In vitro phosphorylation of COOH termini of the epithelial Na⁺channel and its effects on channel activity inXenopusoocytes

Abstract

Recent findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na(+) channel (ENaC). This study reports the in vitro phosphorylation of the COOH termini of ENaC subunits expressed as glutathione S-transferase fusion proteins. Channel subunits were specifically phosphorylated by kinase-enriched cytosolic fractions derived from rat colon. The phosphorylation observed was not mediated by the serum- and glucocorticoid-regulated kinase sgk. For the gamma-subunit, phosphorylation occurred on a single, well-conserved threonine residue located in the immediate vicinity of the PY motif (T630). The analogous residue on beta(S620) was phosphorylated as well. The possible role of gammaT630 and betaS620 in channel function was studied in Xenopus laevis oocytes. Mutating these residues to alanine had no effect on the basal channel-mediated current. They do, however, inhibit the sgk-induced increase in channel activity but only in oocytes that were preincubated in low Na(+) and had a high basal Na(+) current. Thus mutating gammaT630 or betaS620 may limit the maximal channel activity achieved by a combination of sgk and low Na(+).

Interactions of β and γENaC with Nedd4 Can Be Facilitated by an ERK-mediated Phosphorylation

“…Recombinant DNA and Proteins-The carboxyl termini of the rat ␤ and ␥ENaC (␤ 557-638 and ␥ 564 -650) were subcloned downstream GST in the bacterial expression vector pGEX3X as described in a previous study (14). cDNA constructs expressing fusion proteins between GST and the three WW domains of rat Nedd4 were kindly provided by D. Rotin (Hospital for Sick Children, Toronto, Canada) and are described in a previous study (5).…”

“…cDNA constructs expressing fusion proteins between GST and the three WW domains of rat Nedd4 were kindly provided by D. Rotin (Hospital for Sick Children, Toronto, Canada) and are described in a previous study (5). GST fusion proteins were expressed in a protease-deficient Escherichia coli strain and purified on glutathione beads as detailed previously (14). Functional expression in Xenopus oocytes was done using ENaC clones in pSPORT-1, obtained from B. C. Rossier (Institute of Pharmacology, University of Lausanne).…”

“…We have recently demonstrated phosphorylation of the carboxyl termini of ENaC subunits, expressed as glutathione Stransferase (GST) fusion proteins by crude cytosolic fractions (14). The current study characterizes conserved residues phosphorylated in the carboxyl termini of ENaC subunits, explores their physiological role, and identifies the kinase involved.…”

A Novel Pathway of Epithelial Sodium Channel Activation Involves a Serum- and Glucocorticoid-inducible Kinase Consensus Motif in the C Terminus of the Channel's α-Subunit

“…In A6 cells a baseline phosphorylation of the ␣-and ␤-subunits of ENaC was detected, whereas the ␥-subunit was found to be either weakly phosphorylated or not phosphorylated (48). More recently, in vitro studies have confirmed the phosphorylation of certain threo-nine and serine residues of the ␤-and ␥-subunits, whereas no significant phosphorylation was found in the C terminus of the ␣-subunit (33)(34)(35). In this context it is interesting to note that SGK1 has been reported to physically interact with the Cterminal tails of ␣-ENaC and ␤-ENaC in vitro.…”

“…Aldosterone, insulin, and protein kinases A and C have been shown to increase in vivo phosphorylation of the C termini of the ␤-and ␥-subunits of ENaC (32). Moreover, the C termini of ENaC subunits expressed as glutathione S-transferase fusion proteins were found to be phosphorylated by cytosolic fractions derived from rat colon (33). This phosphorylation is thought to involve at least three different types of kinases, including the extracellular-regulated kinase (34) and casein kinase 2 (35).…”