蛋白激脢A媒介之絲氨酸35的磷酸化導致連結組蛋白H1.4從有絲分裂中的染色質上游離之探討

Protein Kinase A-Mediated Serine 35 Phosphorylation Dissociates Histone H1.4 from Mitotic Chromosome

朱祁舜(Chi-Shuen Chu)

指導教授 ： 阮麗蓉

國立臺灣大學/醫學院/分子醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.03261

摘要

真核細胞之DNA由核心組蛋白(Core histones)及連結組蛋白(Linker histone)纏繞成Nucleosomes。組蛋白之轉譯後修飾(Posttranslational modification)構成組蛋白密碼(Histone code)，可調控細胞基因轉錄(Gene transcription) 。相對於核心組蛋白，連結組蛋白之轉譯後修飾及其生理意義並不清楚。本實驗室利用質譜儀鑑定蛋白質轉譯後修飾的策略鑑定出許多在連結組蛋白H1.4上尚未被鑑定的蛋白質轉譯後修飾，其中，我們發現其N端第35個思氨酸 （serine 35）上具有一磷酸化位置，此磷酸化在有絲分裂時候會累積。進一步研究發現蛋白質磷酸激脢A （PKA）為其主要的磷酸化酵素。思氨酸35磷酸化後的H1.4會減低原本H1.4和染色質連結的能力。我們進一步發現此氨基酸對於抑制H1.4基因表現後引發的有絲分裂缺陷是重要的。在抑制PKA活性的實驗中，我們亦發現PKA對於染色質在有絲分裂下的濃縮扮演相當的角色，而這樣的角色有部分是跟H1.4有關的。我們同時發現在思氨酸35鄰近區域有一離氨酸 （lysine 33）會被進行甲基化，SET7，一個組蛋白H3上第四個離氨酸的甲基脢亦會對H1.4進行甲基化。並且，此兩個鄰近的蛋白質轉譯後修飾具有互相調控的機制存在。在此篇研究中，我們闡述了此一H1.4之N端serine 35 磷酸化在細胞分裂的重要性，在發現其鄰近的離氨酸33之甲基化的同時也發現了此兩修飾相互調控的可能。因此，此篇研究提供了更進一步的訊息，作為將來明瞭連結組蛋白密碼之複雜功能之用。

關鍵字

連結組蛋白 ； 蛋白質磷酸激脢A ； 細胞週期 ； 磷酸化 ； 有絲分裂 ； 染色質濃縮

並列摘要

Global histone H1 phosphorylation correlates with cell cycle progression. However, the function of site-specific H1 variant phosphorylation remains unclear. Our mass spectrometry analysis revealed several new modifications on H1. Among them, a novel N-terminal phosphorylation of the major H1 variant H1.4 at serine 35 (H1.4S35ph) was found to accumulate at mitosis. Protein kinase A (PKA) was found to be a kinase for H1.4S35. Importantly, S35-phosphorylated H1.4 contains weaker binding affinity to mitotic chromatin. Moreover, H1.4S35A substitution mutant cannot efficiently rescue the mitotic defect following H1.4 depletion and inhibition of PKA activity increases the mitotic chromatin compaction depending on H1.4. In addition, an adjacent methylation on lysine 33 was also identified. We further demonstrated that SET7, a histone H3K4 methyltransferase, can methylate H1.4K33 both in vivo and in vitro. Finally, a crosstalk between H1.4S35ph and H1.4K33me was characterized. Our results not only indicate that PKA-mediated H1.4S35 phosphorylation interferes H1.4 binding affinity to mitotic chromatin, but also suggest that this phosphorylation is necessary for specific mitotic functions. Our data suggest that the adjacent H1.4K33me might be involved in this regulation.

並列關鍵字

Linker histone ； H1 ； H1.4 ； protein kinase A ； cAMP-dependent protein kinase ； PKA ； Cell cycle ； phosphorylation ； mitosis ； DNA compaction

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