Role of the scaffold protein RACK1 in apical expression of CFTR.
Previous studies from this laboratory demonstrated a role for protein kinase C (PKC)epsilon in the regulation of cAMP-dependent cystic fibrosis transmembrane regulator (CFTR) Cl channel function via binding of PKCepsilon 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 PKCepsilon-RACK1-NHERF1-NHERF1-tubulin with a role in stable expression of CFTR in the apical plasma membrane of epithelial cells.
Auerbach
Michael
M
Department of Pediatrics, Rainbow Babies & children Hospital, Case Western Reserve University, BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948, USA.
Liedtke
Carole M
CM
D000199:Actins / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D046912:Multiprotein Complexes / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D046912:Multiprotein Complexes; D009363:Neoplasm Proteins; D010750:Phosphoproteins; C419368:RACK1 protein, human; D034741:RNA, Small Interfering; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D017923:Sodium-Hydrogen Exchangers; D014404:Tubulin; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D051744:Protein Kinase C-epsilon; D019204:GTP-Binding Proteins