In Xenopus egg extract, the Topo II CTD promotes the recruitment of Haspin kinase to centromeres

s and different Aurora B substrates require different levels of centromeric Aurora B activity. Because H3T3ph is dependent on the kinase activity of Haspin, small molecule inhibitors of Haspin would provide independent means to address these questions. Compared with RNAi-based approaches, inhibitors offer the potential advantages of selective, rapid, and strong temporal inhibition of kinase activity without depleting the protein itself, which might have kinase-independent functions in mitosis and roles at other cell cycle stages. Using high-throughput chemical library screening, we recently identified several Haspin inhibitors.Results Three distinct compounds inhibit H3T3 phosphorylation by Haspin in vitro and in cells We determined IC50 values for inhibition of H3T3 peptide phosphorylation by full-length Haspin of 3 nM for 5-iodotubercidin, 10 nM for LDN-192960, and 100 nM for LDN-211898. These values are consistent with previous studies, and demonstrate that these three Sodium laureth sulfate molecules show a range of potencies for Haspin inhibition in vitro. To determine compound potency for Haspin inhibition in cells, we arrested HeLa cells in mitosis using nocodazole, then added Haspin inhibitors in the continued presence of nocodazole for 1 h. Immunoblotting of cell lysates for H3T3ph showed that all three inhibitors strongly inhibited Haspin, with relative potencies that reflected their in vitro activity. In contrast, none of the inhibitors had a detectable effect on the Aurora B product H3S10ph at these doses. Immunofluorescence analysis confirmed that all three inhibitors reduced H3T3ph in mitotic U2OS cells. Notably, although 1 M 5-iodotubercidin or LDN192960, or 5 M LDN-211898 caused dramatic reductions in H3T3ph, complete loss of detectable H3T3ph required >3 M 5-iodotubercidin, >5 M LDN-192960, or >30 M LDN211898. Haspin inhibitors delocalize the CPC from centromeres but not the central spindle RNAi of Haspin causes premature loss of cohesion in mitosis. However, centromeres remained paired in numerous immunofluorescence experiments with all three Haspin inhibitors, including when spread mitotic chromosomes were examined. We conclude that the inhibitors allow assessment of kinase-dependent functions of Haspin in the absence of premature sister chromatid separation, which had previously confounded direct analysis of the role of this kinase in error correction and the spindle checkpoint. Haspin RNAi causes CPC loss from centromeres, but not the central spindle. Similarly, when added to nocodazole-arrested mitotic cells, all three Haspin inhibitors caused displacement of Aurora B from inner centromeres to a diffuse distribution on chromatin, even when MG132 was included to counter mitotic exit. In contrast, although anaphase was disrupted PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19833994 at high doses of Haspin inhibitors, Aurora B was not lost from central spindles, and CPC formation was not affected. Direct comparison of H3T3ph and Aurora B staining suggested that maximal displacement of preaccumulated centromeric CPC required >3 M 5-iodotubercidin, >10 M LDN-192960, or >100 M LDN-211898, which suggested that even low levels of H3T3ph can maintain a significant population of the CPC at centromeres. These results provide evidence that the kinase activity of Haspin is required for normal centromeric localization of Aurora B, which is consistent with the notion that H3T3ph provides a docking site for the CPC. Haspin inhibitors influence Aurora B activity toward centromeric targets To determine funct