CHARACTERIZATION OF THE MITOTIC SPECIFIC PHOSPHORYLATION SITE OF HISTONE-H1 - ABSENCE OF A CONSENSUS SEQUENCE FOR THE P34(CDC2) CYCLIN-B-KINASE/

P-32-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated in to 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 phospho rylation rate in S and then declining in G(2) and M, 2) peptides reach ing a maximum phosphorylation rate in G(2) and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throug hout S and G(2) and reaching a maximum in M, 4) one peptide that was p hosphorylated only in M, and 5) peptides that had low levels of phosph orylation that remained constant throughout the cell cycle, Amino acid analysis and sequencing demonstrated that the mitotic specific H1 pho sphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETA PAAPAAAPPAEK 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 sequ ence is thought to be required by the p34(cdc2)/cyclin B kinase that h as maximum phosphorylating activity in mitosis, These data indicate th at 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.