Protein Kinase Cε-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

Abstract

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⑀ was required for cAMP-dependent CFTR function. The goal of this study was to determine whether PKC⑀ interacts directly with CFTR. Using overlay assay, immunoprecipitation, pulldown and binding assays, we show that PKC⑀ 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

The Role of the C Terminus and Na+/H+ Exchanger Regulatory Factor in the Functional Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Nonpolarized Cells and Epithelia

“…NHERF overexpression may facilitate CFTR phosphorylation by anchoring the catalytic subunit of PKA via the NHERFezrin macromolecular complex to the vicinity of the channel (28,29,31). Since the activation kinetics of the PKA-stimulated whole cell current were not influenced by NHERF or the dominant negative D1 overexpression (Fig.…”

“…NHERF Potentiates the PKA-dependent Activation of CFTR-NHERF may facilitate CFTR activation at the cell membrane by inducing conformational changes, promoting CFTR phosphorylation by anchoring the catalytic subunit of PKA/protein kinase C to the vicinity of the channel (28,29,31), and/or increasing the channel number (32,34). To examine whether NHERF can have a modulatory effect on the channel activity in intact cells, we compared the effect of NHERF and D1 domain on the PKA-stimulated whole cell chloride current activity.…”

“…Channel activation is facilitated by a conformational change of CFTR induced by binding to the multivalent NHERF and 70-kDa CFTR-associated protein, as found in excised inside-out patches (13,30). Furthermore, NHERF may promote the phosphorylation of CFTR by tethering PKA and protein kinase C⑀ to the vicinity of the channel (28,31).…”

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

“…Immunoblotting of PDZ Adapter Proteins-NHERF and E3KARP were detected using antibodies described previously (20,21) at a dilution of 1:3000 each. PDZK1 and IKEPP were detected using commercially available antibodies (catalogue numbers PA3-16818 and PA3-16819ABR, Golden, CO) at a dilution of 1:1000 each.…”

Interactions of connexins with other membrane channels and transporters

“…High affinity association of multivalent PDZ domain-containing proteins with the conserved C-terminal TRL motif has been demonstrated. For example, the C-terminal of CFTR forms a binding site for the first PDZ domain of EBP50 (ezrin/radixin/moesin-binding phosphoprotein of 50 kDa), an adaptor protein concentrated at the apical membrane of numerous cell types, including airway epithelial cells (Liedtke et al, 2002). EBP50 interacts with ezrin, which acts as an actin binding protein, therefore forming a complex linked to the cytoskeleton.…”