Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells

• Published in: American Journal of Physiology: Cell Physiology Vol. 284; no. 2; pp. C487 - C496
• Main Authors: Liedtke, Carole M, Hubbard, Melinda, Wang, Xiangyun
• Format: Journal Article
• Language: English
• Published: United States 01.02.2003
• Subjects: Acetophenones - pharmacology; Actin Cytoskeleton - drug effects; Actin Cytoskeleton - metabolism; Adrenergic alpha-Agonists - pharmacology; Animals; Benzopyrans - pharmacology; Bridged Bicyclo Compounds, Heterocyclic - pharmacology; Bumetanide - pharmacology; Cell Compartmentation - drug effects; Cell Compartmentation - physiology; Cell Membrane - drug effects; Cell Membrane - metabolism; Cells, Cultured; Cytosol - metabolism; Depsipeptides; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; Epithelial Cells - cytology; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Humans; Immunohistochemistry; Isoenzymes - metabolism; Methoxamine - pharmacology; Peptides, Cyclic - pharmacology; Protein Binding - drug effects; Protein Binding - physiology; Protein Kinase C - metabolism; Protein Kinase C-delta; Protein Structure, Tertiary - physiology; Respiratory Mucosa - cytology; Respiratory Mucosa - drug effects; Respiratory Mucosa - metabolism; Sodium-Potassium-Chloride Symporters - metabolism; Solute Carrier Family 12, Member 2; Thiazoles - pharmacology; Thiazolidines
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00357.2002

Warren Alan Bernbaum, M.D. Center for Cystic Fibrosis Research, Department of Pediatrics, Rainbow Babies & Children Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948 Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)- , which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC- were verified by Western blot analysis of immunoprecipitated proteins. PKC- was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC- . Pulldown of endogenous actin revealed the presence of NKCC1 and PKC- . Binding of recombinant PKC- to NKCC1 was not detected in overlay assays. Rather, activated PKC- bound to actin, and this interaction was prevented by a peptide encoding C2, a C2-like domain based on the amino acid sequence of PKC- . C2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC- in the cytosol and cell periphery. Merged images of cells stained for actin and PKC- indicated colocalization of PKC- and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC- . C2-like domain; coimmunoprecipitation; protein kinase C; rottlerin; jasplakinolide; latrunculin; sodium-potassium-chloride cotransport