Inhibitory Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Anion-transporting Activities by Shank2

Abstract

Accumulating evidence suggests that protein-protein interactions play an important role in transepithelial ion transport. In the present study, we report on the biochemical and functional association between cystic fibrosis transmembrane conductance regulator (CFTR) and a PDZ domain-containing protein Shank2. Exploratory reverse transcription-PCR screening revealed mRNAs for several members of PDZ domain-containing proteins in epithelial cells. Shank2, one of these scaffolding proteins, showed a strong interaction with CFTR by yeast two-hybrid assays. Shank2-CFTR interaction was verified by co-immunoprecipitation experiments in mammalian cells. Notably, this interaction was abolished by mutations in the PDZ domain of Shank2. Protein phosphorylation, HCO 3 ؊ transport and Cl ؊ current by CFTR were measured in NIH 3T3 cells with heterologous expression of Shank2. Of interest, expression of Shank2 suppressed cAMP-induced phosphorylation and activation of CFTR. Importantly, loss of Shank2 by stable transfection of antisense-hShank2 plasmid strongly increased CFTR currents in colonic T84 cells, in which CFTR and Shank2 were natively expressed. Our results indicate that Shank2 negatively regulates CFTR and that this may play a significant role in maintaining epithelial homeostasis under normal and diseased conditions such as those presented by secretory diarrhea.Secretory epithelia perform vectorial transport of salt and water molecules by coordinated actions of the transporters expressed in polarized epithelial membranes. One of the key membrane proteins regulating overall fluid movements is the cystic fibrosis transmembrane conductance regulator (CFTR), 1 which itself has an anion-transporting activity (1-3). Aberrant membrane transport caused by either hypo-or hyper-functioning of CFTR, can be detrimental, and may result in life-threatening diseases, such as cystic fibrosis or secretory diarrhea (4, 5). Therefore, the fine regulation of salt and water transport is essential in epithelial and body homeostasis.Accumulating evidence suggests that protein-protein interaction performs an important role in the regulation of transepithelial ion transport (6). Clustering of ion transporters and associated proteins in microdomains of polarized epithelia can facilitate the effective secretion or absorption of salt and water molecules. In this regard, modular adaptor proteins such as PDZ (PSD-95/discs large/ZO-1) domain-containing proteins have drawn increasing attention due to their ability to form supramolecular complexes (7). We have previously shown that the regulatory interaction between CFTR and Na ϩ /H ϩ exchanger 3 (NHE3) through PDZ-based scaffolds is essential for the coordinated regulation of pancreatic bicarbonate secretion (8). In addition, it was found that a number of membrane transporters and receptors participating in pancreatic fluid formation, such as Na ϩ -HCO 3 Ϫ cotransporters (NBC), purinergic receptors, and secretin receptors have a PDZ-binding motif on their C terminus (9, 10). Therefore, multiple pro...

Serum- and Glucocorticoid-induced Protein Kinase 1 (SGK1) Increases the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Airway Epithelial Cells by Phosphorylating Shank2E Protein

“…Although WT Shank2E moderately enhanced the effect of dexamethasone on CFTR, overexpression of WT Shank2E did not significantly increase the effect of dexamethasone on CFTR compared with dexamethasonetreated cells transfected with the empty vector. A similar observation was made in NIH 3T3 cells in which Shank2 overexpression tended to increase CFTR, but the effect was not significant (38). By contrast, mut-Shank2E attenuated the effect of dexamethasone on whole cell and plasma membrane CFTR (Fig.…”

“…However, this study is notable in that it demonstrates, for the first time, that the Shank2E isoform mediates an increase in cell lysate and plasma membrane CFTR and that this effect is dependent on SGK1 phosphorylation of the first SGK1 consensus site in Shank2E, which is not present in the short Shank2 isoform. Thus, the Shank2-induced increase in plasma membrane CFTR in NIH 3T3 cells heterologously expressing CFTR and Shank2 is unlikely to be mediated by phosphorylation via SGK1 (38).…”

“…The C termini of the sodium-coupled phosphate transporter, NHE3, and CFTR interact with Shank2, and this interaction is required for the Shank2-mediated increase in plasma membrane abundance of these transport proteins (17,(37)(38)(39)(40). However, this study is notable in that it demonstrates, for the first time, that the Shank2E isoform mediates an increase in cell lysate and plasma membrane CFTR and that this effect is dependent on SGK1 phosphorylation of the first SGK1 consensus site in Shank2E, which is not present in the short Shank2 isoform.…”

“…Shank2E contains six N-terminal ankyrin repeats and an SH3 domain, which are absent in the short Shank2 isoform (37,43). Previous studies have shown that Shank2 interacts with CFTR in the rat colon and pancreas as well as in heterologous cells overexpressing both proteins (38,40) and that heterologous expression of Shank2 in NIH 3T3 cells increased plasma membrane CFTR by 39%, although the increase was not statistically significant (38). Both isoforms contain a shared C-terminal SGK1 consensus site, and Shank2E contains an additional site in the N-terminal region that is not present in the short Shank2 isoform (Fig.…”

“…Shank2E Is Expressed in CFBE Cells-Shank2 isoforms are expressed at the apical membrane in epithelial cells in the pancreas, colon, liver, and kidney (38,43). However, expression has not yet been characterized experimentally in airway epithelial cells.…”