Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle
Site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different processes during the cell cycle
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DOI：
10.1007/s00412-009-0228-2
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发表时间：
2009-12-01
期刊：
影响因子：
1.6
通讯作者：
Lindner, Herbert H.
中科院分区：
文献类型：
--
作者：
Talasz, Heribert;Sarg, Bettina;Lindner, Herbert H.
细胞周期相关的组蛋白H1.5磷酸化表现为三种离散的磷酸化形式，在间期仅发生在Ser（17）、Ser（172）和Ser（188）上。在G2晚期和有丝分裂期间，上磷酸化仅发生在Thr（137）或Thr（154）处的苏氨酸上，以构建H1.5的四磷酸化形式，而五磷酸化形式由Thr（10）处的磷酸化产生。为了确定组蛋白H1磷酸化在同步化的Hela细胞核内的动力学和空间分布，我们使用针对磷酸化的Ser（17）、Ser（172）和Thr（10）产生的亲和纯化的多克隆抗体定位了组蛋白亚型H1.5的三个不同的磷酸化位点。使用特异性抗体对同步化的HeLa细胞进行免疫荧光标记显示，H1.5 Ser（17）的磷酸化在G1早期出现在离散斑点处，随后是Ser（172）的磷酸化。Thr（10）磷酸化在前期开始，在中期显示最高磷酸化水平，在染色质去凝聚发生之前明显消失。使用激酶抑制剂staurosporine的实验表明，在不同的磷酸化位点涉及不同的激酶。共定位研究表明，H1.5和H1.2的Ser（172）磷酸化确实共定位于DNA复制和转录位点。这些结果支持这样的想法，即位于细胞核不同区域的H1.5和H1.2的各种位点特异性磷酸化形式与细胞周期期间的不同功能相关。
The cell cycle-associated phosphorylation of histone H1.5 is manifested as three discrete phosphorylated forms, occurring exclusively on Ser(17), Ser(172), and Ser(188) during interphase. During late G2 and mitosis the up-phosphorylation occurs exclusively on threonine at either Thr(137) or Thr(154) to build the tetraphosphorylated forms of H1.5, whereas the pentaphosphorylated forms result from phosphorylation at Thr(10). To determine the kinetic and spatial distribution of histone H1 phosphorylation within the nucleus of synchronized Hela cells we localized three distinct phosphorylation sites of histone subtype H1.5 using affinity-purified polyclonal antibodies generated against phosphorylated Ser(17), Ser(172), and Thr(10). Immunofluorescence labeling of synchronized HeLa cells using the specific antibodies revealed that phosphorylation of H1.5 Ser(17) appeared early in G1 at discrete speckles followed by phosphorylation of Ser(172). Thr(10) phosphorylation started during prophase, showed highest phosphorylation levels during metaphase, and disappeared clearly before chromatin decondensation occurred. Experiments using the kinase inhibitor staurosporine indicate the involvement of different kinases at the various phospho-sites. Colocalization studies revealed that Ser(172) phosphorylation of H1.5 and H1.2 does colocalize to DNA replication and transcription sites. These results favor the idea that the various site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different functions during the cell cycle.