Role for airway epithelial RACK1 in the apical expression of CFTR

We have demonstrated a role for protein kinase C epsilon (PKCε) in the regulation of cAMP‐dependent CFTR Cl channel function via binding of PKCε to RACK1, a receptor for activated C kinase, and of RACK1 to human Na + /H + exchanger regulatory factor (NHERF1). The goal of the present study was to determine the role of RACK in regulating CFTR function in a Calu‐3 airway epithelial cells. Confocal microscopy showed apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which selectively binds to NHERF1, but not RACK1, via a PDZ1 domain, and pulls down endogenous NHERF1, but not RACK1, from Calu‐3 total cell lysate. In binding and pulldown assays, we show inhibition of NHERF1‐tubulin binding by a PDZ1 domain, direct binding of a PDZ2 domain to NHERF1, and pulldown of endogenous NHERF1 by a PDZ2 domain. Downregulation of RACK1 using double‐stranded short hairpin turn silencing RNA reduced the amount of RACK1 by 77.5 % and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR was not affected by treatment with siRACK1. Based on these results, we model a RACK1 proteome consisting of PKC□‐RACK1‐NHERF1‐NHERF1‐tubulin which explains the apical localization of RACK1 and suggests a role for the proteome in stable expression of CFTR in the apical plasma membrane of epithelial cells. Supported by a grant from the NIH, RO1‐67190.