Oncogene (2006) 25, 6901–6914 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Hec1 sequentially recruits Zwint-1 and ZW10 to kinetochores for faithful chromosome segregation and spindle checkpoint control Y-T Lin1,2, Y Chen2,GWu1 and W-H Lee1 1Department of Biological Chemistry, University of California, Irvine, CA, USA and 2Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX, USA Faithful chromosome segregation is essential for main- Introduction taining the genomic integrity, which requires coordination among chromosomes, kinetochores, centrosomes and Accurate chromosome segregation is essential for spindles during mitosis. Previously, we discovered a novel maintaining the integrity of genome. In eukaryotic cells, coiled-coil protein, highly expressed in cancer 1 (Hec1), this process is precisely controlled by coordination which is indispensable for this process. However, the among the centrosomes, spindles, kinetochores and precise underlying mechanism remains unclear. Here, we chromosomes during the M phase progression. If the show that Hec1 directly interacts with human ZW10 process goes awry, chromosome segregation in indivi- interacting protein (Zwint-1), a binding partner of Zeste dual cellswill proceed with poor fidelity and the cellscan White 10 (ZW10) that is required for chromosome gain or lose the whole chromosomes. Such cells will motility and spindle checkpoint control. In mitotic cells, either die or survive with aneuploidy. Surviving cells will Hec1 transiently forms complexes with Zwint-1 and ultimately proliferate without a proper regulation and ZW10 in a temporal and spatial manner. Although the progressively accumulate more gross chromosomal three proteins have variable cell cycle-dependent expres- abnormalitiesto form a tumor (Zhou et al., 1998; sion profiles, they can only be co-immunoprecipitated Dobles et al., 2000; Fodde et al., 2001; Kaplan et al., during M phase. Immunofluorescent study showed that 2001; Michel et al., 2001; Cleveland et al., 2003). Indeed, Hec1 and Zwint-1 co-localize at kinetochores beginning at most solid tumors are aneuploidy and have chromosome prophase and that ZW10 joins them later at prometa- instability (CIN) phenotype (Lengauer et al., 1997; phase. Depletion of Hec1 impairs the recruitment of both Takahashi et al., 1999). Understanding the molecular Zwint-1 and ZW10 to kinetochores, while depletion of mechanism involved in accurate chromosome segrega- Zwint-1 abrogates the kinetochore localization of ZW10 tion will provide an insight into the generation of the but not Hec1. The results suggest that the localization of chromosome instability phenotype in tumor cells. Hec1 at kinetochores is required for the sequential Kinetochoresare composedof multiple proteins recruitment of Zwint-1 and ZW10. Disrupting this associated with the centromeric heterochromatin recruitment by inhibiting the expression of Hec1 or DNA. They physically link chromosomes to the spindle Zwint-1 causes chromosome missegregation, spindle apparatusto mediate a poleward chromosome motion. checkpoint failure, and eventually cell death upon A fully functional kinetochore requiresa complicated cytokinesis. Taken together, these results, at least in yet highly organized assembly of distinct subcomplexes part, provide a molecular basis to explain how Hec1 plays (Cheeseman et al., 2002b). The kinetochore proteins a crucial role for spindle checkpoint control and faithful include intrinsic components such as CENP-A, -B, and chromosome segregation. -C, which bind to the centromere throughout the cell Oncogene (2006) 25, 6901–6914. doi:10.1038/sj.onc.1209687; cycle (Earnshaw and Rothfield, 1985; Earnshaw et al., published online 29 May 2006 1987; Palmer et al., 1987; Craig et al., 1999) and transient components, which come to kinetochores Keywords: mitosis; kinetochores; spindle checkpoint; transiently during different mitotic phases (Craig et al., chromosome segregation and microtubule 1999; Cleveland et al., 2003). Kinetochoresplay a key role in establishing bipolar-microtubule attachment, which depends on those proteins involved in stable kinetochore microtubules (kMTs) formation, such as highly expressed in cancer 1 (Hec1), Nuf2, Spc24 and Spc25 (Desai et al., 2003; McCleland et al., 2003; Bharadwaj et al., 2004; Deluca et al., 2005). The lack of Correspondence: Dr W-H Lee, Department of Biological Chemistry, bipolar-microtubule attachment or tension on kineto- University of California, Irvine, 124 Sprague Hall, Irvine, CA 92697- chorescan activate the spindle checkpoint to delay or 4037, USA. E-mail: [email protected] arrest cell cycle progression until every chromosome has Received 14 December 2005; revised 3 April 2006; accepted 12 April an appropriate