Previously, CK2 was known to regulate cell division, but its specific role in cell division had not been established. In this study, we revealed a mitotic role for CK2 in the regulation of the kinetochore proteins Mif2 and Ndc10. Our results show that CK2 antagonizes SAC signaling. Loss of CK2 function activates SAC and results in a cell cycle arrest with short spindles. Notably, this defect is different from the defects observed in ipl1ts
cells, which do not display a uniform arrest phenotype. In ipl1ts
mutants, budding and cytokinesis are largely normal, while chromosomes fail to segregate properly (Biggins et al., 1999
; Kim et al., 1999
; Biggins and Murray, 2001
). A recent study reported that, in fission yeast, CK2 is required for the SAC through the regulation of Mad2 stability (Shimada et al., 2009
). When CK2 kinase activity was impaired in fission yeast, mitosis occurred despite a spindle defect, a phenotype that is different from what we observed in cka1
cells, where spindle elongation and chromosome segregation were blocked (). It is therefore possible that CK2 regulates mitosis through a different mechanism in fission yeast. Our data suggest that the absence of CK2 activity delays cell cycle progression in a manner dependent on SAC signaling.
We demonstrated that CK2 directly phosphorylates Mif2/CENP-C and Ndc10. CENP-C is a critical bridging component of kinetochores for both mitosis and meiosis. In budding yeast, Mif2 connects Cse4-containing nucleosomes and the Mtw1 complex (Westermann et al., 2003
). During meiosis, Mif2 was found to interact with the monopolin complex, and this interaction was found to be essential for sister-chromatid coorientation in meiosis I (Corbett et al., 2010
). In fission yeast, Cnp3/CENP-C functions as a scaffold for the recruitment of kinetochore proteins specific for mitosis and meiosis (Tanaka et al., 2009
). In Xenopus
egg extracts, CENP-C, together with CENP-N, binds directly to CENP-A nucleosomes and recruits other nonhistone CENPs (Carroll et al., 2010
). More recently, the CENP-C N-terminal region was found to interact directly with the Mis12 complex in Drosophila
and human cells. The high affinity between CENP-C and the Mis12 complex is sufficient to recruit the core outer kinetochore components and serves as the crucial link between the inner and outer kinetochore (Przewloka et al., 2011
; Screpanti et al., 2011
). Thus a number of CENP-C interactions may potentially be subjected to regulation.
Our study of Mif2 indicated that N-terminally clustered Ipl1 phosphorylation of Mif2 is required for Mif2 stability. In human cells, deletion of the N-terminus of CENP-C does not disrupt CENP-C binding to centromeres but instead results in massive accumulation of CENP-C diffusely in the nucleus. It has been hypothesized that the N-terminus of CENP-C serves as a destruction sequence that prevents the accumulation of CENP-C when it is mistargeted (Lanini and McKeon, 1995
). However, how the N-terminus of CENP-C signals for degradation is not clear. We found that Mif2, when not phosphorylated by Ipl1, is quickly degraded. Intriguingly, Ipl1-regulated Mif2 degradation is counterregulated by CK2. Given the important role of Mif2 in kinetochore function, its control by dual regulatory inputs is not surprising. It is now important to test whether human CENP-C is regulated in a similar manner.
Our mutagenesis study dissected the roles of Aurora B and CK2 kinases in Ndc10 regulation. Phospho-mimicking Ndc10 mutations at Ipl1 sites abolished its targeting to the anaphase spindle, but not its localization to kinetochores, whereas the phospho-mimicking mutant of Ndc10 at the CK2 site combined with mutations of the Ipl1 sites additionally affected Ndc10 localization to the kinetochore. The former finding is consistent with two previous observations. First, Ndc10-GFP localizes normally in ipl1–321
cells (Bouck and Bloom, 2005
). Similarly, in our study, substituting alanines for the Ndc10 residues phosphorylated by Ipl1 (ndc10(4A)
) does not affect Ndc10 localization. Second, the Ndc10-containing CBF3 complex displayed a lower microtubule-binding activity in glc7–10
extracts (Sassoon et al., 1999
). Glc7 is a phosphatase whose function counteracts that of Ipl1 kinase. Presumably, in glc7–10
cells, Ndc10 is hyperphosphorylated or constitutively phosphorylated, and therefore loses its ability to bind to microtubules. Our study showed that Ndc10(4D), a constitutively phosphorylated Ndc10, consistently failed to target to anaphase spindles. Localization of Ndc10 to the spindle depends on Ndc10 binding to Bir1, an essential component of the CPC, and the Ndc10-Bir1 interaction is subject to regulation of Cdk1 phosphorylation (Widlund et al., 2006
). In addition to regulation of Ndc10 targeting to spindles by Ipl1, Ndc10 is also sumoylated, and its sumoylation is required for its binding to Bir1 and, hence, Ndc10 spindle localization (Montpetit et al., 2006
). Whether Ipl1 phosphorylation of Ndc10 regulates its sumoylation remains to be tested. It appears that Ndc10 is finely tuned by multiple mechanisms to ensure its proper localization to spindles. In this study, we established the existence of another regulatory mechanism for Ndc10 by CK2. Ndc10 is a CK2 substrate, and CK2 phosphorylation of Ndc10, together with its phosphorylation by Aurora B, regulates Ndc10 localization to kinetochores. In total, our data establish an important, previously unappreciated role for CK2 in mitotic regulation, mediated at least in part by direct phosphorylation of the kinetochore proteins Mif2 and Ndc10.