Carole M. Liedtke

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.

Cl⁻transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl⁻transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-ε)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-δ). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-ε decreased the amount of PKC-ε and reduced cAMP-dependent activation of CFTR but not α₁-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-δ did not affect CFTR function but did block α₁-adrenergic activation of NKCC1 and reduce PKC-δ mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.