibodies. Thus, injection of antibodies did not interfere with mitotic entry. Nonimmune antibodies were diffusely distributed throughout the mitotic cell (Fig. 1 A, row 1, middle) and did not interfere with the localization of hBUBR1 at kinetochores of the unaligned chromosomes (Fig. 1 A, row 1, right). In contrast, the injected hBUBR1 antibodies were concentrated at the kinetochores of the unaligned chromosomes (Fig. 1 A, row 2, middle). The presence of hBUBR1 at kinetochores of these cells was independently confirmed by staining with hBUBR1 antibodies (Fig. 1 A, row 2, right).
When cells were examined at later times after nocodazole treatment, the mitotic index of cells injected with nonimmune IgG remained high, whereas the mitotic index of cells injected with hBUBR1 antibodies was about threefold lower (Fig. 1 B). The decrease in the mitotic index of cells that were injected with the hBUBR1 antibodies was accompanied by a fivefold rise in the number of interphase cells with aberrantly shaped nuclei relative to the negative control (Fig. 1 A, row 3, and B). The flattened morphology as well as the presence of nuclei indicated that these cells exited mitosis (Fig. 1 A, row 3). The presence of paired centromeres, as revealed by staining with an anticentromere autoimmune (ACA) serum, suggested that either the chromosomes did not separate or had separated but re-replicated (Fig. 1 A, row 3, right, inset).
To directly confirm that inhibition of hBUBR1 abrogated the mitotic checkpoint and caused cells to exit mitosis, time-lapse videomicroscopy was used to follow the fate of nocodazole-treated cells that were microinjected with hBUBR1 antibodies. Fig. 1 C shows that within 30 min after the cell entered mitosis, the cell began to flatten out. 2 h later, the presence of a nucleus demonstrated that it had clearly exited mitosis (Fig. 1 C, 150'). At this level of resolution, we could not determine how rapidly cells injected with hBUBR1 antibodies exited mitosis. Uninjected cells on the same coverslip as well as cells injected with nonimmune antibodies remained blocked in mitosis for up to 16 h (data not shown).
To obtain independent verification of the microinjection results, we attempted to disrupt endogenous hBUBR1 function by targeting an hBUBR1 mutant to kinetochores. We created a mutant by deleting nine amino acids (residues 795–803) from subdomain II of the conserved catalytic core of the hBUBR1 kinase. Western blots confirmed that both mutant and wild-type hBUBR1 were expressed at equivalent levels (data not shown). Both mutant and wild-type gfp:hBUBR1 were transfected into synchronized Hela cells shortly after they were released from the G1/S boundary (Fig. 2). Nocodazole was added ∼2 h before the cells were expected to enter mitosis. By 12 h after release from the G1/S boundary, mitotic cells expressing both mutant and wild-type gfp-hBUBR1 at their kinetochores were identified (Fig. 2 A, rows 1 and 2). After overnight incubation in the presence of nocodazole, the mitotic index of cells expressing the mutant gfp:hBUBR1 was ∼2 times lower than for wild-type gfp:hBUBR1–transfected cells (Fig. 2 B). In concordance with the microinjection results, many of the cells expressing the mutant hBUBR1 exited mitosis and formed aberrant shaped nuclei (Fig. 2 A, row 3, and B). The accumulation of the gfp:hBUBR1Δkinase in the cytoplasm of these aberrant interphase cells is consistent with the distribution of endogenous hBUBR1 in interphase cells as previously reported (Chan et al. 1998). The microinjection and transfection experiments demonstrate that hBUBR1 kinase is likely to be an essential component of the mitotic checkpoint.

