Chromosome segregation in mitosis is orchestrated by protein kinase signaling cascades. A biochemical cascade named spindle checkpoint ensures the spatial and temporal order of chromosome segregation during mitosis. Here we report that spindle checkpoint protein MAD1 interacts with NEK2A, a human orthologue of the Aspergillus nidulans NIMA kinase. MAD1 interacts with NEK2A in vitro and in vivo via a leucine zipper-containing domain located at the C terminus of MAD1. Like MAD1, NEK2A is localized to HeLa cell kinetochore of mitotic cells. Elimination of NEK2A by small interfering RNA does not arrest cells in mitosis but causes aberrant premature chromosome segregation. NEK2A is required for MAD2 but not MAD1, BUB1, and HEC1 to associate with kinetochores. These NEK2A-eliminated or -suppressed cells display a chromosome bridge phenotype with sister chromatid inter-connected. Moreover, loss of NEK2A impairs mitotic checkpoint signaling in response to spindle damage by nocodazole, which affected mitotic escape and led to generation of cells with multiple nuclei. Our data demonstrate that NEK2A is a kinetochore-associated protein kinase essential for faithful chromosome segregation. We hypothesize that NEK2A links MAD2 molecular dynamics to spindle checkpoint signaling.Chromosome movements during mitosis are governed by the interaction of spindle microtubules with a specialized chromosome domain located within the centromere. This specialized region, called the kinetochore (1, 2), is the site for spindle microtubule-centromere association. In addition to providing a physical link between chromosomes and spindle microtubules, the kinetochore has an active function in chromosomal segregation through microtubule motors and spindle checkpoint sensors located at or near it (3-5).Several lines of evidence have implicated the kinetochore in generation of a diffusible checkpoint signal that can block cell cycle progression into anaphase until all kinetochores have successfully attached to spindle microtubules. Delayed attachment of one or more chromosomes to the spindle is correlated with a corresponding delay in the onset of anaphase. For mutants that fail to arrest the cell cycle in mitosis after disassembly of microtubules in budding yeast, genetic screen has identified three MAD (mitotic arrest deficiency) and three BUB (budding uninhibited by benomyl) genes (7). Vertebrate homologues of MAD1 (8), MAD2 (9 -10), BUB3 (11-12), BUB1, and BUBR1 (13-15) are spindle checkpoint components transiently associated with kinetochore. Expression of the kinetochore binding domain of murine BUB1 (13) or injection of antibodies against BUBR1 (15) results in premature onset of anaphase, presumably by replacement of the endogenous proteins at kinetochores. Collectively, these data indicate that binding of these spindle checkpoint components at the kinetochores may generate a signal in response to spindle defects and/or aberrant kinetochore protein-protein interactions.Mitosis is orchestrated by signaling cascades that coordinate mitotic p...

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubule and the kinetochore. Our recent ultrastructural studies demonstrated a dynamic distribution of TTK, from the kinetochore to the centrosome, as cell enters into anaphase. Here, we show that a centrosomal protein TACC2 is phosphorylated in mitosis by TTK signaling pathway. TACC2 was pulled down by wild type TTK but not kinase death mutant, suggesting the potential phosphorylation-mediated interaction between these two proteins. Our immunofluorescence studies revealed that both TTK and TACC2 are located to the centrosome. Interestingly, expression of kinase death mutant of TTK eliminated the centrosomal localization of TACC2 but not other centrosomal proteins such as c-tubulin and NuMA, a phenotype seen in TTKdepleted cells. In these centrosomal TACC2-liberated cells, chromosomes were lagging and mis-aligned. In addition, the distance between two centrosomes was markedly reduced, suggesting that centrosomal TACC2 is required for mitotic spindle maintenance. The inter-relationship between TTK and TACC2 established here provides new avenue to study centrosome and spindle dynamics underlying cell divisional control.