bipolar attachment (Li and Nicklas, 2006; published online 29 May 2006 1995; Rieder et al., 1995). A group of evolutionarily Hec1 recruits Zwint-1 and ZW10 to kinetochores Y-T Lin et al 6902 conserved proteins, including Bub1, Bub3, BubR1, Nuf2 complex serves as a scaffold at the kinetochore for Mad1, Mad2 and Mps1 that are known to be essential binding of these proteins (Martin-Lluesma et al., 2002; for spindle checkpoint functions, also localize at McCleland et al., 2003; Meraldi et al., 2004). kinetochores(Hoyt et al., 1991; Li and Murray, 1991; However, how Hec1 playsa critical role at kineto- Weiss and Winey, 1996; Chan et al., 1999; Cleveland chores in mediating faithful chromosome segregation et al., 2003). Furthermore, higher eukaryotic cellshave remainsunclear. Interestingly,one of Hec1 interacting additional spindle checkpoint proteins, such as human proteins, Zwint-1, isolated from the original yeast two- ZW10 interacting protein (Zwint-1), Zeste White 10 hybrid screening, was subsequently shown to interact (ZW10) and Rod, to ensure high fidelity of chromosome with ZW10, the human homologue of Drosophila Zeste segregation (Basto et al., 2000; Chan et al., 2000; White 10 (Starr et al., 2000; Wang et al., 2004). Zwint-1 Williams et al., 2003; Wang et al., 2004). appearsto localize at kinetochoresduring prophase, Hec1 wasoriginally identified through itsinteraction followed by the relocation of ZW10, which either with retinoblastoma protein (Rb) and it localizes at recruitsdynactin and dynein to facilitate chromosome kinetochores(Chen et al., 1997a). Inactivation of Hec1 movement or regulatesspindle checkpoint (Starr et al., by microinjection with a specific antibody leads to a 1998; Chan et al., 2000; Savoian et al., 2000; Sharp et al., severe chromosome missegregation and, consequently, 2000). Thus, it is likely that Hec1 serves as a recruiter at the death of daughter cellsin the following cycle (Chen kinetochores for facilitating chromosome segregation et al., 1997a). Hec1 isconservedevolutionarily from and monitoring spindle checkpoint. In this report, we yeast to human (Zheng et al., 1999; He et al., 2001). The show that Hec1 interacts directly with Zwint-1, which in Saccharomyces cerevisiae homologue of Hec1, also turn bindsZW10. Furthermore, Hec1 isrequired for the known asNdc80/TID3/YIO4, wasinitially character- recruitmentsof Zwint-1 and ZW10 to kinetochores. ized as a protein associated with the spindle pole body Disruption of Zwint-1 and ZW10 recruitment results in and later found to be a kinetochore protein (Wigge chromosome missegregation, spindle checkpoint failure et al., 1998; Zheng et al., 1999). The scHec1 null and subsequently cell death upon cytokinesis. These mutation is lethal but can be rescued by the expression results suggest that Hec1 may participate in the dynamic of human Hec1 (hsHec1) (Zheng et al., 1999). Inactiva- recruitmentsfor capturing key regulatory proteinsonto tion of hsHec1 by a temperature sensitive mutation in kinetochores for faithful chromosome segregation. budding yeast leads to a chromosome missegregation and cell death at the non-permissive temperature; the phenotypes observed in these yeast cells are very similar Results to those observed in mammalian cells injected with neutralizing anti-Hec1 antibodies(Chen et al., 1997a; Hec1 binds ZW10 through Zwint-1 Zheng et al., 1999). These results strongly suggested that Using the coiled-coil region of Hec1 as bait, we Hec1 is an essential protein for proper chromosome previously identified a panel of Hec1-interacting pro- segregation. teins through a yeast two-hybrid screening (Zheng et al., How Hec1 executesitsfunction in chromosome 1999). One such protein was Zwint-1, a known human segregation has been an interesting subject to pursue. ZW10 binding partner (Chan et al., 2000). To examine AsHec1 containsthree coiled-coil domainsthat are the molecular domainsmediating the Zwint-1/Hec1 frequently involved in protein–protein interaction, we interaction, we employed two complementary ap- have used yeast two-hybrid screening and identified proaches. We first performed yeast two-hybrid assays several Hec1-interacting proteins, including structural by using various coiled-coil containing Hec1 derivatives components of mitotic chromosomes, Smc1 and Smc2, fused with the Gal4 DNA-binding domain (DBD) and which have important roles in chromosome cohesion full-length Zwint-1 fused with the Gal4 transactivation and condensation; three subunits of the 26S proteasome domain (TA). Fusion protein containing the first (aa including MSS1 (CIM5/subunit 7), p45/Trip1 (Sug1/ 251–431) or second (aa 361–547) coiled-coil region of CIM3/subunit 8), and p44.5 (subunit 9) and Nek2A, a Hec1, wassufficient