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

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.

mTOR complex-2 activates ENaC by phosphorylating SGK1.

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.

20-HETE inhibits the proliferation of vascular smooth muscle cells via transforming growth factor-beta.

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 arachidonic acid metabolite, has been shown to modulate the growth of vascular smooth muscle cells (VSMCs). We asked whether 20-HETE modulates the proliferation of R22D cells, a clonal VSMC from neonatal rats, by releasing transforming growth factor-beta (TGF-beta). Incubation of R22D cells with 20-HETE for 24 h attenuated [(3)H]thymidine incorporation in a concentration-dependent manner without causing the release of lactate dehydrogenase. 20-HETE also inhibited platelet-derived growth factor (PDGF)-induced [(3)H]thymidine incorporation in R22D cells and human VSMCs. At 5 muM, 20-HETE reduced [(3)H]thymidine incorporation by 34 +/- 6%; anti-TGF-beta neutralizing antibody, but not nonspecific IgG, completely reversed the attenuated [(3)H]thymidine incorporation induced by 20-HETE. In addition, 20-HETE attenuated fetal bovine serum- and PDGF-induced expression of cyclin D1, a downstream effector of TGF-beta(1), which was reversed by anti-TGF-beta antibody. Further studies demonstrated that 20-HETE may increase TGF-beta release to a level high enough to inhibit [(3)H]thymidine incorporation without altering the steady-state mRNA level of TGF-beta. Nevertheless, pretreatment of indomethacin (a cyclooxygenase inhibitor) or paxilline (a potassium channel inhibitor) did not affect the inhibitory effect on DNA synthesis induced by 20-HETE. These results demonstrate for the first time a growth-inhibitory effect induced by 20-HETE, which may be mediated by TGF-beta.

Chloride-dependent calcium transients induced by angiotensin II in vascular smooth muscle cells.

Cl- is essential for the vasoconstrictive response to angiotensin II (ANG II). In vascular smooth muscle cells (VSMC), we determined whether ANG II-induced transient increase in intracellular Ca2+ concentration ([Ca2+]i) is Cl- dependent. After incubating the cells at different extracellular Cl- concentration ([Cl-]e) for 40 min, the ANG II-induced Ca2+ transients at 120 meq/l Cl- were more than twice those at either 80 or 20 meq/l Cl-. Replacing Cl- with bicarbonate or gluconate yielded similar results. In addition, after removal of extracellular Ca2+, ANG II-induced as well as platelet-derived growth factor-induced Ca2+ release exhibited Cl- dependency. The difference of Ca2+ release with high vs. low [Cl-]e was not affected by acutely altering [Cl-]e 1 min before administration of ANG II when [Cl-]i was yet to be equilibrated with [Cl-]e. Pretreatment of a Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid, increased ANG II-induced Ca2+ release and entry at 20 meq/l Cl- but did not alter those at 120 meq/l Cl-. However, after equilibration, a reduced [Cl-]e did not affect thapsigargin-induced Ca2+ release, suggesting that Cl- may not affect the size of intracellular Ca2+ stores. Nevertheless, at high [Cl-], the peak increase of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] induced by ANG II was approximately sixfold that at low [Cl-]. Thus the Cl- -dependent effects of ANG II on Ca2+ transients may be mediated, at least in part, by a Cl- -dependent Ins(1,4,5)P3 accumulation in VSMC.