The Mitotic Checkpoint Kinase NEK2A Regulates Kinetochore Microtubule Attachment Stability

Oncogene (2008) 27, 4107–4114 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE The mitotic checkpoint kinase NEK2A regulates kinetochore microtubule attachment stability JDu1,6, X Cai1,2,6, J Yao1, X Ding2,3,QWu1,2, S Pei1, K Jiang1,2, Y Zhang1, W Wang3, Y Shi1, Y Lai1, J Shen1, M Teng1, H Huang4, Q Fei5, ES Reddy2, J Zhu5, C Jin1 and X Yao1,2 1Hefei National Laboratory for Physical Sciences at Micro-scale, University of Science and Technology of China, Hefei, China; 2Cancer Biology Program, Morehouse School of Medicine, Atlanta, GA, USA; 3Department of Medicine, Beijing University of Chinese Medicine, 4Department of Hematology, the 1st Affiliated Hospital, Zhejiang University, Hongzhou, China and 5Cancer Epigenetics Program, Shanghai Cancer Institute, Shanghai Jiaotong University, Shanghai, China Loss or gain of whole chromosome, the form of Introduction chromosome instability commonly associated with cancers is thought to arise from aberrant chromosome segregation Chromosomal instability (CIN) has been recognized as a during cell division. Chromosome segregation in mitosis is hallmark of human cancer and is caused by continuous orchestrated by the interaction of kinetochores with chromosome missegregation during cell division. Proper spindle microtubules. Our studies showthat NEK2A is a chromosome segregation requires a faithful physical link kinetochore-associated protein kinase essential for faith- between spindle microtubules and centromeric DNA via ful chromosome segregation. However, it was unclear how a protein supercomplex called kinetochore (Cleveland NEK2A ensures accurate chromosome segregation in et al., 2003). In addition to providing a physical link mitosis. Here we show that NEK2A-mediated Hec1 between chromosomes and spindle microtubules, the (highly expressed in cancer) phosphorylation is essential kinetochore has an active function in orchestrating for faithful kinetochore microtubule attachments in chromosome movements through microtubule motors mitosis. Using phospho-specific antibody, our studies show and correcting spindle microtubule attachment to that NEK2A phosphorylates Hec1 at Ser165 during kinetochore via checkpoint sensors located at or near mitosis. Although such phosphorylation is not required it (Yao et al., 2000; Ke et al., 2003; Fang and Fang for assembly of Hec1 to the kinetochore, expression of 2007). Recent studies show that CIN results from high non-phosphorylatable mutant Hec1S165 perturbed chromo- incidence of aberrant kinetochore microtubule attach- some congression and resulted in a dramatic increase in ments such as syntelic attachment (Storchova et al., microtubule attachment errors, including syntelic and 2006), which promotes tumorigenesis in p53-null cells monotelic attachments. Our in vitro reconstitution experi- (Fujiwara et al., 2005). An outstanding question is how ment demonstrated that Hec1 binds to microtubule in low kinetochore microtubule attachment errors are gener- affinity and phosphorylation by NEK2A, which prevents ated and corrected during cell division. aberrant kinetochore-microtubule connections in vivo, The mitotic checkpoint is one pathway that prevents increases the affinity of the Ndc80 complex for micro- segregation errors by controlling the time of anaphase until tubules in vitro. Thus, our studies illustrate a novel all chromosomes achieve proper attachments to the spindle regulatory mechanism in which NEK2A kinase operates a microtubule (Musacchio and Salmon 2007). Another faithful chromosome attachment to spindle microtubule, process to prevent errors is to stabilize and destabilize which prevents chromosome instability during cell division. the kinetochore-microtubule connection (Cleveland et al., Oncogene (2008) 27, 4107–4114; doi:10.1038/onc.2008.34; 2003). Mounting evidence demonstrates that the Ndc80 published online 25 February 2008 complex, comprised of Hec1 (highly expressed in cancer), Nuf2, Spc24 and Spc25, constitutes an important kine- Keywords: chromosome instability; mitosis; kinetochore- tochore-microtubule connection in vivo and in vitro microtubule attachment; Hec1; NEK2A; phosphorylation (Cheeseman et al., 2006; DeLuca et al., 2006). Hec1 was initially identified as an Rb-binding protein and subse- quently demonstrated as an evolutionarily conserved kinetochore protein (Durfee et al., 1993). Our recent study revealed that Ndc80 complex provides a link Correspondence: Dr X Ding, Department of Medicine Beijing between mitotic motor CENP-E and kinetochore (Liu University of Chinese Medicine, Beijing, China; E-mail: et al., 2007). [email protected] or Dr C Jin, Hefei National Laboratory, Hefei, Mitosis is orchestrated by kinase-signaling cascades China; E-mail: [email protected] or