Stability of actin cytoskeleton and PKC-δ 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)-, 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-.