Maiolica Alessio, de Medina-Redondo Maria, Schoof Erwin M, Chaikuad Apirat, Villa Fabrizio, Gatti Marco, Jeganathan Siva, Lou Hua Jane, Novy Karel, Hauri Simon, Toprak Umut H, Herzog Franz, Meraldi Patrick, Penengo Lorenza, Turk Benjamin E, Knapp Stefan, Linding Rune, Aebersold Ruedi
From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, Zurich, Switzerland;
§Department of Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland;
Mol Cell Proteomics. 2014 Jul;13(7):1724-40. doi: 10.1074/mcp.M113.034819. Epub 2014 Apr 14.
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.
近期的发现突出了Haspin激酶活性对于激酶Aurora B在着丝粒上正确定位的重要性。Haspin使组蛋白H3（H3）的苏氨酸（Thr）3位点磷酸化，这为Aurora B定位于有丝分裂染色体的着丝粒提供了信号。迄今为止，组蛋白H3是唯一已确认的Haspin底物。我们综合运用生化、药理学和质谱方法来研究有丝分裂细胞中Haspin抑制的后果。我们对染色质相关蛋白上的3964个磷酸化位点进行了定量，并确定了一个Haspin蛋白 - 蛋白相互作用网络。我们确定了Haspin的共有基序以及该激酶与组蛋白H3尾部的共晶体结构。该结构揭示了一种独特的弯曲底物结合模式，将与Haspin磷酸化苏氨酸相邻的组蛋白H3残基精氨酸（Arg）2和赖氨酸（Lys）4定位到酸性结合口袋中。激酶 - 底物复合物的这种独特构象解释了之前报道的组蛋白H3赖氨酸4位点甲基化对Haspin活性的调节作用。此外，底物识别结构基础和Haspin氨基酸序列偏好的确定有助于鉴定新的Haspin候选底物。特别是，我们验证了组蛋白变体macroH2A的丝氨酸（Ser）137位点磷酸化是Haspin激酶活性的一个靶点。MacroH2A丝氨酸137位点位于一段约40个氨基酸的碱性区域，该区域是稳定核小体外DNA所必需的，这表明丝氨酸137位点磷酸化可能调节macroH2A与DNA的相互作用。总体而言，我们的数据表明Haspin活性会影响参与基因表达调控和剪接的蛋白质的磷酸化状态。
