mTOR Complex-2 Activates ENaC by Phosphorylating SGK1

Abstract

The serum-and glucocorticoid-induced kinase 1 (SGK1) plays a central role in hormone regulation of epithelial sodium (Na ϩ ) channel (ENaC)-dependent Na ϩ transport in the distal nephron. Phosphorylation within a carboxy-terminal domain, designated the hydrophobic motif (HM), determines the activity of SGK1, but the identity of the HM kinase is unknown. Here, we show that the highly conserved serine-threonine kinase mammalian target of rapamycin (mTOR) is essential for the phosphorylation of the HM of SGK1 and the activation of ENaC. We observed that mTOR, in conjunction with rictor (mTORC2), phosphorylated SGK1 and stimulated ENaC. In contrast, when mTOR assembled with raptor in the rapamycin-inhibited complex (mTORC1), it did not phosphorylate SGK1 or stimulate ENaC. Inhibition of mTOR blocked both SGK1 phosphorylation and ENaC-mediated Na ϩ transport, whereas specific inhibition of mTORC1 had no effect.Similarly, small hairpin RNA-mediated knockdown of rictor inhibited SGK1 phosphorylation and Na ϩ current, whereas knockdown of raptor had no effect. Finally, in co-immunoprecipitation experiments, SGK1 interacted selectively with rictor but not with raptor, suggesting selective recruitment of SGK1 to mTORC2. We conclude that mTOR, specifically mTORC2, is the HM kinase for SGK1 and is required for ENaC-mediated Na ϩ transport, thereby extending our understanding of the molecular mechanisms underlying Na ϩ balance.

mTORC2 regulates renal tubule sodium uptake by promoting ENaC activity

“…The recent development of TORKinibs, specific catalytic site inhibitors of mTOR, has allowed for characterization of distinct physiological functions for mTORC1 and mTORC2 (18). Previously, we used the TORKinib PP242 to demonstrate in the mouse cortical collecting duct (CCD) cell line mpkCCD that mTORC2-mediated SGK1 phosphorylation is required for generating ENaC-dependent Na + current (15). To determine whether mTOR activity is required in vivo for renal Na + retention, we treated WT mice with 30 mg/kg PP242 i.p.…”

“…mTOR inhibition led to a significant increase in net Na + excretion and a trend toward increased urine output compared with vehicle-treated mice ( Figure 1, A and B, and Table 1); the latter did not reach statistical significance, possibly due to the short half-life of PP242 lation at a site within its C-terminal hydrophobic motif (S422), followed by phosphorylation at a second site within the activation loop (T256) for complete catalytic activation. Phosphoinositidedependent kinase 1 (PDK1) has been well established as the activation loop kinase, and more recently, mTOR was identified as the hydrophobic motif kinase (12,15,16). mTOR exists in 2 evolutionarily conserved complexes, mTORC1 and mTORC2, and controls a diverse array of physiological processes, including protein translation, proliferation, migration, and metabolism.…”

“…RPS6 is phosphorylated by p70S6K, a direct substrate of mTORC1. Because of the poor quality of the phosphospecific SGK1 hydrophobic motif antibody for detection of the endogenous phosphoprotein (15,20), in order to measure SGK1 phosphorylation, we assessed the disappearance of slower-migrating forms of SGK1 using an antibody against total SGK1. The slower-migrating forms of SGK1 observed 3F).…”

“…Only recently, with the generation of specific catalytic-site inhibitors of mTOR (termed TORKinibs), has it been possible to distinguish between mTORC1 and mTORC2 (18). Using the TORKinib PP242 in addition to genetic depletion of Rictor or Raptor, we previously demonstrated in cultured cells that mTORC2 is the hydrophobic motif kinase for SGK1 and that its activity is required for ENaCdependent Na + reabsorption (15). PP242, unlike rapamycin, equally inhibits the kinase regardless of whether it is in mTORC2 or mTORC1.…”

Regulation of OSR1 and the sodium, potassium, two chloride cotransporter by convergent signals

“…SGKs and the related Akt enzymes are activated by phosphorylation on multiple sites, most prominently a residue in the activation loop by the phosphoinositide-dependent protein kinase and on a second site in a C-terminal hydrophobic motif (25). The kinase that phosphorylates the hydrophobic motif site under many circumstances is the mammalian target of rapamycin complex 2 (mTORC2), which provides an additional phosphatidylinositol-3 kinase (PI3K)-dependent input to these kinases (26)(27)(28)(29)(30)(31)(32)(33).…”