Human Bubr1 Is a Mitotic Checkpoint Kinase That Monitors Cenp-E Functions at Kinetochores and Binds the Cyclosome/APC

Abstract

Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind to kinetochores where they are postulated to be components of the mitotic checkpoint that monitors kinetochore activities to determine if chromosomes have achieved alignment at the spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, and T.J. Yen. 1998. Chromosoma. 107:386–396). In support of this, hBUB1 and the homologous mouse BUB1 have been shown to be important for the mitotic checkpoint (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson, S.D. Markowitz, K.W. Kinzler, and B. Vogelstein. 1998. Nature. 392:300–303; Taylor, S.S., and F. McKeon. 1997. Cell. 89:727–735). We now demonstrate that hBUBR1 is also an essential component of the mitotic checkpoint. hBUBR1 is required by cells that are exposed to microtubule inhibitors to arrest in mitosis. Additionally, hBUBR1 is essential for normal mitotic progression as it prevents cells from prematurely entering anaphase. We establish that one of hBUBR1's checkpoint functions is to monitor kinetochore activities that depend on the kinetochore motor CENP-E. hBUBR1 is expressed throughout the cell cycle, but its kinase activity is detected after cells have entered mitosis. hBUBR1 kinase activity was rapidly stimulated when the spindle was disrupted in mitotic cells. Finally, hBUBR1 was associated with the cyclosome/anaphase-promoting complex (APC) in mitotically arrested cells but not in interphase cells. The combined data indicate that hBUBR1 can potentially provide two checkpoint functions by monitoring CENP-E–dependent activities at the kinetochore and regulating cyclosome/APC activity.

p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase

“…Similarly, upon Noc treatment more M phase cells were accumulated in BUBR1 transfected, but not in BUBR1 K795M -transfected cells, as compared with the vector-transfected control (Table 1), suggesting that BUBR1 kinase activity plays a role in regulating spindle checkpoint. This observation is also consistent with a recent report by Yen's group that hBUBR1 is essential for spindle checkpoint activation (Chan et al, 1999).…”

“…This may be due simply to the fact that more p55CDC is expressed in mitotic cells (Figure 3a, lanes 5 and 6). Chan et al have shown that native hBUBR1 forms two complexes during mitosis (Chan et al, 1999). It would be important to examine which complex contains p55CDC.…”

“…BUBR1 hyperphosphorylation induced by nocodazole is rapid, and recombinant BUBR1 is capable of autophosphorylation (Li et al, 1999). A recent study has demonstrated that hBUBR1 is essential for spindle checkpoint function and that APC may be the target of the spindle checkpoint kinase (Chan et al, 1999).…”

CENP-meta, an Essential Kinetochore Kinesin Required for the Maintenance of Metaphase Chromosome Alignment in Drosophila

“…The checkpoint is mediated through a diffusible signal of Mad2/Cdc20 released from unattached kinetochores and which inhibits activation of the anaphase promoting complex required for the degradation of regulators of chromatid linkage (e.g., Fang, 1999; Morgan 1999). In mammals, the checkpoint kinase BubR1 (Cahill et al 1998; Taylor et al 1998) has been shown to interact with CENP-E (Chan et al 1998, Chan et al 1999), forming a nearly stoichiometric, soluble complex with CENP-E in mitotically arrested cells (Yao et al 2000). Moreover, immunodepletion of CENP-E from frog extracts that can cycle between interphase and mitosis eliminates the ability to activate or maintain the mitotic checkpoint (Abrieu, A., J.A.…”

“…For example, the relocalization of CENP-E to the midzone at anaphase-B may indicate an additional role for this plus-end motor (Wood et al 1997) in spindle elongation and/or cytokinesis. CENP-E has also been implicated as part of, or a target of, the spindle assembly checkpoint based on a physical interaction with the spindle assembly protein hBubR1 (Chan et al 1998, Chan et al 1999; Yao et al 2000). Indeed, in Xenopus extracts CENP-E is required for establishing and maintaining this checkpoint (Abrieu, A., J.A.…”