Histone H3 (H3) phosphorylation at Ser10 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 Thr7, 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 Ser28 phosphorylation. Then we produced a monoclonal antibody, HTA28, using a phosphopeptide corresponding to phosphorylated H3 Set28. This antibody specifically recognized the phosphorylation of H3 Ser28 but not that of glial fibrillary acidic protein Thr7. Immunocytochemical studies with HTA28 revealed that Ser28 phosphorylation occurred in chromosomes predominantly during early mitosis and coincided with the initiation of mitotic chromosome condensation. Biochemical analyses using 32P-labeled mitotic cells also confirmed that H3 is phosphorylated at Ser28 during early mitosis. In addition, we found that H3 is phosphorylated at Ser28 as well as Ser10 when premature chromosome condensation was induced in tsBN2 cells. These observations suggest that H3 phosphorylation at Set28, together with Ser10, is a conserved event and is likely to be involved in mitotic chromosome condensation.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology