Bub1 regulates chromosome segregation in a kinetochore-independent manner

Abstract

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

“…Mad1-Mad2 first catalyzes MCC assembly at interphase nuclear pores [3], then migrates to kinetochores at nuclear envelope breakdown (NEBD) and resumes MCC assembly until bipolar spindle attachment is complete [1,2]. There is significant debate about the factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes [4][5][6][7][8][9]. Through gene editing and livecell imaging, we found that the human Rod-Zw10-Zwilch (RZZ) complex is dispensable for cell viability and initial recruitment of Mad1-Mad2 to kinetochores at NEBD, but then becomes necessary to tether Mad1-Mad2 at kinetochores and sustain SAC arrest in cells challenged with spindle poisons.…”

The Spindle Assembly Checkpoint: Clock or Domino?

“…Recent experiments both in fission yeast and human cells suggest that such a behavior is not only limited to the effector proteins, but can also be found for the more upstream kinase Bub1 (Klebig et al 2009;Windecker et al 2009). A first study based on a structure-function study of human Bub1 mutants in an Bub1 RNAi background, indicates that Bub1 does not need to be located at the kinetochores to control chromosome congression and spindle assembly checkpoint even though there is a minor impairment of each function (Klebig et al 2009).…”

“…Indeed, all three spindle checkpoint kinases, Mps1, BubR1, and Bub1, regulate and sometimes correct defective kinetochore-microtubule attachments (Kops 2009;Kops et al 2010;Musacchio and Salmon 2007). Importantly, Bub1 and BubR1 separation of function mutants have demonstrated that this ability to regulate kinetochoremicrotubule attachments is independent of their capacity to control spindle checkpoint signaling (Elowe et al 2010;Klebig et al 2009;Malureanu et al 2009;Warren et al 2002). BubR1 controls the dynamics of kinetochore-bound microtubules, while Bub1 has been proposed to contribute to the transition of lateral kinetochore-microtubule attachments to end-on attachments during chromosome alignment (Gillett et al 2004;Lampson and Kapoor 2005;Meraldi and Sorger 2005).…”

“…A first study based on a structure-function study of human Bub1 mutants in an Bub1 RNAi background, indicates that Bub1 does not need to be located at the kinetochores to control chromosome congression and spindle assembly checkpoint even though there is a minor impairment of each function (Klebig et al 2009). The same is true in S. pombe, where bub3 mutants, which fail to localize Bub1 to kinetochores, are still able to mount a Bub1-dependent checkpoint response and to control Shugoshin localization at centrosomes through Bub1 (Windecker et al 2009).…”

“…The same is true in S. pombe, where bub3 mutants, which fail to localize Bub1 to kinetochores, are still able to mount a Bub1-dependent checkpoint response and to control Shugoshin localization at centrosomes through Bub1 (Windecker et al 2009). This behavior is not restricted to Bub1, since BubR1 does not need to localize to kinetochores to control chromosome alignment (Klebig et al 2009). These data suggest that Bub1 and BubR1 are not mere scaffold proteins at the kinetochores and that their binding to kinetochores only increases the efficiency by which they control kinetochoremicrotubule attachment and spindle checkpoint signaling.…”