Goto H, Tomono Y, Ajiro K, Kosako H, Fujita M, Sakurai M, Okawa K, Iwamatsu A, Okigaki T, Takahashi T, Inagaki M
Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.
J Biol Chem. 1999 Sep 3;274(36):25543-9. doi: 10.1074/jbc.274.36.25543.
Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryotes and was recently shown to play an important role in chromosome condensation in Tetrahymena. When producing monoclonal antibodies that recognize glial fibrillary acidic protein phosphorylation at Thr(7), we obtained some monoclonal antibodies that cross-reacted with early mitotic chromosomes. They reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate. With microsequencing, this phosphoprotein was proved to be H3. Mutational analysis revealed that they recognized H3 Ser(28) phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Ser(28). This antibody specifically recognized the phosphorylation of H3 Ser(28) but not that of glial fibrillary acidic protein Thr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using (32)P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser(28) during early mitosis. In addition, we found that H3 is phosphorylated at Ser(28) as well as Ser(10) when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Ser(28), together with Ser(10), is a conserved event and is likely to be involved in mitotic chromosome condensation.
组蛋白H3（H3）的丝氨酸10位点（Ser(10)）磷酸化在真核生物有丝分裂期间发生，最近研究表明其在嗜热四膜虫的染色体凝聚过程中发挥重要作用。在制备识别胶质纤维酸性蛋白苏氨酸7位点（Thr(7)）磷酸化的单克隆抗体时，我们获得了一些能与早期有丝分裂染色体发生交叉反应的单克隆抗体。它们在有丝分裂细胞裂解液中特异性地与15 kDa的磷酸化蛋白发生反应。通过微量测序，证明该磷酸化蛋白为H3。突变分析表明，它们识别H3的丝氨酸28位点（Ser(28)）磷酸化。随后，我们使用对应于磷酸化H3丝氨酸28位点的磷酸肽制备了一种单克隆抗体HTA28。该抗体特异性识别H3丝氨酸28位点的磷酸化，而不识别胶质纤维酸性蛋白苏氨酸7位点的磷酸化。用HTA28进行的免疫细胞化学研究表明，丝氨酸28位点的磷酸化主要发生在有丝分裂早期的染色体中，与有丝分裂染色体凝聚的起始同时发生。使用32P标记的有丝分裂细胞进行的生化分析也证实，在有丝分裂早期H3的丝氨酸28位点发生了磷酸化。此外，我们发现，当在tsBN2细胞中诱导早熟染色体凝聚时，H3丝氨酸28位点以及丝氨酸10位点均发生了磷酸化。这些观察结果表明，H3丝氨酸28位点的磷酸化与丝氨酸10位点的磷酸化一样，是一个保守事件，可能参与有丝分裂染色体凝聚过程。
