endogenous Bub1. In contrast to earlier studies, Bub1Mad3, which binds Bub3, accu mulates normally at kinetochores. Another surprising observation was that, despite an intact kinase domain, H2AT121 phosphorylation was markedly dimin ished. Consistently, Aurora B distributed weakly along chromosome arms in Bub1/ MEFs expressing Bub1Mad3, and Sgo1 kinetochore asso ciation was lost. The same defects were observed in Bub1/ MEFs expressing Bub1 lacking catalytic activity or the entire kinase domain. Because deletion of the N terminus of Bub1 profoundly reduced H2A phosphorylation in vivo and because the kinase domain is intact, we monitored the in vitro kinase activity of HABub1Mad3. Bub1 was immunoprecipitated with the HA tag from Bub1/ cells expressing HABub1Mad3 or HABub1 and Bub1F/F cells as a control. Immunoprecipitates were incubated with recombinant histone H2A in the pres ence of ATP. Whereas wildtype Bub1 was sufficient to phosphorylate H2A in vitro, there was little detectable in corporation of 32P into H2A with HABub1Mad3, indicating the N terminus is required for H2A phosphoryla tion in vitro. Collectively, these data uncover a novel require ment for Mad3 homology domain in activating Bub1 kinase activity. Finally, we note that consistent with in vitro kinase assays on Bub1 protein precipitated from wildtype and Bub1KD/KD MEFs, HABub1D892N precipitated from Bub1/ MEFs with antiHA antibody failed to phosphorylate histone H2A substrate in vitro, providing additional evidence that Bub1D892N is catalytically deficient. Phosphorylated histone H2A and H3 stably bind Aurora B but not Sgo1 It has been proposed that H2A phosphorylation at T121 by Bub1 creates a mark for inner centromeric localization of Sgo1, in which one of its proposed functions is to load the chromo somal 
passenger complex. We recently reported that phosphorylation of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834673 H2AT121 is not restricted to centromeres and spreads to chromo some arms in MEFs derived from Bub1 transgenic Bub1 kinase integrates diverse Aurora B functions Ricke et al. 939 mice that overexpress wildtype Bub1. Interestingly, we observed that phosphorylated H2AT121 at chromosome arms of Bub1T264 MEFs is not sufficient to load Sgo1 or Aurora B. Several studies have pointed to the impor tance of H3T3 phosphorylation in inner centromeric Aurora B localization, which prompted us to examine whether 940 JCB VOLUME 199 NUMBER 6 2012 phosphorylation of H2AT121 and H3T3 together might serve as a binding site for Sgo1, Sgo2, or Aurora B. To this end, we transduced wildtype and Bub1T264 MEFs with a lentivirus en coding doxycycline inducible Haspin kinase. Over expression of Haspin in wildtype MEFs resulted in robust H3T3 phosphorylation along the entire chromosome but was insuffi cient to delocalize Sgo1, Sgo2, or Aurora B. In contrast, Bub1 kinase integrates diverse Aurora B functions Ricke et al. 941 overexpression of Haspin in Bub1T264 MEFs localized Aurora B in a detergentresistant manner along chromosomes, indicative of binding comparable with inner centromeric Aurora B. Strikingly, neither Sgo1 nor Sgo2 loaded onto chromosome arms, suggesting that neither shugoshin serves as an adaptor for Aurora B. Collectively, these data indicate that phosphorylated H2AT121 and H3T3 act in concert to create a highaffinity binding site for shugoshinindependent loading of Aurora B at inner centromeres. To obtain additional evidence for this hypothesis, we arti ficially targeted Sgo1 to centromeres by BAY-41-2272 transducing wildtype
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