hBUBR1 Is Essential for a Normal Mitosis
We have previously shown that hBUBR1 can form a complex with the kinetochore motor CENP-E (Chan et al. 1998). If hBUBR1 can monitor kinetochore microtubule interactions mediated by CENP-E as part of its normal checkpoint function, disruption of hBUBR1 might cause cells to exit mitosis prematurely. Alternatively, hBUBR1 might regulate CENP-E functions that are important for chromosome alignment. In this case, loss of hBUBR1 should interfere with CENP-E functions at the kinetochore and block cells in mitosis with unaligned chromosomes (Schaar et al. 1997). To test the importance of hBUBR1 during normal mitosis, the fates of synchronized cells that were either injected with hBUBR1 antibodies or transfected with the gfp:hBUBR1Δkinase were determined. Approximately 12 h after release from the G1/S boundary, cells that were injected with nonimmune IgG were found in prometaphase and metaphase with the majority being at metaphase (Fig. 3 A, row 1). Immunofluorescence staining revealed that the metaphase cells exhibited detectable levels of hBUBR1 at their kinetochores despite the presence of nonimmune antibodies throughout the cell (Fig. 3 A, row 1, middle and right). For cells injected with the hBUBR1 antibody, most of the mitotic cells were in prometaphase. The injected hBUBR1 antibodies were found throughout the cell including kinetochores (Fig. 3 A, row 2, middle). The presence of hBUBR1 antibodies at the kinetochores did not interfere with CENP-E localization (Fig. 3 A, row 2, right). Although a few metaphase cells injected with hBUBR1 were seen, we consistently identified anaphase cells that contained lagging chromosomes (Fig. 3 A, row 3, left). These cells appeared to be in late anaphase as they contained a mature cleavage furrow that was concentrated with CENP-E (Fig. 3 A, row 3, right). The presence of lagging chromosomes suggested that these cells exited mitosis in the presence of unaligned chromosomes and gave rise to the high frequency of aberrantly divided cells that accumulated at later time points (Fig. 3 A, row 4, and B). The nuclei of the cells injected with hBUBR1 antibodies were abnormally shaped and DNA was invariably found in between the two divided cells (Fig. 3 A, row 4, left). The DNA in between the cells was derived from chromosomes because they contained centromeres as determined by ACA staining (Fig. 3 A, row 5, right, inset). However, we cannot distinguish whether the foci of ACA staining represent separated centromeres or fragmented centromeres. This cut phenotype was specific for hBUBR1 antibodies as this was not observed in uninjected cells or cells injected with nonimmune antibodies. In fact, hBUBR1 antibody injection did not produce any normal telophase cells (Fig. 3 B).
Similar results were obtained when synchronized Hela cells were transfected with the mutant hBUBR1Δkinase. In transfected cells that had progressed into mitosis, the mutant hBUBR1 was concentrated at kinetochores (Fig. 3 C, top right). At later times, there was a large increase in aberrantly divided cells with the cut phenotype (Fig. 3 C, bottom left). Both mock and wild-type gfp:hBUBR1–transfected cells divided normally and did not exhibit the cut phenotype (data not shown). The combined data suggest that disruption of hBUBR1 function caused the cells to exit mitosis in the presence of unaligned chromosomes.

hBUBR1 Monitors CENP-E Kinetochore Functions
We have previously shown that disruption of CENP-E functions at kinetochores prevented chromosome alignment and arrested cells in mitosis for >12 h (Schaar et al. 1997). To test whether hBUBR1 is required for this checkpoint, we coinjected hBUBR1 and CENP-E antibodies into synchronized Hela cells, and then compared the fates of these cells to those that were injected with CENP-E antibodies alone. Injection of CENP-E antibodies blocked the assembly of CENP-E onto kinetochores (Fig. 1 A, left and middle) but did not interfere with kinetochore localization of hBUBR1 (Fig. 4 A, left gallery, row 1) or the distribution of other centromere components that were recognized by ACA (Fig. 4 A, left gallery, row 2). As shown previously, the mitotic block induced by injection of CENP-E antibodies is the direct result of disruption of kinetochore function and not bipolar spindle formation (Fig. 4 A, left gallery, row 3).
