B kinase activity, we depleted endogenous Ska1 in HeLa cells stably expressing mutant versions of RNAi-resistant GFP-Ska1 lacking either a basic patch in the MT-binding surface of Ska1 or the entire MT-binding domain. These mutants both make the full Ska complex MT-binding deficient in vitro without affecting complex formation; in intact cells, they fail to localize to spindle MTs but not KTs. Although Ska1-depleted cells expressing wild-type Ska1 showed Hec1-pS44, KNL1-pS24, MCAK KT, H3-pS10, and Aurora B pT232 levels comparable to those of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19835880 control cells, expression of Ska1R155A, R236A, R245A and Ska1MTBD failed to restore wild-type levels of these Aurora B activity markers. These results indicate that the Ska complex regulates Aurora B activity in an MT-dependent manner. The Ska complex promotes mitotic Aurora B activity redli et al. 81 To further explore a dependence on MTs, we examined H3S10 phosphorylation in Ska-depleted cells that had been treated with high doses of nocodazole before mitotic entry. Under these conditions, the Ska complex was dispensable for H3-S10 phosphorylation. AMI-1 site Notably, however, levels of H3-pS10 were significantly lower after premitotic nocodazole treatment compared with DMSO-treated prometaphase cells, consistent with a requirement of spindle MTs for Aurora B activation in prometaphase. In further support of this notion, we found that when mitotic cells in which Aurora B was initially inhibited with ZM, but reactivation of the kinase was allowed upon washout of the inhibitor, Aurora B activity toward histone H3-pS10 recovered faster in the presence of MTs and absence of Aurora B centromere enrichment. This suggests that spindle MTs can contribute to Aurora B activity also independently of facilitating its centromere targeting. Collectively, these results support the hypothesis that spindle MTs facilitate Aurora B activation and that the Ska complex, in turn, depends on MTs to functionally interact with Aurora B. The Ska complex stimulates Aurora B kinase activity in vitro Finally, we asked whether the Ska complex could directly promote the catalytic activity of Aurora B. To test this hypothesis, we examined Aurora B activation in vitro. Aurora B was preincubated in the presence of its primary activator, the INCENP IN-Box domain, with the reconstituted Ska complex or equimolar amounts of BSA before – ATP and recombinant histone H3 as substrate were added to initiate the kinase reaction. Compared with preincubation with BSA, the Ska complex markedly enhanced the rate of histone H3 phosphorylation as well as Aurora B autophosphorylation. Similar results were obtained when BSA was omitted, and BSA had a negligible effect on Aurora B autophosphorylation, ruling out that BSA interfered with Aurora B activation. Likewise, incubation of the Ska complex with -ATP and histone H3, but without Aurora B and INCENP790918, did not increase incorporation of 32P into histone H3, confirming the absence of copurifying bacterial kinases. Together, these results show that the Ska complex is able to stimulate the kinase activity of Aurora B within the CPC core complex. They further suggest that Ska directly interacts with the CPC core complex. Consistently, the Ska complex associated with Aurora B in vitro, and we reproducibly found the CPC subunits Aurora B, INCENP, and Survivin in endogenous Ska1 immunoprecipitates from mitotic HeLa S3 cells. However, we failed to clearly detect Ska complex subunits in reverse immunoprecipitat