Document Detail


Involvement of NH2 terminus of PKC-delta in binding to F-actin during activation of Calu-3 airway epithelial NKCC1.
MedLine Citation:
PMID:  15590896     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Nicole D Smallwood; Bryan S Hausman; Xiangyun Wang; Carole M Liedtke
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Publication Detail:
Type:  Journal Article     Date:  2004-12-08
Journal Detail:
Title:  American journal of physiology. Cell physiology     Volume:  288     ISSN:  0363-6143     ISO Abbreviation:  Am. J. Physiol., Cell Physiol.     Publication Date:  2005 Apr 
Date Detail:
Created Date:  2005-03-11     Completed Date:  2005-04-19     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  100901225     Medline TA:  Am J Physiol Cell Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  C906-12     Citation Subset:  IM    
Affiliation:
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.
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MeSH Terms
Descriptor/Qualifier:
Actins / metabolism*
Amino Acid Sequence
Animals
Binding Sites / physiology
Binding, Competitive / physiology
Cell Line
Enzyme Activation / physiology
Humans
Molecular Sequence Data
Peptides / metabolism
Protein Binding / physiology
Protein Kinase C / chemistry*,  metabolism*
Respiratory Mucosa / metabolism*
Sodium-Potassium-Chloride Symporters / metabolism*
Chemical
Reg. No./Substance:
0/Actins; 0/Peptides; 0/Sodium-Potassium-Chloride Symporters; 0/sodium-potassium-chloride cotransporter 1 protein; EC 2.7.11.13/Protein Kinase C

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