BUBR1 phosphorylation is regulated during mitotic checkpoint activation

Eukaryotic cells have evolved a mechanism that delays the progression of mi tosis until condensed chromosomes are properly positioned on the mitotic sp indle. To understand the molecular basis of such monitoring mechanism in hu man cells, we have been studying genes that regulate the mitotic checkpoint . Our early studies have led to the cloning of a full-length cDNA encoding MAD3-like protein (also termed BUBR1/MAD3/SSK1). Dot blot analyses show tha t BUBR1 mRNA is expressed in tissues with a high mitotic index but not in d ifferentiated tissues. Western blot analyses show that in asynchronous cell s, BUBR1 protein primarily exhibits a molecular mass of 120 kDa, and its ex pression is detected in most cell lines examined. In addition, BUBR1 is pre sent during various stages of the cell cycle. As cells enter later S and G( 2), BUBR1 levels are increased significantly. Nocodazole-arrested mitotic c ells obtained by mechanical shake-off contain BUBR1 antigen with a slower m obility on denaturing SDS gels. Phosphatase treatment restores the slowly m igrating band to the interphase state, indicating that the slow mobility of the BUBR1 antigen is attributable to phosphorylation. Furthermore, purifie d recombinant His6-BUBR1 is capable of autophosphorylation. Our studies ind icate that BUBR1 phosphorylation status is regulated during spindle disrupt ion. Considering its strong homology to BUB1 protein kinase, BUBR1 may also play an important role in mitotic checkpoint control by phosphorylation of a critical cellular component(s) of the mitotic checkpoint pathway.