Characterization of the Mitotic Specific Phosphorylation Site of Histone H1

Abstract

32P-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated into 15 phosphopeptides by high performance liquid chromatography. These phosphopeptides were grouped into five classes having different cell cycle phosphorylation kinetics: 1) peptides reaching a maximum phosphorylation rate in S and then declining in G 2 and M, 2) peptides reaching a maximum phosphorylation rate in G 2 and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throughout S and G 2 and reaching a maximum in M, 4) one peptide that was phosphorylated only in M, and 5) peptides that had low levels of phosphorylation that remained constant throughout the cell cycle. Amino acid analysis and sequencing demonstrated that the mitotic specific H1 phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is phosphorylated on both the Ser and Thr, does not contain the consensus sequence (S/T)PXZ (where X is any amino acid and Z is a basic amino acid). This sequence is thought to be required by the p34 cdc2 /cyclin B kinase that has maximum phosphorylating activity in mitosis. These data indicate that this kinase either does not have an obligatory requirement for the consensus sequence in vivo as generally believed or that it is not the enzyme responsible for the mitotic specific H1 phosphorylation.For over 27 years, the phosphorylation of histone H1 has been thought to play a role in controlling the cell cycle (Ord and Stocken, 1968). To examine this possibility our laboratory used synchronized CHO 1 cells to determine the cell cycle kinetics of histone phosphorylation during cell proliferation (see review by Gurley et al. (1978a)). In those studies it was found that histone H2A and H4 phosphorylations were cell cycle-independent and probably not involved in cell cycle control, while histone H1 and H3 phosphorylations were cell cycle-dependent and, therefore, more likely to have a role in cell cycle control.

Mitosis-Specific Hyperphosphorylation of Epstein-Barr Virus Nuclear Antigen 2 Suppresses Its Function

“…A number of proteins are subjected to mitosis-specific phosphorylation, which in most cases is involved with p34 cdc2 kinase (22,36,40). To test whether the p34 cdc2 kinase might be involved in phosphorylation of EBNA-2, we carried out in vitro kinase assays by using purified p34 cdc2 kinase.…”

Histone H1 and its isoforms: Contribution to chromatin structure and function

“…A correlation between the phosphorylation of H1 histones and mitotic chromosome condensation was further demonstrated by treatment of mammalian cells with the kinase inhibitor staurosporine and with the DNA-binding dye Hoechst 33342, respectively. Both substances not only prevented H1 histone phosphorylation, but also entry into mitosis (Th'ng et al, 1994;Gurley et al, 1995). When murine cells were blocked in mitosis with nocodazole, the addition of staurosporine caused rapid H1 histone dephosphorylation accompanied by chromosome decondensation (Th'ng et al, 1994).…”

Histone H1 Phosphorylation Occurs Site-specifically during Interphase and Mitosis

“…When investigating Chinese hamster ovary cells Gurley et al (11) found that in interphase histone H1 is phosphorylated exclusively in the C-terminal region and that during mitosis only it is also phosphorylated in the N-terminal end. With regard to H1 interphase phosphorylation the authors supposed that there is no qualitative cell cycle specificity as to which site is phosphorylated first and that the number of phosphates per H1 molecule is more important than which sites are phosphorylated.…”

“…The p34cdc2/cyclin B kinase, having maximum activity at mitosis (10), was expected to be the enzyme responsible for phosphorylation of the mitotic-specific serine and threonine sites of histone H1. Surprisingly, in Chinese hamster ovary cells mitotic-specific phosphorylation of both serine and threonine residues was found to be located at the end of the N-terminal region of H1, which has no (S/T)PXZ consensus sequences (11). Only few data exist about site-specific phosphorylation during interphase.…”

“…Only few data exist about site-specific phosphorylation during interphase. Gurley et al (11) assumed that there is no absolute cell cycle-specific site of phosphorylation and that during interphase, the number of phosphates per H1 molecule is more important than which sites are phosphorylated. On the other hand, Mizzen et al (12) demonstrated that phosphorylation of macronuclear H1 in the ciliated protozoan Tetrahymena occurs hierarchically during interphase.…”