Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC188

Poster Communications

Cyclic AMP-induced phosphorylation of endogenous Nedd-4/2 and surface abundance of epithelial channel subunits (α-, β- and γ-ENaC) in H441 human airway epithelial cells

N. A. Ismail1, S. M. Wilson1

1. Centre for cardiovascular and Lung Biology, University of Dundee, Dundee, United Kingdom.

  • Fig. 1. (A) Confluent cells on 10 cm Petri dishes were lysed in the presence of protease and phosphatase inhibitors so that Nedd-4/2 could be immunopurified from 1 mg aliquots of extracted protein and subject to Western analysis using antibodies against the Ser221-, Thr246- and Ser327-phosphorylated and total forms of this protein. (B) Abundance of α-, β- and γ-ENaC in surface-exposed protein fraction isolated by biotinylation / streptavidin-binding. Dex: 0.2 μM dexamethasone for ~24 h; cAMP: 10 μM forskolin, 100 μM isobutylmethylxanthine, 1 mM N6,2’-O-dibutyryl adenosine 3’5’-cyclic monophosphate for the final 20 min of this incubation period.

H441 human distal airway epithelial cells expresses an endogenous Na+ conductance identical to that associated with α-, β- and γ-ENaC co-expression. This conductance is quiescent in hormone-deprived cells but can be activated by dexamethasone, and this glucocorticoid-induced Na+ conductance (GNa) is further augmented by brief (20 min) activation of protein kinase A (PKA) (Clunes et al., 2004). Glucocorticoids seem to control ENaC via serum and glucocorticoid-inducible kinase 1 (SGK1), a kinase (Lang et al., 2006) that phosphorylates residues (Ser221, Thr246, Ser327) in Nedd-4/2, a ubiquitin ligase that normally targets ENaC for internalization / degradation. By phosphorylating Nedd-4/2, SGK1 seems to prevent this interaction with ENaC thus allowing the channels to remain in the membrane leading to a rise in GNa and stimulation of Na+ transport. Moreover, since PKA can also phosphorylate Nedd-4/2-Ser221, -Thr246 and -Ser327, it has been suggested that cAMP-coupled agonists also augment GNa by increasing the surface abundance of α-, β- and γ-ENaC (Snyder et al., 2004). We have therefore explored the effects of PKA activation upon the phosphorylation of endogenous Nedd-4/2 and the surface abundance of α-, β- and γ-ENaC in H441 cells. Dexamethasone (0.2 μM, 24 h) had little effect upon Nedd-4/2 phosphorylation, but did evoke phosphorylatin of this protein after only 3 h stimulation (n = 3, not shown), a finding which accords with data showing that dexamethasone activates SGK1 transiently (Ismail et al. 2011). The cAMP-elevating drugs evoked phosphorylation of the cAMP response element binding protein Ser133 confirming activation of PKA, and this response was accompanied by a increase in the abundance of Ser221-, Thr246- and Ser327-phosphorylated Nedd-4/2 both in hormone-deprived and dexamethasone-stimulated cells. Interestingly, this response was always accompanied by a marked increase in the overall abundance of Nedd-4/2(Fig. 1). Dexamethasone (24 h) increased the surface abundance of α-ENaC with no such effect upon β- or γ-ENaC (see also Ismail et al. 2011). Activating PKA independently of dexamethasone (n = 4) mimicked the effects of glucocorticoid stimulation by increasing the surface amount of α-ENaC with no effect on the β- or γ-subunits (Fig. 4B). In 5 out of 11 experiments, exposing dexamethasone-stimulated cells to cAMP did not affect the surface abundance of α-, β- or γ-ENaC (Fig. 1B), but increased surface expression of β- and γ-ENaC, but not α-ENaC, was seen in 6 instances. Whilst it clearly phosphorylates Nedd-4/2 (Fig 1A) the PKA-induced increases in GNa (Clunes et al. 2004) do not necessarily correlate with changes to the surface expression of ENaC subunits.

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