Lee Il-Ha, Dinudom Anuwat, Sanchez-Perez Angeles, Kumar Sharad, Cook David I
Discipline of Physiology, School of Medical Science, Faculty of Medicine, University of Sydney, Sydney, New South Wales 2006, Australia.
J Biol Chem. 2007 Oct 12;282(41):29866-73. doi: 10.1074/jbc.M701923200. Epub 2007 Aug 22.
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.
上皮钠通道（ENaC）在跨上皮钠（Na⁺）吸收中起重要作用；因此其功能对于维持Na⁺和液体平衡以及调节血压至关重要。胰岛素是调节ENaC活性的激素之一。在本研究中，我们调查了两种相关蛋白激酶，Akt（也称为蛋白激酶B）和血清及糖皮质激素依赖激酶（Sgk），对在表达ENaC的Fisher大鼠甲状腺细胞中胰岛素诱导的ENaC活性的作用。Akt1或Sgk1的过表达显著增加了ENaC活性，而Akt1的显性负性构建体Akt1（K179M）的表达则降低了ENaC的基础活性。通过短干扰RNA抑制Akt1和Sgk1的内源性表达不仅抑制了ENaC，还破坏了胰岛素以及胰岛素下游效应物磷脂酰肌醇3激酶和PDK1对ENaC的刺激作用。相反，Akt1或Sgk1的过表达增加了细胞膜上ENaC的表达，并克服了Nedd4-2对ENaC的抑制作用。此外，Nedd4-2上Akt1和Sgk1的共有磷酸化位点Ser（342）和Ser（428）的突变完全消除了Sgk1和Akt1对Nedd4-2作用的抑制效果。这些数据共同表明，Akt和Sgk都是胰岛素信号通路的组成部分，该信号通路通过一个涉及抑制Nedd4-2的调节系统上调ENaC的膜表达，从而增加Na⁺吸收。
