cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na(+) channel through convergent phosphorylation of Nedd4-2.
The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.
Snyder
Peter M
PM
Department of Internal Medicine, and Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. psnyder@blue.weeg.uiowa.edu
Olson
Diane R
DR
Kabra
Rajesh
R
Zhou
Ruifeng
R
Steines
Jennifer C
JC
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D006801:Humans; D017874:Immediate-Early Proteins; D017136:Ion Transport; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012964:Sodium; D015222:Sodium Channels; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human