| Research Abstract |
Our group previously showed that a GTPase activating protein, MgcRacGAP, plays critical roles both in cellular differentiation and cytokinesis (Kawashima et al.,Blood,2000:Hirose et al.,J.Biol.Chem.,2001:Minoshima et al.,Dev.Cell,2003:Tonozuka et al.,Blood 2004:Kawashima et al.,J.Cell.Biol.,2006). In the present study, we identified a protein that interacts with MgcRacGAP by yeast two-hybrid screening. Because this protein localizes to the centromere throughout the cell cycle, we named it CENP-50 (CENtromere Protein, molecular weight 50 KDa). Recently, CENP-50 was also described as KLIP1/MLF1IP. We identified CENP-50 as a novel kinetochore component. We found that CENP-50 is a constitutive component of the centromere that colocalizes with CENP-A and CENP-H throughout the cell cycle in vertebrate cells. To determine the precise role of CENP-50, we examined its role in centromere function by generating a loss-offunction mutant in the chicken DT40 cell line. The CENP-50 knockout was not l
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ethal ; however, the growth rate of cells with this mutation was slower than that of wild-type cells. We observed that the time for CENP-50-deficient cells to complete mitosis was longer than that for wild-type cells. Centromeric localization of CENP-50 was abolished in both CENP-H- and CENP-I-deficient cells. Co-immunoprecipitation experiments revealed that CENP-50 interacted with the CENP-H/CENP-I complex in chicken DT40 cells. We observed severe mitotic defects in CENP-50-deficient cells with apparent premature sister chromatid separation when the mitotic checkpoint was activated, indicating that CENP-50 is required for recovery from spindle damage. Observed premature sister chromatid separation in CENP-50-deficient cells suggested that CENP-50 is required for maintenance of sister chromatid adhesion (Minoshima et al.,Mol.Cell.Biol.,2005). In addition, we have recently identified the region of CENP-50 required for localization to the centromere, and did functional analysis of this region (manuscript in preparation). Less
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