HUMAN BUB1 - A PUTATIVE SPINDLE CHECKPOINT KINASE CLOSELY LINKED TO CELL-PROLIFERATION

Eukaryotic cells have evolved a mechanism that delays the onset of ana phase until chromosomes are properly positioned on the spindle. To und erstand the molecular basis of such surveillance mechanism in human ce lls, we have cloned a full-length cDNA encoding a putative mitotic che ckpoint kinase termed hBub1, Sequence comparison reveals that hBub1 is a structurally conserved protein, sharing 23% amino acid residue iden tity with BUB1 of budding yeast. In addition, the NH2-terminal portion (161 amino acids) of hBub1 shows a significant homology to yeast MADE , a protein also known to be involved in the mitotic checkpoint respon se pathway. Northern blot analyses show that the hBub1 mRNA level is a bundantly expressed in tissues or cells with a high mitotic index. Whe n Dami cells undergo terminal differentiation after treatment with pho rbol ester, hBub1 expression in this cell line is down-regulated rapid ly. The hBub1 protein level is low in G1 and remains relatively consta nt in S, G(2), and M phases. Immunofluorescence analysis shows that hB ub1 protein colocalizes with a centromere-kinetochore antigen CREST in interphase, mitotic prophase, and nocodazole-treated cells. Antibody electroporation experiments show that hBub1 is an important component of the spindle checkpoint pathway. Furthermore, fluorescence in situ h ybridization analysis maps the hBub1 gene to chromosome 2q12-13, Our s tudies suggest that hBub1 expression is restricted to proliferating ce lls and appears to be involved in regulating cell cycle progression, T he molecular cloning of hBub1 cDNA will facilitate the study of its ro le in spindle checkpoint control as well as its potential role in cert ain genetic disorders.