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

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Abstract
Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-δ, 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-δ were verified by Western blot analysis of immunoprecipitated proteins. PKC-δ was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-δ. Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-δ. Binding of recombinant PKC-δ to NKCC1 was not detected in overlay assays. Rather, activated PKC-δ bound to actin, and this interaction was prevented by a peptide encoding δC2, a C2-like domain based on the amino acid sequence of PKC-δ. δC2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-δ in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-δ indicated colocalization of PKC-δ and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-δ.

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