Phosphorylation by cdc2 kinase modulates DNA binding activity of high mobility group I nonhistone chromatin protein.
Chromatin high mobility group protein I (HMG-I) is a mammalian nonhistone protein that has been demonstrated both in vitro and in vivo to preferentially bind to A.T-rich sequences of DNA. Recently the DNA-binding domain peptide that specifically mediates the in vitro interaction of high mobility group protein (HMG)-I with the narrow minor groove of A.T-DNA has been experimentally determined. Because of its predicted secondary structure, the binding domain peptide has been called "the A.T hook" motif. Previously we demonstrated that the A.T hook of murine HMG-I protein is specifically phosphorylated by purified mammalian cdc2 kinase in vitro and that the same site(s) are also phosphorylated in vivo in metaphase-arrested cells. We also found that the DNA binding affinity of short synthetic binding domain peptides phosphorylated in vitro by cdc2 kinase was significantly reduced compared with unphosphorylated peptides. Here we extend these findings to intact natural and recombinant HMG-I proteins. We report that the affinity of binding of full-length HMG-I proteins to A.T-rich sequences is highly dependent on ionic conditions and that phosphorylation of intact proteins by cdc2 kinase reduces their affinity of in vitro binding to A.T-DNA by about 20-fold when assayed near normal mammalian physiological salt concentrations. Furthermore, in cell synchronization studies, we demonstrated that murine HMG-I proteins are phosphorylated in vivo in a cell cycle-dependent manner on the same amino acid residues modified by purified cdc2 kinase in vitro. Together these results strongly support the assertion that HMG-I proteins are natural substrates for mammalian cdc2 kinase in vivo and that their cell cycle-dependent phosphorylation by this enzyme(s) significantly modulates their DNA binding affinity, thereby possibly altering their biological function(s).
Department of Biochemistry/Biophysics, Washington State University, Pullman 99164-4660.
M S
MS
Nissen
T A
TA
Langan
R
R
Reeves
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010449:Peptide Mapping; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D014357:Trypsin
D002868:Chromosomal Proteins, Non-Histone; D006609:High Mobility Group Proteins; D011994:Recombinant Proteins; D004247:DNA; D016203:CDC2 Protein Kinase; D014357:Trypsin