Differential regulation of CENP-A and histone H3 phosphorylation in G2/M.
Differential regulation of CENP-A and histone H3 phosphorylation in G2/M.
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发表时间：
2001-02
影响因子：
4
通讯作者：
Samantha G. Zeitlin;Barber Cm;Allis Cd;K. Sullivan
中科院分区：
文献类型：
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作者：
Samantha G. Zeitlin;Barber Cm;Allis Cd;K. Sullivan
在DNA复制之后，细胞浓缩它们的染色体以便在有丝分裂期间分离它们。缩合过程以及随后的分离需要组蛋白H3在丝氨酸10处的磷酸化。组蛋白H3磷酸化在染色体臂中的H3磷酸化之前在着丝粒周围灶中在G2期间开始。着丝粒蛋白A（CENP-A）是一种在着丝粒上发现的组蛋白H3样蛋白，含有一个与H3丝氨酸10相似的序列基序，提示CENP-A磷酸化可能与组蛋白H3磷酸化的臂间起始有关。为了验证这一假设，我们产生了针对CENP-A的推定磷酸化位点的肽抗体。ELISA、western blot和免疫细胞化学分析表明，CENP-A在共有基序处被磷酸化。同时共检测表明CENP-A和组蛋白H3的磷酸化在G2/M中是单独的事件。CENP-A磷酸化发生在组蛋白H3磷酸化的臂间起始和全基因组阶段之后。定量免疫细胞化学显示CENP-A磷酸化开始于前期，并在前中期达到最高水平。CENP-A磷酸表位反应性在后期丢失，并且在末期细胞中变得不可检测。在G2中，在组蛋白H3完全磷酸化之前，检测到CENP-A焦点加倍的前动粒的复制。组蛋白H3家族蛋白质的有丝分裂磷酸化显示出严格的空间和时间控制，发生在三个阶段：（1）臂间H3磷酸化，（2）染色体臂H3磷酸化和（3）着丝粒处的CENP-A磷酸化。这些观察结果揭示了G2进展的新的细胞学标志。
After DNA replication, cells condense their chromosomes in order to segregate them during mitosis. The condensation process as well as subsequent segregation requires phosphorylation of histone H3 at serine 10. Histone H3 phosphorylation initiates during G2 in pericentric foci prior to H3 phosphorylation in the chromosome arms. Centromere protein A (CENP-A), a histone H3-like protein found uniquely at centromeres, contains a sequence motif similar to that around H3 Ser10, suggesting that CENP-A phosphorylation might be linked to pericentric initiation of histone H3 phosphorylation. To test this hypothesis, we generated peptide antibodies against the putative phosphorylation site of CENP-A. ELISA, western blot and immunocytochemical analyses show that CENP-A is phosphorylated at the shared motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate events in G2/M. CENP-A phosphorylation occurs after both pericentric initiation and genome-wide stages of histone H3 phosphorylation. Quantitative immunocytochemistry reveals that CENP-A phosphorylation begins in prophase and reaches maximal levels in prometaphase. CENP-A phosphoepitope reactivity is lost during anaphase and becomes undetectable in telophase cells. Duplication of prekinetochores, detected as the doubling of CENP-A foci, occurs prior to complete histone H3 phosphorylation in G2. Mitotic phosphorylation of histone H3-family proteins shows tight spatial and temporal control, occurring in three phases: (1) pericentric H3 phosphorylation, (2) chromosome arm H3 phosphorylation and (3) CENP-A phosphorylation at kinetochores. These observations reveal new cytological landmarks characteristic of G2 progression.