The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage l Show more
The kinetochore is a supramolecular structure essential for microtubule attachment and the mitotic checkpoint. Human blinkin/human Spc105 (hSpc105)/hKNL1 was identified originally as a mixed-lineage leukemia (MLL) fusion partner and later as a kinetochore component. Blinkin directly binds to several structural and regulatory proteins, but the precise binding sites have not been defined. Here, we report distinct and essential binding domains for Bub1 and BubR1 (here designated Bubs) at the N terminus of blinkin and for Zwint-1 and hMis14/hNsl1 at the C terminus. The minimal binding sites for Bub1 and BubR1 are separate but contain a consensus KI motif, KI(D/N)XXXF(L/I)XXLK. RNA interference (RNAi)-mediated replacement with mutant blinkin reveals that the Bubs-binding domain is functionally important for chromosome alignment and segregation. We also provide evidence that hMis14 mediates hNdc80 binding to blinkin at the kinetochore. The C-terminal fragment of blinkin locates at kinetochores in a dominant-negative fashion by displacing endogenous blinkin from kinetochores. This negative dominance is relieved by mutations of the hMis14 binding PPSS motif on the C terminus of blinkin or by fusion of the N sequence that binds to Bub1 and BubR1. Taken together, these results indicate that blinkin functions to connect Bub1 and BubR1 with the hMis12, Ndc80, and Zwint-1 complexes, and disruption of this connection may lead to tumorigenesis. Show less
Centromeric DNA forms two structures on the mitotic chromosome: the kinetochore, which interacts with kinetochore microtubules, and the inner centromere, which connects sister kinetochores. The assemb Show more
Centromeric DNA forms two structures on the mitotic chromosome: the kinetochore, which interacts with kinetochore microtubules, and the inner centromere, which connects sister kinetochores. The assembly of the inner centromere is poorly understood. In this study, we show that the human Mis14 (hMis14; also called hNsl1 and DC8) subunit of the heterotetrameric hMis12 complex is involved in inner centromere architecture through a direct interaction with HP1 (heterochromatin protein 1), mediated via a PXVXL motif and a chromoshadow domain. We present evidence that the mitotic function of hMis14 and HP1 requires their functional association at interphase. Alterations in the hMis14 interaction with HP1 disrupt the inner centromere, characterized by the absence of hSgo1 (Shugoshin-like 1) and aurora B. The assembly of HP1 in the inner centromere and the localization of hMis14 at the kinetochore are mutually dependent in human chromosomes. hMis14, which contains a tripartite-binding domain for HP1 and two other kinetochore proteins, hMis13 and blinkin, is a cornerstone for the assembly of the inner centromere and kinetochore. Show less