Katsumi Kitagawa Lab :: The Research Institute at Nationwide Children's Hospital

Katsumi Kitagawa Lab :: Research Description

The spindle checkpoint signaling mechanism   

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.
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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.


Sgt1-Hsp90 complex is required for the assembly of kinetochore protein complexes in human cells.

A human homolog of Sgt1 (HsSgt1A) can rescue the yeast sgt1-null mutant from the lethality of its phenotype. This rescue strongly suggests that the functions of Sgt1 are highly conserved in yeast and humans. To examine how depletion of HsSgt1 proteins in mammalian cells affects chromosome segregation in those cells, we used RNA interference (in collaboration with Dr. Andrea Musacchio). We also performed experiments in which deletion mutants of human Sgt1 were overexpressed by the cells. We found that Sgt1 depletion or overexpression of the mutant Sgt1 causes mitotic defects and that in Sgt1-depleted HeLa cells, some previously characterized outer or central kinetochore proteins are not present at kinetochores.

Our immunoprecipitation-mass spectrometry analyses have shown that, like yeast Sgt1, human Sgt1 interacts with Hsp90, Hsp70, and Skp1. The Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG, a geldanamycin derivative), which is also a novel anticancer drug being tested clinical trials, induces a mitotic delay in human cells in culture. We found that the mitotic delay caused by 17-AAG depends on the spindle checkpoint, and a significant number of 17-AAG–treated cells had misaligned chromosomes at metaphase. Several known kinetochore proteins were not present at the kinetochores in 17-AAG–treated cells. We also found that Hsp90 is required from late S phase to early G2 phase for the recruitment of kinetochore proteins. Thus, we discovered a novel anticancer mechanism of 17-AAG. Synthetic lethality occurred in response to 17-AAG treatment and application of human Sgt1 siRNA. Our results strongly suggest that the Sgt1-Hsp90 complex is required for the assembly of kinetochore protein complexes in human cells.
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The novel mitotic cell death induced by a defect in the spindle checkpoint The spindle checkpoint protects cells from aneuploidy by monitoring the status of kinetochore-microtubule attachment. Substantial aneuploidy is thought to result when cells have defects in the checkpoint pathway and in kinetochore-microtubule attachment. We found in humans a novel type of caspase-independent apoptosis that occurs during mitosis, which we named CIMD (caspase-independent mitotic death). Simultaneous human Bub1 depletion and treatment of cells with nocodazole (a microtubule-depolymerizing drug), paclitaxel (Taxol, a microtubule-stabilizing drug), or 17-AAG induced DNA fragmentation at early mitosis (Figure 2).
Figure 2. CIMD (caspase-independent mitotic death). HeLa cells that were BUB1-depleted and 17-AAG–treated exhibit DNA fragmentation during mitosis. Forty-eight hours after HeLa cells were transfected with MAD2, BUB1, or Luc siRNA, they were incubated with 17-AAG (+17AAG, 500 nM) for 24 h at 37 degrees celcius. Fixed samples were stained using an in situ cell death detection system that contained TMR red (red signal; Roche), an anti–phosphorylated histone H3 (p-H3) mouse monoclonal antibody (Abcam), and FITC–conjugated secondary antibodies (green signal; Jackson ImmunoResearch). DNA was stained with DAPI to visualize prophase and metaphase cells. Samples were analyzed by fluorescence microscopy, and images were captured by using Plan-APOCHROMAT 63X (Zeiss; magnification, x63). The scale bar represents 10 mcm.

CIMD is the cell death mechanism protecting cells from aneuploidy

However, human Mad2 depletion did not induce this DNA fragmentation. This novel mitotic cell death appeared to be independent of caspase activation. We found that AIF (apoptosis-inducing factor) and EndoG (endonuclease G), which are effectors of caspase-independent cell death, are released from mitochondria during the activation of CIMD and that the DNA fragmentation depends on AIF and EndoG. We also found that CIMD depends on p73 (a homolog of p53) but not p53. Therefore, our results suggest that human Bub1 is a crucial factor in regulating cell death during mitosis, and we propose that CIMD protects cells from aneuploidy by inducing the death of cells that are prone to substantial chromosome missegregation.
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We have shown that survived cells that escape CIMD have abnormal nuclei, and we have determined the molecular mechanism by which BUB1 depletion activates CIMD. BUB3 (a BUB1 interactor and a spindle checkpoint protein) interacts with p73 (a homolog of p53) specifically in cells wherein CIMD occurs. BUB3 that is freed from BUB1 associates with p73 on which Y99 is phosphorylated by c-Abl tyrosine kinase, resulting in the activation of CIMD. These results strongly support the hypothesis that CIMD is the cell death mechanism protecting cells from aneuploidy by inducing the death of cells prone to substantial chromosome missegregation (Figure 3).
Figure 3.  Pathway showing how and at what points the treatments used in our studies act on our proposed model. The anticancer drug 17-AAG, MT inhibitors, and cold shock induce defects in the kinetochore–MT attachment, which lead to CIMD in Bub1-deficient cells. (#1) This assay identifies inhibitors that are similar in action to these anticancer agents. (#2) Bub1 inhibitors (that can induce CIMD in mitotically dividing cancer cells in combination with 17-AAG and MT inhibitors) are likely to work synergistically as anticancer drugs with 17-AAG or MT inhibitors.

Katsumi Kitagawa Lab :: Current Lab Members

Rashid Abdulle - Lab Manager

Yohei Niikura - Post Doctoral Student

Hiroo Ogi - Post Doctoral Student

Risa Kitagawa - Post Doctoral Student / Scientific Lab Specialist

Nationwide Children's Hospital
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