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RECEPTORS AND SIGNAL TRANSDUCTION

Involvement of NH₂ terminus of PKC- in binding to F-actin during activation of Calu-3 airway epithelial NKCC1

Willard A. Bernbaum Cystic Fibrosis Research Center, Department of Pediatrics, Rainbow Babies and Children’s Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio

Submitted 1 October 2004 ; accepted in final form 7 December 2004

Direct binding of nonmuscle F-actin and the C2-like domain of PKC- (C2-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 C2-like domain. Truncations of the C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His₆-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 His₆ probe for expression of the His₆ tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC- with nonmuscle F-actin. Peptides from the NH₂-terminal region, but not the COOH-terminal region, of the C2-like domain blocked binding of activated PKC- to F-actin. The C2-like domain and three NH₂-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC₅₀ values ranging from 1.2 to 2.2 nmol (6–11 µM). NH₂-terminal C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH₂ terminus of the C2-like domain encodes a 1-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 1-sheet region of the C2-like domain in airway epithelial cells.

truncation; protein kinase C-; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter

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