The early phase of the stimulatory effect of aldosterone on sodium reabsorption in renal epithelia is thought to involve activation of apical sodium channels. However, the genes initiating this effect are unknown. We used a combination of polymerase chain reaction-based subtractive hybridization and differential display techniques to identify aldosterone-regulated immediate early genes in renal mineralocorticoid target cells. We report here that aldosterone rapidly increases mRNA levels of a putative Ser/Thr kinase, sgk (or serum-and glucocorticoid-regulated kinase), in its native target cells, i.e. in cortical collecting duct cells. The effect occurs within 30 min of the addition of aldosterone, is mediated through mineralocorticoid receptors, and does not require de novo protein synthesis. The fulllength sequences of rabbit and mouse sgk cDNAs were determined. Both cDNAs show significant homology to rat and human sgk (88 -94% at the nucleotide level, and 96 -99% at the amino acid level). Coexpression of the mouse sgk in Xenopus oocytes with the three subunits of the epithelial Na ؉ channel results in a significantly enhanced Na ؉ current. These results suggest that sgk is an immediate early aldosterone-induced gene, and this protein kinase plays an important role in the early phase of aldosterone-stimulated Na ؉ transport.

The serum-and glucocorticoid-induced kinase (sgk) is a serine and threonine kinase that stimulates amiloridesensitive sodium transport in Xenopus oocytes. Because aldosterone induces phosphorylation on serine/threonine (Ser/Thr) residues in the carboxyl termini of ␤ and ␥ subunits of epithelial sodium channels (ENaCs) and causes an increase in the sgk transcript in mammalian and amphibian renal epithelial cells, it seems likely that sgk mediates the action of aldosterone to stimulate sodium transport. Experiments were performed in Xenopus oocytes to determine the mechanism by which sgk increases sodium conductance by examining its effect on phosphorylation, kinetics, and membrane abundance of ENaC. Our results demonstrate that deletions of the carboxyl termini of the three subunits do not inhibit sgk-induced sodium current, indicating that the effect of sgk is not mediated via phosphorylation within the carboxyl termini of ENaC. They also show no evidence that sgk reduces the removal of ENaC from the plasma membrane because mutations of tyrosine residues in the sequences necessary for endocytosis and degradation did not affect the response to sgk. Further studies performed with the patch-clamp technique indicated that sgk did not increase the open probability or changed the kinetics of ENaC. These studies, however, showed a 3-fold increase in the abundance of ENaC in the plasma membrane in the presence of sgk compared with control. Together, the experiments indicate that sgk stimulates electrogenic sodium transport by increasing the number of ENaCs at the cell surface and suggest that sgk may mediate the early increase in aldosterone-induced sodium current.

Highlights d SGK1 regulates autophagy in both C. elegans and mammalian cells d Elevated autophagy and mPTP opening shorten lifespan in sgk-1/mTORC2 mutant worms d SGK-1 phosphorylates mPTP component VDAC1 on Ser104, promoting its degradation d Loss of SGK function exaggerates mPTP-dependent hepatic ischemia/reperfusion injury

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is an AGC kinase that regulates membrane sodium channel expression in renal tubular cells in an mTORC2-dependent manner. We hypothesized that SGK1 might represent a novel mTORC2-dependent regulator of T cell differentiation and function. Here we demonstrate that upon activation by mTORC2, SGK1 promoted TH2 differentiation by negatively regulating the NEDD4-2 E3 ligase-mediated destruction of transcription factor JunB. Simultaneously, SGK1 repressed the production of interferon-γ (IFN-γ) by controlling the expression of the long isoform of transcription factor TCF-1. Consistent with these findings, mice with a selective deletion of SGK1 in T cells were resistant to experimentally induced asthma, generated robust amounts of IFN-γ in response to viral infections and more readily rejected tumors.

Results on the subcellular localization of the mineralocorticoid receptor (MR) have been controversial. To determine the subcellular distribution and trafficking of the MR in living cells after binding of agonists and antagonists, we expressed a MR-green f luorescent protein (GFP) chimera in mammalian cells lacking endogenous MR. The GFP-tagged MR (GFP-MR) remained transcriptionally active, as determined in cotransfection experiments with the MR-responsive reporter, TAT3-LUC. The subcellular localization of GFP-MR was monitored by f luorescence time-lapse microscopy. In the absence of hormone, MR was present both in the cytoplasm and nucleus. Aldosterone induced a rapid nuclear accumulation of the MR. Aldosterone-bound GFP-MR was concentrated in prominent clusters within the nucleus, whereas GFP-MR did not form clusters in the absence of hormone. Similar subnuclear distribution was observed with corticosterone, another MR agonist. In the presence of the MR antagonists spironolactone or ZK91587 the rate of nuclear translocation was significantly slower and the final nuclearto-cytoplasmic ratio in steady state was significantly lower than with aldosterone. In addition, MR antagonists did not induce formation of nuclear GFP-MR clusters. MR antagonists also were able to disrupt pre-existing nuclear clusters formed in the presence of aldosterone. GFP-MR clusters were retained in nuclear matrix preparations after in vivo crosslinking. These data strongly suggest that hormoneactivated MRs accumulate in dynamic discrete clusters in the cell nucleus, and this phenomenon occurs only with transcriptionally active mineralocorticoids.The mineralocorticoid receptor (MR) is a member of the steroid-thyroid receptor superfamily. These receptors are ligand-dependent transcription factors generally located in the nucleus even in the absence of the hormone (see ref. 1 for review). The glucocorticoid receptor (GR), however, is an exception, because it is mainly cytoplasmic in the absence of ligand, and nuclear translocation occurs only after hormone binding (1). The subcellular localization of the MR is still unclear. Because the MR shows significant sequence homology to the GR, one would expect it to also be cytoplasmic in the absence of hormone. However, previous data on its localization are controversial. On the one hand, earlier biochemical and immunocytochemical studies found the unliganded MR to be cytoplasmic (2, 3). Similar findings were reported for the recombinant MR overexpressed in Sf9 insect cells and mouse macrophages (4). On the other hand, immunohistochemistry of kidney sections from adrenalectomized animals revealed both nuclear and cytoplasmic staining (2, 5, 6). In addition, a study with enucleated GH3 cells localized MR entirely to the nucleus (7). All previous studies were done by using fixed and permeabilized cells and antibodies, and methodological differences might have contributed to the controversial results. Furthermore, these studies did not permit examination of the in vivo kinetics of receptor t...