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+) channel (ENaC)-dependent Na+ 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+ transport, whereas specific inhibition of mTORC1 had no effect. Similarly, small hairpin RNA-mediated knockdown of rictor inhibited SGK1 phosphorylation and Na+ 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+ transport, thereby extending our understanding of the molecular mechanisms underlying Na+ balance.

Figure 1.

Rapamycin-resistant SGK1 phosphorylation is modulated by mTOR. (A) mpkCCD cells were grown on collagen-coated Transwell polycarbonate membranes and treated with aldosterone and insulin for 4 hours, followed by treatment with inhibitors as shown for 1 hour. Lysates were prepared and analyzed by immunoblot using antibodies against holo-SGK1, phospho-HM, Akt phospho-S473, and phospho-p70S6K, as shown. α-Tubulin is shown as loading control. (B) Phosphatase treatment eliminates mobility-shifted phospho-SGK1 band. mpkCCD cells were grown on Transwell filters and treated with aldosterone and insulin as in A, and whole-cell lysates were treated with λ-phosphatase (λ-PPase) before Western blotting analysis with antibodies, as shown. (C) Immunodetection of HM phosphorylated SGK1 using a commercially available antibody (S422; Santa Cruz Biotechnology). HEK-293 cells were transfected with FLAG-SGK1 or vector control; incubated with insulin for 4 hours; and treated with inhibitors for 1 hour at concentrations of 20 μM for LY, 0.3 μM for PP242, and 0.1 μM for rapamycin. FLAG-SGK1 was enriched by immunoprecipitation with anti-FLAG antibody (see text for details) and immunoblotted as shown.

Figure 2.

SGK1 phosphorylation is inhibited by knockdown of the expression of rictor. (A) FLAG-SGK1 plasmid was transfected into HEK-293 cells; 24 hours later, cells were infected with recombinant lentiviruses harboring rictor shRNA or control shRNA. After another 24 hours, cells were lysed and analyzed by Western blot using antibodies against rictor; shown is representative blot and quantitative analysis of at least three independent experiments. (B) FLAG-SGK1 was immunoprecipitated from cell lysates in A and analyzed by immunoblot using S422 antibody, as in Figure 1C. The ratio of the signal from anti–p-Sgk1 against anti–holo-Sgk1 was determined and normalized for SGK1 phosphorylation. Shown is a representative blot and quantitative analysis. In each graph, values were significantly different (P < 0.01) by unpaired t test. Quantification was performed in at least three independent experiments.

Figure 3.

Raptor knockdown has no effect on SGK1 phosphorylation. (A) FLAG-SGK1 plasmid was transfected into HEK-293 cells; after 24 hours, cells were infected with recombinant lentiviruses harboring raptor or control shRNA. After another 24 hours, cells were lysed and analyzed by Western blotting using antibodies against raptor. (B) SGK1 was immunoprecipitated from cell lysates in A and analyzed for HM phosphorylation. The ratio of the signal from anti–p-Sgk1 against anti–holo-Sgk1 was determined and normalized for SGK1 phosphorylation. In each graph, values were significantly different (P < 0.01) by unpaired t test, except where shown as NS. Quantification was performed in at least three independent experiments.

Figure 4.

Rapamycin-resistant ENaC-dependent Na⁺ current is modulated by mTOR. (A) mpkCCD cells were grown on Transwell filters, incubated with aldosterone and insulin for 4 hours, and treated with inhibitors for 1 hour at concentrations of 20 μM for LY, 0.3 μM for PP242, and 0.1 μM for rapamycin. Amiloride-sensitive equivalent current was measured by mini–volt-ohm meter (see the Concise Methods section). (B) Concentration dependence of Na⁺ current inhibition by PP242. Cells were treated with aldosterone and insulin as in A and incubated for 2 hours with concentrations of PP242 shown, and equivalent current was determined.

Figure 5.

ENaC-dependent Na⁺ current is inhibited by knockdown of rictor expression. (A) mpkCCD cells were infected with recombinant lentiviruses harboring rictor shRNA or control shRNA. Infected cells were plated on Transwell filters, and, after reaching high electrical resistance (24 to 72 hours), amiloride-sensitive ENaC-dependent Na⁺ currents were measured from 12 Transwell filters for each group in three independent experiments. (B) Infected cells were lysed, and rictor level was determined by Western blot. Values in bar graphs are significantly different (P < 0.01) by unpaired t test in at least three independent experiments.

Figure 6.

Raptor knockdown has no effect on ENaC-dependent Na⁺ current. (A) mpkCCD cells were infected with recombinant lentiviruses harboring raptor or control shRNA and plated on Transwell filters, and amiloride-sensitive ENaC-dependent Na⁺ currents were measured, as in Figure 5. (B) Infected cells were lysed, and raptor level was determined by Western blot. Values in bar graphs are significantly different (P < 0.01) by unpaired t test in at least three independent experiments. (C) Infected cells were lysed and probed by Western blot using antibodies against the raptor substrates p-p70S6K and p-4EBP1, respectively.

Figure 7.

SGK1 associates preferentially with mTORC2. A plasmid harboring FLAG-SGK1 was transfected into HEK-293 cells. A GFP-containing plasmid was used as negative control (NC). Forty-eight hours after transfection, the cells were lysed and immunoprecipitated for SGK1 with anti-FLAG antibody. Bound proteins were recovered and analyzed by Western blot using antibodies against rictor, mTOR, raptor, or the FLAG tag, respectively.

Figure 8.

SGK1 activation is mediated by mTORC2 in the regulation of ENaC. SGK1 associates with mTORC2 (delineated by dashed oval), which phosphorylates S422 in the HM. HM phosphorylated SGK1 interacts with PDK1, which phosphorylates T256 (small red circles represent phosphate groups, and red arrows represent phosphorylation events).