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Am J Physiol Cell Physiol 284: C487-C496, 2003. First published October 16, 2002; doi:10.1152/ajpcell.00357.2002
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Vol. 284, Issue 2, C487-C496, February 2003

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

Carole M. Liedtke, Melinda Hubbard, and Xiangyun Wang

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)-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 delta C2, a C2-like domain based on the amino acid sequence of PKC-delta . delta C2 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 .

C2-like domain; coimmunoprecipitation; protein kinase C; rottlerin; jasplakinolide; latrunculin; sodium-potassium-chloride cotransport


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