MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2
MAP kinase-mediated phosphorylation of distinct pools of histone H3 at S10 or S28 via mitogen- and stress-activated kinase 1/2
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DOI：
10.1242/jcs.02373
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发表时间：
2005-05-15
影响因子：
4
通讯作者：
Mahadevan, LC
中科院分区：
文献类型：
--
作者：
Dyson, MH;Thomson, S;Mahadevan, LC
ERK 和 p38 MAP 激酶通过下游丝裂原和应激激活激酶 1/2 (MSK1/2) 发挥作用，在核小体亚组分（包括 Fos 和 Jun 处的核小体亚组分）上引发组蛋白 H3 磷酸化，同时进行基因诱导。 H3 尾部的 S10 和 S28 均已被证明是体内磷酸受体。两个磷酸化表位都以相似的时间进程出现，并且都出现在对 TSA 诱导的超乙酰化高度敏感的 H3 尾上，相似之处可能表明 MSK1/2 磷酸化同一 H3 尾上的两个位点。事实上，在体外重组组蛋白八聚体上，MSK1 有效地磷酸化同一 H3 尾部的两个位点。然而，连续免疫沉淀研究表明，针对磷酸化 S10-H3 的抗体几乎可以恢复所有该表位，而不会耗尽磷酸化 S28-H3，反之亦然，表明这两个磷酸化表位在体内并不位于同一 H3 尾部。共聚焦免疫细胞化学证实了完整小鼠细胞核中两个磷酸表位的清晰物理分离。最后，我们使用基于转染的实验来测试可能解释这种差异靶向的模型。尽管异位激酶被外部刺激完全激活，但 MSK1 的过度表达和离域不会导致体内靶向的破坏。这些研究揭示了间期小鼠细胞核染色质内不同 H3 尾部的 S10 和 S28 磷酸化靶向水平显着。讨论了这种精确定位的可能模型。
ERK and p38 MAP kinases, acting through the downstream mitogen- and stress-activated kinase 1/2 (MSK1/2), elicit histone H3 phosphorylation on a subfraction of nucleosomes - including those at Fos and Jun - concomitant with gene induction. S10 and S28 on the H3 tail have both been shown to be phospho-acceptors in vivo. Both phospho-epitopes appear with similar time-courses and both occur on H3 tails that are highly sensitive to TSA-induced hyperacetylation, similarities which might suggest that MSK1/2 phosphorylates both sites on the same H3 tails. Indeed, on recombinant histone octamers in vitro, MSK1 efficiently phosphorylates both sites on the same H3 tail. However, sequential immunoprecipitation studies show that antibodies against phosphorylated S10-H3 recover virtually all this epitope without depletion of phosphorylated S28-H3, and vice versa, indicating that the two phospho-epitopes are not located on the same H3 tail in vivo. Confocal immunocytochemistry confirms the clear physical separation of the two phospho-epitopes in the intact mouse nucleus. Finally, we used transfection-based experiments to test models that might explain such differential targeting. Overexpression and delocalisation of MSK1 does not result in the breakdown of targeting in vivo despite the fact that the ectopic kinase is fully activated by external stimuli. These studies reveal a remarkable level of targeting of S10 and S28 phosphorylation to distinct H3 tails within chromatin in the interphase mouse nucleus. Possible models for such exquisite targeting are discussed.