Involvement of NH 2 terminus of PKC-δ in binding to F-actin during activation of Calu-3 airway epithelial NKCC1

• Published in: American Journal of Physiology: Cell Physiology 2005-04, Vol.288 (4), p.C906-C912
• Main Authors: Smallwood, Nicole D., Hausman, Bryan S., Wang, Xiangyun, Liedtke, Carole M.
• Format: Article
• Language: English
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00484.2004

Direct binding of nonmuscle F-actin and the C2-like domain of PKC-δ (δC2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid δC2-like domain. Truncations of the δC2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His 6 -tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His 6 probe for expression of the His 6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC-δ with nonmuscle F-actin. Peptides from the NH 2 -terminal region, but not the COOH-terminal region, of the δC2-like domain blocked binding of activated PKC-δ to F-actin. The δC2-like domain and three NH 2 -terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC 50 values ranging from 1.2 to 2.2 nmol (6–11 μM). NH 2 -terminal δC2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH 2 terminus of the δC2-like domain encodes a β1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the β1-sheet region of the δC2-like domain in airway epithelial cells.