NEK2A Interacts with MAD1 and Possibly Functions as a Novel Integrator of the Spindle Checkpoint Signaling

Abstract

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...

The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability

“…Our recent study has confirmed the critical role of NEK2A in chromosome segregation in mammalian cells (Lou et al, 2004;Fu et al, 2007). However, NEK2A is not required for assembly of Hec1 to kinetochore (Figures 2A-C), suggesting that other regulatory mechanisms exist to govern the assembly of Ndc80 complex.…”

“…Our recent studies revealed that NEK2A is a kinetochore-associated kinase active in spindle checkpoint signaling (Lou et al, 2004;Fu et al, 2007). To delineate the regulatory function of NEK2A in kinetochore protein-protein interactions, we conducted a quick search for NEK2A-binding proteins using a 'highcontent' far-western assay and had identified an NEK2A-Sgo1 interaction (Fu et al, 2007).…”

“…We have recently showed that NEK2A kinase activity is essential for faithful chromosome segregation (Chen et al, 2002;Lou et al, 2004;Fu et al, 2007). To elucidate the mechanistic insight underlying NEK2A regulation in mitosis, we examined the function of NEK2-mediated Hec1 phosphorylation in microtubule-kinetochore attachment.…”

Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells

“…Our data show that Nek2 knockdown cells display multipolar centrosomes and aberrant mitotic figures. Nek2 is known to phosphorylate mitotic checkpoint proteins such as Mad2 and Cdc20 (34), and to interact with the spindle assembly checkpoint protein Mad1 (31). Nek2 also contributes to the microtubule reorganization necessary for the G2/M transition by phosphorylating several centrosomal substrates, including c-Nap1, rootletin, PP1, Nlp, centrobin and HMGA2 (28,29).…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…It remains unclear whether the SAC component, Mps1p, also plays a role in centrosome duplication in higher eukaryotes [Fischer et al, 2004;Fisk et al, 2003;Liu et al, 2003;Stucke et al, 2004;Stucke et al, 2002] as it does in budding [Winey et al, 1991] but not in fission yeast [He et al, 1998]. The mammalian Ndc80p complex [McCleland et al, 2003], the ROD and ZW10 kinetochore proteins [Basto et al, 2000;Chan et al, 2000], the outer kinetochore protein Zwint-1 [Obuse et al, 2004;Wang et al, 2004a], and the kinetochore-associated NEK2A protein [Lou et al, 2004] are also required for the spindle checkpoint, indicating that the kinetochore's role in SAC signaling is evolutionarily conserved, although no ROD, ZW10, or Zwint-1 homologues have been identified in yeast. The mammalian survivin/Aurora B complex regulates BubR1p and Mad2p localization to kinetochores [Lens et al, 2003] and, like the budding yeast Ipl1p/Aurora, is required for SAC function when insufficient tension is applied across sister kinetochores [Carvalho et al, 2003;Hauf et al, 2003;Lens et al, 2003].…”