ol, in cells that were infected with lentivirus encoding mRFP-H2B. Importantly, Bub1T85 and Bub1T264 MEFs were equally able to maintain an arrest in response to nocodazole or taxol, similar to wild type. Consistent with this, we found that kinetochore localization of core mitotic checkpoint proteins that accumulate at unattached kinetochores, including BubR1, Cdc20, Mad2, and Cenp-E, was normal in Bub1 transgenic MEFs. Together, these data suggested that Bub1 overexpression does not interfere with mitotic checkpoint signaling or kinetochore assembly, and imply that the chromosome segregation defects observed in Bub1 transgenic MEFs are invisible from either checkpoint detection or resolution, and not due to rapid anaphase onset. Bub1 overexpression leads to aberrant substrate phosphorylation Selective loss of Bub1 kinase activity in HeLa cells has been shown to promote chromosome misalignment, suggesting that Bub1’s catalytic activity plays a role in microtubulekinetochore attachment. To test whether Bub1 overexpression leads to aberrant catalytic activity, we made use of the recent discovery that Bub1 kinase phosphorylates histone H2A at threonine 121 in humans, a site that is conserved in mouse. In wild-type MEFs, pT121-H2A antibody stained kinetochores in prophase and prometaphase. This staining remained detectable in metaphase but at reduced intensity, and was completely abolished when cells progressed to anaphase. In prophase, H2A phosphorylation at centromeres was much higher in Bub1T264 MEFs than in wild-type MEFs. Furthermore, in prometaphase, H2A phosphorylation was not only much higher at centromeres, but now also occurred along chromosome arms. In metaphase, H2A phosphorylation persisted at both locations, but was less abundant than in prometaphase. In anaphase, no H2A phosphorylation was detectable, similar to wildtype MEFs. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834673 The pattern of H2A phosphorylation in Bub1T85 MEFs mirrored that of Bub1T264 MEFs, although the amount of phosphorylation at centromeres and chromosome arms was typically lower than in Bub1T264 MEFs, suggesting that Bub1 overexpression correlates with aberrant Bub1 catalytic activity. Consistently, by Western blotting we observed Oncogenic 
Bub1 hyperactivates Aurora B kinase Ricke et al. 1053 1054 JCB VOLUME 193 NUMBER 6 2011 more phosphorylated H2A in an asynchronous population of cells in Bub1T264 cells compared with wild type. Kawashima et al. reported H2A phosphorylation of the entire chromosome by ectopically expressed Bub1 kinase domain fused to H2B results in relocation of centromereassociated Sgo1 to chromosome arms. However, we found that Sgo1 was properly localized to the centromeric regions of Bub1T85 MEFs in prometaphase, when H2A phosphorylation was high along chromosome arms. Although Bub1T264 MEFs had slightly reduced Sgo1 levels at inner centromeric regions, there was no detectable increase in Sgo1 staining along chromosome arms in any Bub1T264 cell analyzed. An inverse relationship between Bub1 and Sgo1 abundance at kinetochores has previously been reported, although the exact nature of the relationship is currently unclear. Overexpression of Bub1 results in Aurora B MedChemExpress DMXB-A hyperactivation Earlier in vitro studies using Xenopus oocyte extracts have suggested that Bub1 not only phosphorylates H2A but also INCENP, although the precise residues targeted by Bub1 remain to be identified. INCENP is a component of the chromosome passenger complex, which further consists of Aurora B, Borealin, Survivin
