Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC188
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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