Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1.
Direct binding of nonmuscle F-actin and the C2-like domain of PKC-delta (deltaC2-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 deltaC2-like domain. Truncations of the deltaC2-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-delta with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the deltaC2-like domain blocked binding of activated PKC-delta to F-actin. The deltaC2-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 microM). NH2-terminal deltaC2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the deltaC2-like domain encodes a beta1-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 beta1-sheet region of the deltaC2-like domain in airway epithelial cells.
Smallwood
Nicole D
ND
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, OH 44106-4948, USA.
Hausman
Bryan S
BS
Wang
Xiangyun
X
Liedtke
Carole M
CM
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
D000199:Actins; D010455:Peptides; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D011493:Protein Kinase C