Cells coinjected with CENP-E and hBUBR1 antibodies entered mitosis. Immunofluorescence staining confirmed that hBUBR1 remained at kinetochores (Fig. 4 A, right gallery, row 1, right) of the injected cells. 16 h after release from the G1/S boundary, we counted cells that were either in mitosis, telophase, aberrantly divided cut, or apoptosis. Cells injected with just CENP-E antibodies were arrested in mitosis with unaligned chromosomes (Fig. 4 B). No cells were found to be apoptotic, in telophase, or cut. Examination of the cells that were coinjected with hBUBR1 and CENP-E antibodies showed an ∼5-fold reduction in the number of mitotic cells relative to cells injected with CENP-E alone (Fig. 4 B). The reduction of mitotic cells was accompanied by a large increase in the number of aberrantly divided cells that exhibited the cut phenotype (Fig. 4 A, right gallery, rows 2 and 3) along with a slight increase in apoptotic cells. Normal telophase cells that were coinjected with the hBUBR1 and CENP-E antibodies were not found. These data showed that hBUBR1 is an essential component of the mitotic checkpoint that is sensitive to kinetochore functions that were specified by CENP-E.

Characterization of hBUBR1 Protein Expression and Kinase Activity
Having established that hBUBR1 is an essential component of the mitotic checkpoint, we wanted to investigate its mechanism of action by characterizing its expression and kinase activity as a function of the cell cycle. hBUBR1 was immunoprecipitated from K562 erythroleukemic cells and Hela cells that were separated into different phases of the cell cycles by centrifugal elutriation. Portions of the immunocomplexes were used to determine steady-state levels of protein and kinase activity. We assayed the immunoprecipitated hBUBR1 for autokinase activity as a recent study showed that a transfected hBUBR1 exhibited autokinase activity (Taylor et al. 1998). However, hBUBR1 isolated from K562 and Hela (data not shown) exhibited no detectable autokinase activity (Fig. 5 A, top) or kinase activities towards exogenous substrates (data not shown) even though it was expressed throughout the cell cycle of both cell types (Fig. 5 A, middle and bottom). hBUBR1 levels remained fairly constant throughout the cell cycle of K562 cells, whereas hBUBR1 levels in Hela cells fluctuated with the cell cycle. hBUBR1 level was lowest in G1 and steadily increased as cells progressed towards mitosis.
If hBUBR1 is a mitosis-specific kinase, its activity would have escaped detection because the vast majority of the cells that were elutriated into the late fractions were in G2 and only a very minor fraction was in mitosis. Therefore, we compared the hBUBR1 kinase activity between interphase and mitotically arrested Hela cells. hBUBR1 autokinase activity remained undetectable in interphase cells, but was highly active in mitotically arrested cells (Fig. 5 B). The same profile of hBUBR1 kinase activity was obtained with exogenous substrates such as myelin basic protein and a carboxyl-terminal fragment of CENP-E (data not shown). The increase in hBUBR1 kinase activity in mitotic cells was not due to increased expression of the protein or its association with the hBUB3 subunit relative to interphase cells (Fig. 5 B, bottom). However, hBUBR1 mobility was reduced in mitotically blocked cells relative to the interphase cells (Fig. 5 B, middle right). The upshift was due to phosphorylation as the electrophoretic mobility of hBUBR1 was no longer retarded after phosphatase treatment (Fig. 5 C, compare lanes 1 and 2). In vivo labeling experiments showed that hBUBR1 was also phosphorylated during interphase but became hyperphosphorylated in mitosis (data not shown). To further test if the hyperphosphorylation of hBUBR1 was related to the mitotic checkpoint, we compared hBUBR1 in mitotically blocked cells to cells that were released from the block and had exited mitosis (Fig. 5 C, right). The majority of hBUBR1 was hyperphosphorylated in mitotically blocked cells relative to normal mitotic cells. 1 h after their release from the mitotic block, hBUBR1 was no longer hyperphosphorylated (Fig. 5 C, compare lanes 4 and 5).
