These studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.

Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. Here, we demonstrate that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, we identify S676 as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, we show that this site is phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions.[Keywords: BubR1; tension; phosphorylation; spindle assembly checkpoint] Supplemental material is available at http://www.genesdev.org. The BubR1 protein was first identified as an essential and evolutionarily conserved component of the spindle assembly checkpoint (SAC). During mitosis, this checkpoint inhibits the anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, so that chromosome segregation is triggered only after bipolar attachment to the spindle microtubules (MTs) has been achieved (Musacchio and Hardwick 2002;Bharadwaj and Yu 2004;Taylor et al. 2004). SAC dysfunction and the consequential premature APC/C activation can result in aneuploidy, a hallmark of human solid tumors (Kops et al. 2005). Throughout prophase and prometaphase, SAC components including BubR1 are enriched at kinetochores (KTs), complex proteinaceous structures assembled on centromeric DNA, where a "wait anaphase" signal is thought to be generated. KTs represent the major point of contact between mitotic spindle MTs and chromosomes (Maiato et al. 2004;Chan et al. 2005). Several proteins and multiprotein complexes, including BubR1 ( In addition, KT-associated proteins contribute to maintain SAC activity until the last chromosome has undergone bipolar attachment. Although the exact nature of SAC signaling is not well understood, biochemical and genetic evidence indicate that the source of this signal is at the centromere/KT, where tension generated upon amphitelic attachment is sensed (Pinsky and Biggins 2005;Baumann et al. 2007). How such a mechanical property is transduced into a biochemical signaling cascade remains unclear, but there is strong evidence that phosphorylation plays a key role (Gorbsky 1995).Along with APC/C-mediated proteolyis, M-phase progression is controlled through phosphorylation by mitotic kinases. In addition to the key regulator cyclindependent kinase 1 (Cdk1), Polo-like ...

The survival of eukaryotes depends on the accurate coordination of mitosis with cytokinesis. Key for the coordination of both processes is the chromosomal passenger complex (CPC) comprising Aurora-B, INCENP, survivin, and borealin. The translocation of the CPC from centromeres to the spindle midzone, a structure composed of antiparallel microtubules, at anaphase onset is critical for the completion of cytokinesis. In mammalian cells, the mitotic kinesin Mklp2 is essential for recruitment of the CPC to the spindle midzone. However, the mechanism regulating the binding of Mklp2 to microtubules has remained unknown. Here, we demonstrate that Mklp2 and the CPC mutually depend on each other for midzone localization; i.e., Mklp2 is mislocalized in INCENP-RNAi cells and vice versa. Remarkably, INCENP is required for localization of Mklp2 to the ends of stable microtubules in cells with low Cdk1 activity. In vitro assays revealed that the association between the CPC and Mklp2 is negatively regulated by Cdk1. Collectively, our data suggest that anaphase onset triggers the association between the CPC and Mklp2 and that this association targets the CPC-Mklp2 complex to the ends of stable microtubules in the spindle midzone.

The guanine nucleotide-exchange factor (GEF) Ect2 is essential for cytokinesis. Here we studied the subcellular localization of Ect2 and examined the consequences of either depleting or overexpressing Ect2 in human cells. We show that in mitotic cells Ect2 localizes to the central spindle and to the cell cortex. The latter association is mediated through a PH domain in Ect2 and central spindle localization requires the MKlp1-MgcRacGAP and MKlp2–Aurora-B complexes. Ect2 directly interacts with MKlp1-MgcRacGAP through its BRCT domain, whereas MKlp2–Aurora-B probably exerts a regulatory role in Ect2 central spindle targeting. Depletion of Ect2 impaired cleavage furrow formation and RhoA and Citron kinase failed to accumulate at the cleavage furrow. Ect2 displacement from the central spindle revealed that physiological levels of this protein in this location are not crucial for RhoA activation and cytokinesis. In cells overexpressing appropriate N-terminal Ect2 fragments, RhoA and Citron kinase localized to the cleavage furrow and ingression occurred, but abscission failed. This failure could be correlated with the persistence of these fragments at structures surrounding the midbody, suggesting that abscission requires the displacement of Ect2 from the contractile ring and its re-import into the nucleus.