Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase
Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase
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DOI：
10.1074/mcp.m113.034819
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发表时间：
2014-07-01
影响因子：
7
通讯作者：
Aebersold, Ruedi
中科院分区：
文献类型：
--
作者：
Maiolica, Alessio;de Medina-Redondo, Maria;Aebersold, Ruedi
最近的发现突出了Haspin激酶活性对于激酶Aurora B在着丝粒的正确定位的重要性。Haspin磷酸化组蛋白H3（H3）的Thr（3），为Aurora B定位于有丝分裂染色体的着丝粒提供信号。迄今为止，组蛋白H3是唯一确认的Haspin底物。我们使用生物化学，药理学和质谱方法的组合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Haspin蛋白-蛋白相互作用网络。我们确定了Haspin共有基序和激酶与组蛋白H3尾的共晶结构。该结构揭示了一种独特的弯曲底物结合模式，将组蛋白H3残基Arg（2）和Lys（4）定位在Haspin磷酸化苏氨酸附近的酸性结合口袋中。激酶-底物复合物的这种独特构象解释了所报道的组蛋白H3的Lys（4）甲基化对Haspin活性的调节。此外，底物识别的结构基础和Haspin的氨基酸序列偏好的鉴定有助于鉴定新的候选Haspin底物。特别地，我们验证了组蛋白变体macroH2A的Ser（137）的磷酸化作为Haspin激酶活性的靶标。MacroH2A Ser（137）存在于一段约40个氨基酸的碱性延伸中，该延伸是稳定人核小体DNA所需的，这表明Ser（137）的磷酸化可能调节macroH2A和DNA的相互作用。总之，我们的数据表明，Haspin活性影响参与基因表达调控和剪接的蛋白质的磷酸化状态。
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.