Modulation of the Chromatin Phosphoproteome by the Haspin Protein

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphory-lates Thr3 of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chro-mosomes. To date, histone H3 is the only confirmed Has-pin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chro-matin-associated proteins and identified a Haspin pro-tein-protein interaction network. We determined the Has-pin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the his-tone H3 residues Arg2 and Lys4 adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate com-plex explains the reported modulation of Haspin activity by methylation of Lys4 of the histone H3. In addition, the identification of the structural basis of substrate recogni-tion and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin sub-strates. In particular, we validated the phosphorylation of Ser137 of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser137 resides in a basic stretch of about 40 amino acids that is required to stabi-lize extranucleosomal DNA, suggesting that phosphoryla-tion of Ser137 might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing. Molecular &amp