Dr X Yao, Cellular Dynamics, that govern the spatiotemporal control of accurate Hefei, China; E-mail: [email protected] 6These authors contributed equally to this work. chromosome segregation. The key switch for the onset Received 30 August 2007; revised 2 January 2008; accepted 6 January of mitosis is the archetypal cyclin-dependent kinase, 2008; published online 25 February 2008 Cdc2. Besides Cdc2, there are three protein serine/ NEK2A regulates chromosome stability JDuet al 4108 threonine kinase families, the Polo kinases, Aurora phosphorylation-related as l-phosphatase (PPase) treat- kinases and the never in mitosis A-related kinases ment caused Hec1 to shift to the faster migrating form, (NEK) (Nigg, 2001; Fry 2002; Ke et al, 2003), which indicating that Hec1 is phosphorylated in mitosis. In participates in mitotic progression. Aneuploidy and contrast, GFP-Hec1S165A from mitotic cells displays no chromosome instability are two of the most common mobility shift, suggesting that Ser165 is a major abnormalities in cancer cells, which arise from aberrant phosphorylation site in mitosis. The phosphorylation of chromosome segregation during mitosis. Emerging GFP-Hec1 in mitosis was also confirmed with phospho- evidence suggests that aberrant regulation of mitotic Ser165 antibody (Figure 1d). Thus, we conclude that kinases including NEK2 is implicated in solid tumor Hec1S165 is phosphorylated by NEK2A in mitosis. formation (Hayward and Fry 2006). However, it has remained elusive as whether and how perturbation of NEK2A signaling results in aberrant kinetochore Phosphorylation of Hec1 is essential for faithful plasticity and chromosome instability. chromosome segregation We have recently showed that NEK2A kinase activity To examine whether NEK2A-mediated phosphoryla- is essential for faithful chromosome segregation (Chen tion is essential for Hec1 localization to the kinetochore, et al., 2002; Lou et al, 2004; Fu et al, 2007). To elucidate plasmids expressing GFP-tagged wild-type, non-phos- the mechanistic insight underlying NEK2A regulation in phorylatable (Hec1S165A) and phospho-mimicking Hec1 mitosis, we examined the function of NEK2-mediated (Hec1S165E) were transfected into HeLa cells, and fusion Hec1 phosphorylation in microtubule-kinetochore at- proteins were scored for their kinetochore localization tachment. Our study revealed that NEK2A-mediated relative to CENP-H under fluorescence microscopy. As phosphorylation of Hec1 is essential for a stable shown in Figure 2A, the kinetochore distribution of kinetochore-microtubule connection, which prevents GFP-Hec1 proteins (wild type, Hec1S165A and Hec1S165E) CIN during cell division. was readily observed as the merged images show the co- distribution of GFP-Hec1 proteins with CENP-H (Figures 2A, d; 2B, d0 and 2C, d00), suggesting that Results exogenous Hec1 proteins are able to assemble onto the kinetochore regardless of the phosphorylation status of Ser165 of Hec1 is phosphorylated by NEK2A in mitosis Ser165. These data suggest that Hec1S165 phosphorylation Our recent studies revealed that NEK2A is a kineto- is not required for kinetochore targeting of Hec1, chore-associated kinase active in spindle checkpoint consistent with the architect of Ndc80 complex resolved signaling (Lou et al, 2004; Fu et al, 2007). To delineate by structural biology (Ciferri et al, 2007). the regulatory function of NEK2A in kinetochore While the distribution pattern of all three GFP-Hec1 protein–protein interactions, we conducted a quick proteins (wild type, S165A and S165E) was similar search for NEK2A-binding proteins using a ‘high- (Figures 2A, b; B, b0 and C, b00), the GFP-Hec1S165A- content’ far-western assay and had identified an expressing cells contain more lagging chromosomes that NEK2A-Sgo1 interaction (Fu et al, 2007). This assay were readily apparent (Figure 2B, b0). As the kineto- validated that NEK2A binds to Hec1 (Supplementary chore position relative to the pole has been previously Figure 1). proposed as an accurate reporter for judging chromo- If Hec1S165 is a cognate substrate of NEK2A (Chen some misalignment (Lampson and Kapoor, 2005), we et al, 2002), suppression of NEK2A would abolish the measured this distance in >100 kinetochore pairs in phosphorylation of Hec1S165 in vivo. As shown in which both kinetochores were in the same focal plane, in Figure 1a, while siRNA treatment has suppressed both wild-type, S165A and S165EHec1-expressing cells. NEK2A protein accumulation (lower panel) it did not Kinetochore positions were surveyed by the distance alter Hec1 levels (upper panel). Probing the same blot from the nearest pole along the pole–pole axis, and with a phospho-Hec1S165-specific antibody (Chen et al, normalized for pole–pole distance (Figure 2D). Our

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