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PROTEIN AND VESICLE TRAFFICKING, CYTOSKELETON

Role of the scaffold protein RACK1 in apical expression of CFTR

Warren Alan Bernbaum, M.D., Center for Cystic Fibrosis Research, Departments of Pediatrics at Rainbow Babies & Children Hospital and of Physiology & Biophysics, Case Western Reserve University, Cleveland, Ohio

Submitted 2 August 2006 ; accepted in final form 2 April 2007

Previous studies from this laboratory demonstrated a role for protein kinase C (PKC) in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKC to RACK1, a receptor for activated C kinase, and of RACK1 to human Na⁺/H⁺ exchanger regulatory factor (NHERF1). In the present study, we investigated the role of RACK1 in regulating CFTR function in a Calu-3 airway epithelial cell line. Confocal microscopy and biotinylation of apical surface proteins demonstrate apical localization of RACK1 independent of actin. Mass spectrometric analysis of NHERF1 revealed copurification of tubulin, which, in in vitro binding assays, selectively binds to NHERF1, but not RACK1, via a PDZ1 domain. In binding and pulldown assays, we show direct binding of a PDZ2 domain to NHERF1, pulldown of endogenous NHERF1 by a PDZ2 domain, and inhibition of NHERF1-tubulin binding by a PDZ1 domain. Downregulation of RACK1 using double-stranded silencing RNA reduced the amount of RACK1 by 77.5% and apical expression of biotinylated CFTR by 87.4%. Expression of CFTR, NHERF1, and actin were not altered by treatment with siRACK1 or by nontargeting control silencing RNA, which, in addition, did not affect RACK1 expression. On the basis of these results, we model a RACK1 proteome consisting of PKC-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.

silencing RNA; downregulation; biotinylation; tubulin; NHERF1; tailless cystic fibrosis transmembrane regulator; PDZ domain