In the previous experiments, hBUBR1 kinase activity was determined in cells that had already been blocked in mitosis for many hours. As a checkpoint kinase, hBUBR1 should respond to spindle defects even after cells have reached metaphase. To test this, we isolated mitotic Hela cells by mechanical shakeoff, and then exposed them to nocodazole for various times. Microscopic analysis revealed that >90% of the mechanically detached cells were in metaphase with some prometaphase and very few (<1%) anaphase cells. hBUBR1 was immunoprecipitated from cell lysates that were harvested at different times of incubation with nocodazole, and then assayed for kinase activities and protein expression (Fig. 6 A). Comparison of hBUBR1 kinase activity between metaphase cells and those that were exposed to nocodazole showed that kinase activity was rapidly stimulated within 15 min of nocodazole treatment (Fig. 6 A). At this time, the spindle was absent and chromosomes were unaligned (data not shown). hBUBR1 kinase activity reached a peak at 30 min after nocodazole treatment and gradually declined. By 4 h, hBUBR1 kinase activity had decayed to a level that was slightly above that of normal metaphase cells. During the course of the experiment, cells were confirmed to be blocked in mitosis by phase-contrast microscopy and expression levels of cyclin B (Fig. 6 B, bottom). Western blots of the hBUBR1 immunoprecipitates revealed that the levels of hBUBR1 and hBUB3 remained fairly constant over the course of the experiment (Fig. 6 B). The increase of kinase activity was not due to changes in levels of hBUBR1 or its subunit, hBUB3. However, we detected an increase in the level of hyperphosphorylated hBUBR1 (as determined by retarded migration) between metaphase cells and those that were incubated in nocodazole (Fig. 6 B, compare top bands of 0 min and 120 min). The increase in the level of hyperphosphorylated hBUBR1 did not correlate with its peak kinase activities (Fig. 6a and Fig. B, compare 30 min and 120 min time points).
The hBUBR1 kinase activities described above were derived from the soluble pool of proteins and excluded those that were associated with kinetochores. As the kinetochore is a critical component of the checkpoint, we probed the insoluble fractions that were enriched in chromosomes for hBUBR1 to see how this population responded to spindle disruption (Fig. 6 C). Western blots revealed that hBUBR1 levels increased slightly after 30 and 60 min in nocodazole, but returned to the initial level after 2 and 4 h of nocodazole treatment. The most dramatic change was that hBUBR1 was quantitatively hyperphosphorylated within 15 min of spindle disruption and this high level of phosphorylation was maintained throughout the block.

hBUBR1 Associates with the Cyclosome/APC
Next, we examined the native size of hBUBR1 between interphase and mitotically blocked cells. Gel filtration chromatography revealed that hBUBR1 exists primarily in an ∼500-kD complex in interphase cells (Fig. 7 A, top). In mitotically arrested cells, hBUBR1 was associated with the ∼500-kD complex, but was also found in a larger complex that overlapped with the cyclosome/APC (Fig. 7 A, bottom). The possibility that hBUBR1 was associated with the cyclosome/APC was tested by immunoprecipitating hBUBR1 from the fractions containing the cyclosome/APC and probing for the presence of the APC subunits hscdc27, hscdc16 (Tugendreich et al. 1995), and APC7 (Yu et al. 1998). The results showed that all three of the APC subunits, coimmunoprecipitated with hBUBR1 in the mitotically blocked lysates (Fig. 7 B, middle). Although some hBUBR1 was detected in the cyclosome/APC containing fractions in interphase cells (Fig. 7 A, top), this was not associated with the cyclosome/APC (Fig. 7 B, left), and most likely resulted from smearing of the 500-kD complex in the gel filtration column. This finding along with the absence of cyclosome/APC subunits in immunoprecipitates from nonimmune IgG demonstrated that the cyclosome/APC specifically associates with hBUBR1 only in mitosis.
We estimated the proportion of the cellular pool of the cyclosome/APC that was associated with hBUBR1 in mitotically arrested cells by comparing the relative levels of various APC subunits in hBUBR1 immunoprecipitates and the remaining supernatant. When ∼30% of the total pool of hBUBR1 was immunoprecipitated from mitotic lysates, roughly 16–20% of the total pool of hscdc16, hscdc27, and APC7 was also found in the precipitate. Assuming that the remaining pool of hBUBR1 that was not immunoprecipitated can also associate with the APC, we can extrapolate that ∼50% of the total pool of APC can be associated with hBUBR1. Since the gel filtration data show that ∼50% of the total pool of hBUBR1 comigrates with the APC, we estimate that about 25% of the total pool of APC is associated with hBUBR1 in mitosis.