CHARACTERIZATION OF THE KINETOCHORE BINDING DOMAIN OF CENP-E REVEALS INTERACTIONS WITH THE KINETOCHORE PROTEINS CENP-F AND HBUBR1

We have identified a 350-amino acid domain in the kinetochore motor CE NP-E that specifies kinetochore binding in mitosis but not during inte rphase. The kinetochore binding domain was used in a yeast two-hybrid screen to isolate interacting proteins that included the kinetochore p roteins CENP-E, CENP-F, and hBUBR1, a BUB1-related kinase that was fou nd to be mutated in some colorectal carcinomas (Cahill, D.P., C. Lenga uer, J. Yu, G.J. Riggins, J.K. Wilson, S.D. Markowitz, K.W. Kinzler, a nd B. Vogelstein. 1998. Nature. 392:300-303). CENP-F, hBUBR1, and CENP -E assembled onto kinetochores in sequential order during late stages of the cell cycle. These proteins therefore define discrete steps alon g the kinetochore assembly pathway. Kinetochores of unaligned chromoso me exhibited stronger hBUBR1 and CENP-E staining than those of aligned chromosomes. CENP-E and hBUBR1 remain colocalized at kinetochores unt il mid-anaphase when hBUBR1 localized to portions of the spindle midzo ne that did not overlap with CENP-E. As CENP-E and hBUBR1 can coimmuno precipitate with each other from HeLa cells, they may function as a mo tor-kinase complex at kinetochores. However, the complex distribution pattern of hBUBR1 suggests that it may regulate multiple functions tha t include the kinetochore and the spindle midzone.