During mitosis of human cells, the kinase Bub1 orchestrates chromosome segregation through phosphorylating histone H2A and the anaphase-promoting complex/cyclosome activator Cdc20. Bub1-mediated H2A-T120 phosphorylation (H2A-pT120) at kinetochores promotes centromeric sister-chromatid cohesion, whereas Cdc20 phosphorylation by Bub1 contributes to spindle checkpoint signaling. Here, we show that phosphorylation at the P+1 substrate-binding loop of human Bub1 enhances its activity toward H2A but has no effect on its activity toward Cdc20. We determine the crystal structure of phosphorylated Bub1. A comparison between structures of phosphorylated and unphosphorylated Bub1 reveals phosphorylation-triggered reorganization of the P+1 loop. This activating phosphorylation of Bub1 is constitutive during the cell cycle. Enrichment of H2A-pT120 at mitotic kinetochores requires kinetochore targeting of Bub1. The P+1 loop phosphorylation of Bub1 appears to occur through intramolecular autophosphorylation. Our study provides structural and functional insights into substrate-specific regulation of a key mitotic kinase and expands the repertoire of kinase activation mechanisms.
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