Identification of the linker histone H1 as a protein kinase Cepsilon-binding protein in vascular smooth muscle

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. PKCε 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 PKCε-binding proteins in vascular smooth muscle of the rat aorta. Proteins of ~32 and 34 kDa in the Triton-insoluble fraction were found to bind PKCε in a phospholipid/diacylglycerol-dependent manner. Although of similar molecular weight to RACK-1, a known PKCε-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 PKCε-binding proteins were further purified from the Tritoninsoluble 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 PKCε in the presence of phospholipid and diacylglycerol but absence of Ca2+. The interaction of PKCε with histone H1 was specific since no interaction was observed with histones H2A, H2S or H3S. Bound PKCε phosphorylated histone H1 in a phospholipid/diacylglycerol-dependent but Ca2+-independent manner. Ca2+-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 PKCε 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.Mingcai Zhao, Cindy Sutherland, David P. Wilson, Jingti Deng, Justin A. MacDonald, and Michael P. Wals