Identification of a novel phosphorylation site on histone H3 coupled with mitotic chromosome condensation

Histone H3 (H3) phosphorylation at Ser(10) occurs during mitosis in eukaryo tes and was recently shown to play an important role in chromosome condensa tion in Tetrahymena. When producing monoclonal antibodies that recognize gl ial fibrillary acidic protein phosphorylation at Thr(7), we obtained some m onoclonal antibodies that cross-reacted with early mitotic chromosomes. The y reacted with 15-kDa phosphoprotein specifically in mitotic cell lysate, W ith microsequencing, this phosphoprotein was proved to be H3, Mutational an alysis revealed that they recognized H3 Ser(28) phosphorylation. Then we pr oduced a monoclonal antibody, HTA28, using a phosphopeptide corresponding t o phosphorylated H3 Ser(28), This antibody specifically recognized the phos phorylation of H3 Ser(28) but not that of glial fibrillary acidic protein T hr(7). Immunocytochemical studies with HTA28 revealed that Ser(28) phosphor ylation occurred in chromosomes predominantly during early mitosis and coin cided with the initiation of mitotic chromosome condensation, Biochemical a nalyses using P-32-labeled mitotic cells also confirmed that H3 is phosphor ylated at Ser(28) during early mitosis. In addition, we found that H3 is ph osphorylated at Ser(28) as well as Ser(10) when premature chromosome conden sation was induced in tsBN2 cells. These observations suggest that H3 phosp horylation at Ser(28), together with Ser(10), is a conserved event and is l ikely to be involved in mitotic chromosome condensation.