Protein kinase C epsilon-dependent regulation of cystic fibrosis transmembrane regulator involves binding to a receptor for activated C kinase (RACK1) and RACK1 binding to Na+/H+ exchange regulatory factor.
Protein kinase C (PKC) regulation of cystic fibrosis transmembrane regulator (CFTR) chloride function has been demonstrated in several cell lines, including Calu-3 cells that express native, wild-type CFTR. We demonstrated previously that PKC epsilon was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC epsilon interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC epsilon does not bind to CFTR, but does bind to a receptor for activated C kinase (RACK1), a 37-kDa scaffold protein, and that RACK1 binds to Na(+)/H(+) exchange regulatory factor (NHERF1), a binding partner of CFTR. In vitro binding assays demonstrate dose-dependent binding of PKC epsilon to RACK1 which is inhibited by an 8-amino acid peptide based on the sequence of the sixth Trp-Asp repeat in RACK1 or by an 8-amino acid sequence in the V1 region of PKC epsilon, epsilon V1-2. A 4-amino acid sequence INAL (70-73) expressed in CFTR shares 50% homology to the RACK1 inhibitory peptide, but it does not bind PKC epsilon. NHERF1 and RACK1 bind in a dose-dependent manner. Immunofluorescence and confocal microscopy of RACK1 and CFTR revealed colocalization of the proteins to the apical and lateral regions of Calu-3 cells. The results indicate the RACK1 binds PKC epsilon and NHERF1, thus serving as a scaffold protein to anchor the enzyme in proximity to CFTR.
Liedtke
Carole M
CM
Warren Alan Bernbaum, M.D. Center for Cystic Fibrosis Research, Department of Pediatrics, Rainbow Babies and Children Hospital and Case Western Reserve University, Cleveland, Ohio 44106-4948, USA. cxl7@po.cwru.edu
Yun
C H Chris
CH
Kyle
Nicole
N
Wang
Dandan
D
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins; D006801:Humans; D015151:Immunoblotting; D007313:Insecta; D007527:Isoenzymes / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers
C505032:CFTR protein, human; D007527:Isoenzymes; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D010455:Peptides; D010750:Phosphoproteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D019204:GTP-Binding Proteins