mSIN1 protein mediates SGK1 protein interaction with mTORC2 protein complex and is required for selective activation of the epithelial sodium channel

Abstract

The mammalian target of rapamycin (mTOR) plays a central role in the regulation of a number of cellular processes including growth, metabolism, and ion transport. mTOR is found in two multiprotein complexes, mTORC1 and mTORC2, which phosphorylate distinct substrates and regulate distinct cellular processes. SGK1 is an mTORC2 substrate, which is a key regulator of epithelial Na(+) transport mediated by the epithelial sodium channel. Although it is known that SGK1 physically interacts with mTORC2, it is unknown which mTORC2 component mediates this interaction or whether this interaction plays a physiologically relevant role in specific activation of SGK1. Here we identify mSIN1 as the mTORC2 component that mediates interaction with SGK1 and demonstrate that this interaction is required for SGK1 phosphorylation and epithelial sodium channel activation. We used the yeast two-hybrid system coupled with random mutagenesis to identify a mutant mSIN1 (mSIN1/Q68H), which does not interact with SGK1. Expression of this mutant does not restore SGK1 phosphorylation to wild-type levels in mSIN1-deficient murine embryo fibroblasts. Furthermore, in kidney epithelial cells, mSIN1/Q68H has a dominant-negative effect on SGK1 phosphorylation and on SGK1-dependent Na(+) transport. Interestingly, this interaction appears to be specific in that another mTORC2 substrate, Akt, does not interact with mSIN1, and its phosphorylation and activity are unaffected by the Q68H mutation. These data support the conclusion that mTORC2 uses distinct strategies to phosphorylate different substrates and suggest a mechanism for mTORC2 specificity in the regulation of diverse cellular processes.

FIGURE 1.

In vitro interaction between mSIN1 and SGK1. A, mSIN1 and the mSIN1/Q68H mutant were expressed as MBP fusions in Escherichia coli and purified on amylose resin. The purified mSIN1 protein, the mutant protein, and the native MBP protein are shown after Coomassie Blue staining. B, wild-type or Q68H mutant mSIN1 fusion protein or native MBP was bound to amylose resin and incubated with in vitro translated SGK1 protein. Bound proteins were recovered by boiling and analyzed by immunoblot (IB) for SGK1. Shown is a representative blot of two independent experiments.

FIGURE 2.

Effect of mSIN1/Q68H on assembly of mTORC2 and interaction of mTORC2 with SGK1. A, FLAG-tagged mSIN1 (wild-type or Q68H mutant) was co-transfected with HA-tagged SGK1 in HEK 293T cells, lysed, and subjected to immunoprecipitation with anti-FLAG antibody followed by immunoblotting (IB), as described under “Experimental Procedures.” Blots were probed with antibodies against mTOR, Rictor, and Raptor, respectively. B, blots as in A were probed with anti-HA antibody to detect SGK1. C, blots probed with anti-p-SGK1 antibody. D, mpkCCD cells were transduced with recombinant adenoviruses harboring FLAG-tagged wild-type mSIN1 or mSIN1/Q68H mutant. mSIN1 immunoprecipitation was carried out using anti-FLAG antibody, and immunoblots were probed as shown.

FIGURE 3.

Attenuation of SGK1 phosphorylation in mSIN1-deficient MEFs harboring mSIN1/Q68H. A, HA-tagged SGK1 expression plasmid was transfected into mSIN1-deficient MEFs along with wild-type mSIN1 or the mSIN1/Q68H mutant. 48 h after transfection, the cells were lysed, and immunoprecipitation was carried out using agarose beads cross-linked with anti-HA antibodies. Immunoblotting (IB) was subsequently performed using antibodies as shown. WCL, whole cell lysates. B, HA-tagged Akt plasmid was transfected into mSIN1-deficient MEFs along with wild-type or mutant mSIN1, as in A, and Akt was immunoprecipitated with anti-HA antibody. Immunoblots were probed with antibodies as shown. pAkt, phosphorylated Akt. C, wild-type mSIN1 or the mSIN1/Q68H mutant was transfected into mSIN1-deficient MEFs. Immunoblotting was performed on whole cell lysates using antibodies against PKCα phospho-S657 and total PKCα. Note the dependence of PKCα phosphorylation (pPKC) and expression, as described previously (38).

FIGURE 4.

Effect of mSIN1/Q68H on SGK1 HM phosphorylation and physical interaction with mTORC2. A, HA-tagged SGK1 plasmid was transfected into HEK 293T cells along with wild-type mSIN1 or mSIN1/Q68H. 48 h after transfection, transfected cells were lysed and immunoprecipitated with anti-HA antibody and immunoblotted (IB) for phospho-S422 SGK1, total SGK1, mSIN1, and Rictor, respectively. B, densitometric quantitation of phospho-SGK1 (three independent experiments). Values were significantly different in the presence of wild-type versus mutant mSIN1 (p < 0.01) by Student's unpaired t test. Error bars indicate S.E.

FIGURE 5.

Effect of mSIN1/Q68H on Akt regulation by mTORC2. A, Akt physically associates with mTORC2 in the presence of either wild-type or Q68H mutant mSIN1. HEK 293T cells were transfected with FLAG-tagged wild-type or mutant mSIN1, and after 48 h, cells were lysed and immunoprecipitated with the anti-FLAG antibody. Immunoblots (IB) were probed with anti-Akt antibody. B, mSIN1/Q68H does not have dominant-negative activity on Akt S473 phosphorylation. Wild-type or mutant mSIN1 was transfected into HEK 293T cells, and after 48 h, cells were lysed and probed with anti-Akt phospho-S473 antibody. The mTOR inhibitor, PP242, was used as positive control. C, Akt-mediated glucose uptake is not inhibited by mSIN1/Q68H in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were transduced with recombinant adenoviruses harboring wild-type mSIN1 or the mSIN1/Q68H mutant. Transduced cells were stimulated with insulin, and glucose uptake was monitored by incubation with ³H-labeled 2-deoxy glucose (n = 3). There was no significant difference in glucose uptake between cells expressing wild-type and mutant mSIN1 (by Student's unpaired t test). PP242-treated cells were significantly different from cells expressing wild-type or mutant mSIN1 (p < 0.01 by unpaired t test). Error bars indicate S.E. D, transduced 3T3-L1 cells were lysed, and expression of mSIN1 and the mSIN1/Q68H mutant was determined by Western blotting. moi, multiplicity of infection.

FIGURE 6.

Inhibition of ENaC-dependent Na⁺ current by mSIN1/Q68H. A, mpkCCD cells were transduced with recombinant adenoviruses harboring mSIN1 or the mSIN1/Q68H mutant. Transduced cells were plated on Transwell filters, and after reaching high electrical resistance (24–72 h), amiloride-sensitive equivalent current was measured by Evometer. Values in the bar graph are significantly different (p < 0.01) by Student's unpaired t test in three independent experiments. Error bars indicate S.E. B, transduced cells were lysed, and expression of mSIN1 and the mSIN1/Q68H mutant was determined by Western blotting (IB). moi, multiplicity of infection.