Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2.
The epithelial sodium channel (ENaC) plays an important role in transepithelial Na(+) absorption; hence its function is essential for maintaining Na(+) and fluid homeostasis and regulating blood pressure. Insulin is one of the hormones that regulates activity of ENaC. In this study, we investigated the contribution of two related protein kinases, Akt (also known as protein kinase B) and the serum- and glucocorticoid-dependent kinase (Sgk), on insulin-induced ENaC activity in Fisher rat thyroid cells expressing ENaC. Overexpression of Akt1 or Sgk1 significantly increased ENaC activity, whereas expression of a dominant-negative construct of Akt1, Akt1(K179M), decreased basal activity of ENaC. Inhibition of the endogenous expression of Akt1 and Sgk1 by short interfering RNA not only inhibited ENaC but also disrupted the stimulatory effect on ENaC of insulin and of the downstream effectors of insulin, phosphatidylinositol 3-kinase and PDK1. Conversely, overexpression of Akt1 or Sgk1 increased expression of ENaC at the cell membrane and overcame the inhibitory effect of Nedd4-2 on ENaC. Furthermore, mutation of consensus phosphorylation sites on Nedd4-2 for Akt1 and Sgk1, Ser(342) and Ser(428), completely abolished the inhibitory effect of Sgk1 and Akt1 on Nedd4-2 action. Together these data suggest that both Akt and Sgk are components of an insulin signaling pathway that increases Na(+) absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2.
Lee
Il-Ha
IH
Discipline of Physiology, School of Medical Science, Faculty of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia.
Dinudom
Anuwat
A
Sanchez-Perez
Angeles
A
Kumar
Sharad
S
Cook
David I
DI
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004594:Electrophysiology / Q000379:methods; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005786:Gene Expression Regulation; D005938:Glucocorticoids / Q000378:metabolism; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000502:physiology*; D000099184:Serum-Glucocorticoid Regulated Kinases
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005938:Glucocorticoids; D017874:Immediate-Early Proteins; D007328:Insulin; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618479:NEDD4L protein, rat; C000618484:Nedd4 protein, rat; C000618461:Nedd4l protein, mouse; C514862:Akt1 protein, mouse; C494920:Akt1 protein, rat