Thr11磷酸化组蛋白H3在MCF-7细胞有丝分裂中的动态分布

组蛋白H3在氨基末端Ser10、Ser28、Thr11和Thr3等氨基酸残基的磷酸化修饰是一类在时间上和空间上与细胞有丝分裂相关的翻译后修饰事件。为了研究Thr11位点磷酸化作用的功能,利用SDS-PAGE、Western Blot、间接免疫荧光标记技术和激光共聚焦显微技术检测分析了人乳腺癌细胞(MCF-7)中Thr11磷酸化组蛋白H3在有丝分裂过程中的动态分布,以研究其在有丝分裂过程中的功能。结果显示:在MCF-7细胞中,组蛋白H3 Thr11的磷酸化发生在早前期细胞染色体的着丝粒处,成点状分布,继而在早中期达到最高水平,并以点状集中在赤道板上,在有丝分裂后期开始脱磷酸化,并于末期完成脱磷酸化。事实表明,H3 Thr11的磷酸化与细胞有丝分裂过程存在着时间和空间上的相关性。Thr11磷酸化H3只存在于着丝粒表明它可能参与有丝分裂期间功能性动原体的组成。这与Ser10磷酸化H3的分布及可能的功能截然相反。

STUDIES OF DYNAMIC DISTRIBUTION OF Thr11-PHOSPHO-RYLATED H3 IN MCF-7 AT MITOSIS

The phosphorylations of histone H3 on Ser10,Ser28,Thr11 and Thr3 of the amino terminal are the events that related to mitosis of the cell.To study the function of Thr11 phosphorylation of histone H3,indireet immunofluorescence labeling and laser confocal were employed with the antibody that was specific for Thr11-phosphorylated histone H3.The celluar dynamic distribution of this protein was examined at mitosis in MCF-7 cell.Our results showed that Thr11-phosphorylation of H3 initiated at centromere at early prophase in MCF-7 cells.The fluorescence signal on Thr11-phorphoslated histone H3 reached the strongest at early metaphase at the centromere punctated in the central of mitotic cell.The dephosphorylation of Thr11- phorphoslated histone H3 was completed in anaphase.The behavior of Thr11 phosphorylation and dephosphorylation in mitotic cell of MCF-7 were different from that of Ser10 phorphorylation and dephosphorylation.The results suggest that the Thr11 phosphorylation of histone H3 has a specific function in mitosis different from Ser10 phosphorylation.There was a precise spatial and temporal correlation between H3 phosphorylation of Thr11 and initial stages of chromatin condensation. Thr11-phorphoslated histone H3 located on centromeres suggest that it maybe included in the active kinetochore during mitosis.