Cyclic Adenosine 3′, 5′-Monophosphate-Dependent Phosphorylation of HMG 14 Inhibits Its Interactions with Nucleosomes

Abstract

The high mobility group protein HMG 14, which is preferentially associated with nucleosomes containing active gene sequences, is phosphorylated on different sites according to the tissue and stimulus being studied. In the thyroid, HMG 14 displays TSH-dependent phosphorylation that is mediated by cAMP-dependent protein kinase (A-kinase). We have, therefore, studied how phosphorylation of HMG 14 on its major and minor A-kinase sites (Ser-6 and -24) affects its interactions with nucleosomes and various forms of DNA, since this could reflect a means of regulating its function of binding to active chromatin. Approximately twice as much Ser-6 phospho- and 4 times as much Ser-6,24 diphospho-HMG 14 were required to produce the same degree of nucleosome band displacement as that caused by native unphosphorylated HMG 14. Phosphorylation also reduced the ability of HMG 14 to protect the ends of nucleosomal DNA from thermal denaturation. When the electrophoretic mobility of naked DNA was examined, the Ser-6 phospho-HMG 14 was about half as effective as native HMG 14 in retarding the various forms of double stranded DNA, and Ser-6,24 diphospho-HMG 14 was even less effective. Our data demonstrate that electrostatic interactions between DNA and basic amino acids in two highly conserved regions (residues 1-5 and 16-27) can be modulated by phosphorylation at Ser-6 and Ser-24. The ability of mammalian HMG 14, but not HMG 17, to display hormone-dependent phosphorylation may indicate a route for differentially modulating their binding to transcriptionally active chromatin.

Mitotic Phosphorylation Prevents the Binding of HMGN Proteins to Chromatin

“…Footprinting (1), cross-linking (4,16,60), and site-directed mutagenesis (48) experiments suggest that the interaction of HMGN proteins with nucleosome cores involved multiple precise, but weak, interactions. The interaction of the proteins with nucleosomes is also affected by posttranslational modifications (3,25,55). Indeed, we find that phosphorylation of serine 6 in the N-terminal region of HMGN1 weakens the binding of the protein to chromatin, but to a lesser degree than that previously noted by Spaulding et al (55).…”

“…HMGN1 contains 10 serine residues located at positions 6,7,20,24,44,45,85,88, and 98, while HMGN2 contains only two serines located at positions 24 and 28. Earlier studies on the location of phosphorylated residues in HMGN1 and -N2 gave conflicting results; however, recent studies suggest that the major phosphorylation sites of this family are serine 6 of HMGN1 and the two serine residues in the conserved NBD, located at positions 20 and 24 in HMGN1 and at positions 24 and 28 in HMGN2 (2,39,55). We generated antibodies against these sites: anti-S6P specific for human HMGN1 phosphorylated at serine 6 and anti-NBD2P specific for HMGN1 and -N2 phosphorylated in the NBD.…”

“…Several potential phosphorylation sites in HMGN1 and -N2 have been previously identified (reviewed in references 20 and 30); however, recent studies indicate that serine 6 in HMGN1 and the two conserved serines in the NBD region of the HMGN1 and -N2 protein family are the only in vivo phosphorylation sites (2,39,55). Even a detailed analysis of okadaic acid-treated cells failed to reveal additional phosphorylation sites in these proteins (39).…”

“…Both HMGN1 and HMGN2 are acetylated (3,25,56,57) and phosphorylated (2,13,39,55; see also references 20 and 30), modifications which are known to affect their target interactions and cellular localization (3,25,39,40,44,55).…”

Alleviation of Histone H1-Mediated Transcriptional Repression and Chromatin Compaction by the Acidic Activation Region in Chromosomal Protein HMG-14

“…Such a mode of access for HMG-14, in particular, is consistent with a potential role in the rapid regulation of gene expression in response to a variety of extracellular signals. As opposed to HMG-17, HMG-14 is specifically and rapidly phosphorylated in response to both mitogenic stimuli and thyroid-stimulating hormone (6,56) and is rapidly acetylated in response to estrogen (10,47).…”