Errors in cell division result in aneuploidy, which is commonly observed in genetic disorders and cancer. Most of these errors are prevented by cell cycle checkpoints, which are cellular control systems that allow certain events to proceed only after the completion of specific events prior to them in the sequence. The spindle checkpoint, for example, is a surveillance system that can delay mitosis by preventing the activation of the anaphase-promoting complex (also called the cyclosome) if spindle organization is defective or chromosomes are incorrectly attached to the spindle. Genes encoding spindle checkpoint components were first isolated from the budding yeast Saccharomyces cerevisiae: they include the mitotic arrest-deficient (MAD) genes MAD1, MAD2, and MAD3; the budding uninhibited by benzimidazole (BUB) genes BUB1, BUB2, and BUB3; and the monopolar spindle gene MPS1. Mutated human homologs of BUB1 have been found in subtypes of colorectal cancer that exhibit chromosome instability.
Kinetochores play an important role in relaying the checkpoint signal – thought to arise from unattached kinetochores or the absence of tension on kinetochores – to the spindle checkpoint pathway. The kinetochore is a complex of proteins bound to the centromere (CEN) DNA–protein complex CBF3, which forms the core of the kinetochore. CBF3 is bound to an essential conserved CEN DNA element (CDEIII) and consists of 4 essential proteins: Ndc10, Cep3, Ctf13, and Skp1. Despite the importance of the spindle checkpoint signaling mechanism, the molecular link between the kinetochore and the spindle checkpoint has not been well characterized.
Figure 1. The spindle checkpoint protects cells from chromosome missegregation caused by mitotic errors. When the checkpoint is activated by defects in kinetochore–microtubule attachment, it arrests the cell cycle by inhibiting the anaphase-promoting complex (APC). APC inhibition causes securin to accumulate, which in turn inhibits separase activity that targets cohesin. As a result, cohesion is maintained and sister chromatids remain bound together. Therefore, APC inhibition by the spindle checkpoint arrests cells in mitosis, thereby preventing aneuploidy. Defects in the mitotic spindle checkpoint result in cell death or aneuploidy, which may lead to tumorigenesis.
We have found from studies on yeast that Bub1, a component of the mitotic checkpoint kinase and the spindle checkpoint, binds to Skp1, a core kinetochore component in budding yeast, and associates with CEN DNA via Skp1. We have found the first biochemical evidence that Bub1 associates with centromere DNA via Skp1. Our genetic and biochemical data strongly suggest that Skp1 needs to interact with Bub1 for the mitotic delay induced by kinetochore tension defects but not by spindle depolymerization, kinetochore assembly defects, or Mps1 overexpression. Although the molecular signal from kinetochores has not been identified, our findings have considerably advanced the knowledge of the molecular mechanism underlying the spindle checkpoint signal pathway. We are further investigating the primary signal from kinetochores.
The kinetochore assembly mechanism links to the anticancer mechanism of the Hsp90 inhibitor 17-AAG The kinetochore is essential for maintaining the high fidelity of chromosome transmission during cell division. In budding yeast, Sgt1, with Skp1, activates Ctf13 (the F-box core kinetochore protein) and is therefore required for the assembly of CBF3. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast and also that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide (TPR) motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that the binding of Hsp90 to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.