Recent discoveries have highlighted the importance of
Haspin kinase activity for the correct positioning of the
kinase Aurora B at the centromere. Haspin phosphorylates
Thr3 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 Arg2 and Lys4 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 Lys4 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
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 stabilize
extranucleosomal DNA, suggesting that phosphorylation
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.