SGK Regulation of Epithelial Sodium Transport

University Of California, San Francisco, San Francisco CA

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Abstract

DESCRIPTION (provided by applicant): Hormone-regulated Na+ transport in the kidney tubules is critical to the control of blood pressure and other vital processes in health and disease. The serine-threonine kinase SGK1 is one of the most intensively studied regulators of epithelial Na+ transport, and has been particularly recognized for its importance in mediating the effects of aldosterone on the epithelial sodium channel (ENaC). SGK1 is under dual control: its expression is controlled by aldosterone through effects on gene transcription, and its activity is controlled by phosphorylation involving a network of kinase cascades. Recent evidence has identified mammalian target of rapamycin (mTOR) as the kinase mediating a key gateway phosphorylation event required for SGK1 activation. We hypothesize that SGK1 physically interacts with specific component(s) of a multi-protein complex containing mTOR, which is essential for the gateway phosphorylation at serine 422 (S422). We further hypothesize that SGK1 together with this mTOR-containing complex is recruited to another protein complex, where SGK1 regulates ENaC by phosphorylating targets such as Nedd4-2. With these hypotheses in mind, our specific aims for this competing renewal are to: 1: Determine the mechanistic basis of mTOR physical association with, and phosphorylation of SGK1. There are two mTOR-containing multi-protein complexes, mTORC1 and mTORC2, which have distinct components and substrates, and regulate distinct cellular processes. We will: A) Examine the physical interactions of individual mTORC1 & 2 components with SGK1 using the yeast two hybrid assay and in vitro interaction. B) Identify domains and specific amino acids within SGK1 and mSin1, which mediate their interaction. C) Determine if physical interaction between mSin1 and SGK1 is necessary for SGK1 to be phosphorylated by mTORC2. D) Examine mTORC2 regulation of SGK1 relatives, Akt, SGK2, and SGK3. 2: Characterize the functional effects of mTOR and SGK1 on ENaC in cultured cortical collecting duct (CCD) cells. This aim will establish the functional role of the above characterized physical interactions and SGK1 modifications in controlling ENaC-mediated Na+ transport in cultured cells. We will: A) Assess the functional role of SGK1 as a mediator of mTOR-dependent activation of ENaC in a CCD cell line, using a combination of small molecule inhibitors of mTOR and SGK1, and genetic manipulation. B) Assess the functional implications of SGK1 interaction with mTORC2 by characterizing the effect of mutants on SGK1 phosphorylation and ENaC current. C) Characterize the effect of cellular conditions and hormonal milieu on mTOR-dependent activation of SGK1 and ENaC. 3: Characterize the role of mTOR and SGK1 in Na+ homeostasis in vivo. In order to advance these findings into native tissues, we will: A) Examine the effects of mTOR activators and inhibitors on Na+ balance, blood pressure, and ENaC subcellular localization in wild type and SGK1-null mice. B) Examine mTOR regulation of amiloride-inhibitable transepithelial Na+ current in isolated perfused CCD harvested from aldosterone-treated mice. C) Examine the role of mTOR in regulation of amiloride-inhibitable currents in isolated CCD using patch clamp.