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Am J Physiol Cell Physiol (October 16, 2002). doi:10.1152/ajpcell.00357.2002
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Articles in PresS, published online ahead of print October 16, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00357.2002
Submitted on August 1, 2002
Accepted on October 8, 2002

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

Carole M Liedtke1*, Melinda Hubbard1, and Xiangyun Wang1

1 Pediatrics, Case Western Reserve University, Cleveland, OH, USA

* To whom correspondence should be addressed. E-mail: CXL7{at}PO.CWRU.EDU.

Activation of airway epithelial NKCC1 requires increased activity of PKC-{delta}, which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signalling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization using jasplakinolide increased activity of NKCC1 whereas inhibition of actin polymerization using latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions between 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} indicate 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}.




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