Total Polo localized normally to kinetochores following INCENP PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19862565 knockdown. This is consistent with the observation that Polo localization to this region precedes that of INCENP. Importantly, we could still readily detect active PoloT182ph at centrosomes of cells following INCENP knockdown. These experiments reveal that INCENP is required for T182 phosphorylation and activation of Polo Aglafoline site Kinase at inner centromeres in early mitosis. Activation of centrosomal Polo does not require INCENP. Polo T-Loop Phosphorylation Is Required for Mitotic Progression But Not For Mitotic Entry In order to investigate the function of Polo T182 phosphorylation in mitosis, we established stable cell lines allowing inducible expression of PoloWT-GFP or PoloT182A-GFP. Endogenous Polo could be depleted in those cells by RNAi against the 39UTR of the native transcript. Expression of PoloWT-GFP rescued the viability and proliferation of cells depleted of endogenous Polo. However, expression of PoloT182A-GFP did not, and cells died. Thus, Polo T-loop phosphorylation is essential for viability. Polo-depleted cells accumulated in mitosis, exhibiting phenotypes similar to those observed for the first polo mutants. Expression of PoloWT-GFP restored mitotic progression in cells depleted of endogenous Polo, but expression of PoloT182A-GFP did not. Cells expressing only PoloT182A-GFP accumulated in prometaphase/metaphase, often with Aurora B Activates Polo Kinase at Centromeres 5 Aurora B Activates Polo Kinase at Centromeres 6 Aurora B Activates Polo Kinase at Centromeres unaligned chromosomes. Interestingly, while the loss of Polo led to an increase in monopolar spindles, substitution of endogenous Polo with PoloT182A-GFP did not. This suggests that T-loop phosphorylation of Polo may be dispensable for its role in bipolar spindle assembly. The observation that INCENP-dependent activation of Polo by phosphorylation at T182 at centromeres/kinetochores is required for chromosome alignment in prometaphase is consistent with the known role of Polo in regulating MedChemExpress HC-030031 kinetochore function. Aurora B Activity Is Required for Polo Kinase Activation at Centromeres Because the best known role of INCENP is to activate Aurora B kinase in the CPC, we next asked whether Aurora B has a role in Polo T-loop phosphorylation at centromeres. Drosophila Polo T182 is preceded by a conserved stretch of basic residues resembling the consensus site for Aurora kinases . Indeed, Drosophila Aurora B complexed with a fragment of INCENP can directly phosphorylate Polo in vitro. A T182A mutation in the Polo used as a substrate reproducibly reduced its phosphorylation by about one half. Thus, Polo T182 is a major phosphorylation site for Aurora B. Similar results were obtained using human Aurora B on GST-PoloWT or GST-PoloT182D. Kinase inhibition studies suggest that Aurora B is responsible for PoloT182 phosphorylation in vivo. Binucleine 2 is the only specific Aurora B kinase inhibitor described to date that is effective in Drosophila cells. When DMel-2 cells were treated with 20 mM Binucleine 2 for 2 h, H3S10ph was undetectable in mitotic cells and INCENP and Aurora B were dispersed in clumps on the chromosomes. Both of these phenotypes are characteristic of the loss of Aurora B function. Aurora B kinase activity is required for Polo activation at kinetochores, and levels of kinetochore-associated PoloT182ph were greatly reduced in Binucleine 2-treated mitotic cells. In contrast, we observed no obvious differ.Total Polo localized normally to kinetochores following INCENP PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19862565 knockdown. This is consistent with the observation that Polo localization to this region precedes that of INCENP. Importantly, we could still readily detect active PoloT182ph at centrosomes of cells following INCENP knockdown. These experiments reveal that INCENP is required for T182 phosphorylation and activation of Polo kinase at inner centromeres in early mitosis. Activation of centrosomal Polo does not require INCENP. Polo T-Loop Phosphorylation Is Required for Mitotic Progression But Not For Mitotic Entry In order to investigate the function of Polo T182 phosphorylation in mitosis, we established stable cell lines allowing inducible expression of PoloWT-GFP or PoloT182A-GFP. Endogenous Polo could be depleted in those cells by RNAi against the 39UTR of the native transcript. Expression of PoloWT-GFP rescued the viability and proliferation of cells depleted of endogenous Polo. However, expression of PoloT182A-GFP did not, and cells died. Thus, Polo T-loop phosphorylation is essential for viability. Polo-depleted cells accumulated in mitosis, exhibiting phenotypes similar to those observed for the first polo mutants. Expression of PoloWT-GFP restored mitotic progression in cells depleted of endogenous Polo, but expression of PoloT182A-GFP did not. Cells expressing only PoloT182A-GFP accumulated in prometaphase/metaphase, often with Aurora B Activates Polo Kinase at Centromeres 5 Aurora B Activates Polo Kinase at Centromeres 6 Aurora B Activates Polo Kinase at Centromeres unaligned chromosomes. Interestingly, while the loss of Polo led to an increase in monopolar spindles, substitution of endogenous Polo with PoloT182A-GFP did not. This suggests that T-loop phosphorylation of Polo may be dispensable for its role in bipolar spindle assembly. The observation that INCENP-dependent activation of Polo by phosphorylation at T182 at centromeres/kinetochores is required for chromosome alignment in prometaphase is consistent with the known role of Polo in regulating kinetochore function. Aurora B Activity Is Required for Polo Kinase Activation at Centromeres Because the best known role of INCENP is to activate Aurora B kinase in the CPC, we next asked whether Aurora B has a role in Polo T-loop phosphorylation at centromeres. Drosophila Polo T182 is preceded by a conserved stretch of basic residues resembling the consensus site for Aurora kinases . Indeed, Drosophila Aurora B complexed with a fragment of INCENP can directly phosphorylate Polo in vitro. A T182A mutation in the Polo used as a substrate reproducibly reduced its phosphorylation by about one half. Thus, Polo T182 is a major phosphorylation site for Aurora B. Similar results were obtained using human Aurora B on GST-PoloWT or GST-PoloT182D. Kinase inhibition studies suggest that Aurora B is responsible for PoloT182 phosphorylation in vivo. Binucleine 2 is the only specific Aurora B kinase inhibitor described to date that is effective in Drosophila cells. When DMel-2 cells were treated with 20 mM Binucleine 2 for 2 h, H3S10ph was undetectable in mitotic cells and INCENP and Aurora B were dispersed in clumps on the chromosomes. Both of these phenotypes are characteristic of the loss of Aurora B function. Aurora B kinase activity is required for Polo activation at kinetochores, and levels of kinetochore-associated PoloT182ph were greatly reduced in Binucleine 2-treated mitotic cells. In contrast, we observed no obvious differ.