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extracts, spindle assembly depends on Aurora B activation through enrichment on chromosomes and likely involves phosphorylation of HC-067047 substrates at a distance on spindle microtubules through a similar diffusion-based mechanism. Surprisingly, in HeLa cells, Aurora B substrates at centromeres and on chromatin are still phosphorylated after depletion of the mitotic kinase Haspin, which inhibits Aurora B localization to centromeres. However, concentration of Aurora B in other chromosomal regions in these cells may be sufficient for kinase activity, similar to what we observed for concentration by LacIINCENP. Our findings explain how Aurora B phosphorylates substrates at micrometer scale distances from centromeres, such as on chromatin and on spindle microtubules. At kinetochores, however, phosphorylation of outer kinetochore substrates depends on their distance from the inner centromere on submicrometer scales. We propose that phosphorylation of these substrates depends on their position in a diffusion-based gradient of kinase activity centered at the inner centromere. An alternative model is that outer kinetochore substrates may also be phosphorylated, while the CPC remains bound to the inner centromere, and it remains unclear to what extent Aurora B can phosphorylate kinetochore substrates without diffusion. The phosphorylation gradient that we observed extends over micrometer distances from centromeres on the chromosome arms, similar to a gradient previously observed in anaphase, which raises the question of how phosphorylation of kinetochore substrates is regulated on submicrometer distance scales. Localized phosphatase activities may provide an answer. Outer kinetochore substrates are dephosphorylated at metaphase, whereas chromatin substrates remain fully phosphorylated, which indicates that phosphatase activity is higher at kinetochores PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834673 than on chromatin, consistent with PP1 localization to kinetochores. These observations suggest that local phosphatase activity may shape a phosphorylation gradient at kinetochores to generate spatial patterns on submicrometer distance scales. Several mechanisms regulate PP1 and PP2A localization at centromeres and kinetochores, and these phosphatases could locally sharpen a phosphorylation gradient by both inactivating Aurora B and directly dephosphorylating substrates. Our findings provide a foundation for understanding how the combination of kinase and phosphatase activities generates a spatial phosphorylation gradient at kinetochores. Cells were transfected with plasmid DNA using Fugene for HeLa cells or Effectene for U2OS cells, following the manufacturer’s instructions. HeLa cells were used for all experiments, except for the LacI-INCENP experiments, in which we used the U2OS-LacO cell line. Plasmids CBDBD-INCENPmCherry encodes the centromeric CBDBD, truncated INCENP, and C-terminal fusion mCherry in vector pcDNA3.1. CBFL-INCENPmCherry and LacI-INCENPmCherry were generated by replacing CBDBD with either CBFL or with LacI. We swapped GFP for mCherry to create the CBDBDINCENP-GFP and CBDBDINCENP-GFP constructs used for FRAP experiments. The INCENPTSS/AAA mutant was created by PCR. The design of the Aurora B phosphorylation sensors is based on a protein kinase C sensor, a CFP/YFP fluorescence resonance energy transfer pair with a substrate peptide and an FHA2 phospho-Thrbinding domain in between. An N-terminal targeting domain localizes the sensor to centromeres, chromatin, or kinetochores as previous