Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells.
Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-delta, 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-delta were verified by Western blot analysis of immunoprecipitated proteins. PKC-delta was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-delta. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-delta. Binding of recombinant PKC-delta to NKCC1 was not detected in overlay assays. Rather, activated PKC-delta bound to actin, and this interaction was prevented by a peptide encoding deltaC2, a C2-like domain based on the amino acid sequence of PKC-delta. deltaC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-delta in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-delta indicated colocalization of PKC-delta and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-delta.
Liedtke
Carole M
CM
Warren Alan Bernbaum, M.D. Center for Cystic Fibrosis Research, Department of Pediatrics, Rainbow Babies & Children Hospital, Cleveland, Ohio 44106-4948, USA. cxl7@po.cwru.edu
Hubbard
Melinda
M
Wang
Xiangyun
X
D000098:Acetophenones / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D047630:Depsipeptides; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000378:metabolism*; D008729:Methoxamine / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D017434:Protein Structure, Tertiary / Q000502:physiology; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000098:Acetophenones; D000316:Adrenergic alpha-Agonists; D001578:Benzopyrans; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010456:Peptides, Cyclic; C578877:SLC12A2 protein, human; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D013844:Thiazoles; D053778:Thiazolidines; D002034:Bumetanide; C057531:jasplakinolide; C085746:rottlerin; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D008729:Methoxamine; C037068:latrunculin B