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Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin 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 chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin 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 histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) 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.

Original publication

DOI

10.1074/mcp.M113.034819

Type

Journal article

Journal

Mol Cell Proteomics

Publication Date

07/2014

Volume

13

Pages

1724 - 1740

Keywords

Amino Acid Sequence, Aurora Kinase B, Cell Line, Tumor, Chromatin, Gene Expression Regulation, HEK293 Cells, HeLa Cells, Histones, Humans, Intracellular Signaling Peptides and Proteins, Methylation, Mitosis, Nuclear Proteins, Phosphoproteins, Phosphorylation, Protein Interaction Maps, Protein-Serine-Threonine Kinases, RNA-Binding Proteins, Serine-Arginine Splicing Factors, Transcription, Genetic