title: Identification of the linker histone H1 as a protein kinase Cepsilon-binding protein in vascular smooth muscle.
abstract: A variety of anchoring proteins target specific protein kinase C (PKC) isoenzymes to particular subcellular locations or multimeric signaling complexes, thereby achieving a high degree of substrate specificity by localizing the kinase in proximity to specific substrates. PKCepsilon is widely expressed in smooth muscle tissues, but little is known about its targeting and substrate specificity. We have used a Far-Western (overlay) approach to identify PKCepsilon-binding proteins in vascular smooth muscle of the rat aorta. Proteins of approximately 32 and 34 kDa in the Triton-insoluble fraction were found to bind PKCepsilon in a phospholipid/diacylglycerol-dependent manner. Although of similar molecular weight to RACK-1, a known PKCepsilon-binding protein, these proteins were separated from RACK-1 by SDS-PAGE and differential NaCl extraction and were not recognized by an antibody to RACK-1. The PKCepsilon-binding proteins were further purified from the Triton-insoluble fraction and identified by de novo sequencing of selected tryptic peptides by tandem mass spectrometry as variants of the linker histone H1. Their identity was confirmed by Western blotting with anti-histone H1 and the demonstration that purified histone H1 binds PKCepsilon in the presence of phospholipid and diacylglycerol but absence of Ca(2+). The interaction of PKCepsilon with histone H1 was specific since no interaction was observed with histones H2A, H2S or H3S. Bound PKCepsilon phosphorylated histone H1 in a phospholipid/diacylglycerol-dependent but Ca(2+)-independent manner. Ca(2+)-dependent PKC was also shown to interact with histone H1 but not other histones. These results suggest that histone H1 is both an anchoring protein and a substrate for activated PKCepsilon and other PKC isoenzymes and likely serves to localize activated PKCs that translocate to the nucleus in the vicinity of specific nuclear substrates including histone H1 itself. Since PKC isoenzymes have been implicated in regulation of gene expression, stable interaction with histone H1 may be an important step in this process.
authors.affiliation: Smooth Muscle Research Group and Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, 330 Hospital Drive N.W., Calgary, AB T2N 4N1, Canada.
authors.forename: Mingcai
authors.initials: M
authors.lastname: Zhao
authors.forename: Cindy
authors.initials: C
authors.lastname: Sutherland
authors.forename: David P
authors.initials: DP
authors.lastname: Wilson
authors.forename: Jingti
authors.initials: J
authors.lastname: Deng
authors.forename: Justin A
authors.initials: JA
authors.lastname: Macdonald
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051744:Protein Kinase C-epsilon; D051381:Rats; D017207:Rats, Sprague-Dawley; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D004075:Diglycerides; D006657:Histones; D007527:Isoenzymes; D010743:Phospholipids; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D002118:Calcium