Sodium homeostasis in terrestrial and freshwater vertebrates is controlled by the corticosteroid hormones, principally aldosterone, which stimulate electrogenic Na ؉ absorption in tight epithelia. Although aldosterone is known to increase apical membrane Na ؉ permeability in target cells through changes in gene transcription, the mechanistic basis of this effect remains poorly understood. The predominant early effect of aldosterone is to increase the activity of the epithelial sodium channel (ENaC), although ENaC mRNA and protein levels do not change initially.Rather, the open probability and͞or number of channels in the apical membrane are greatly increased by unknown modulators. To identify hormone-stimulated gene products that modulate ENaC activity, a subtracted cDNA library was generated from A6 cells, a stable cell line of renal distal nephron origin, and the effect of candidates on ENaC activity was tested in a coexpression assay. We report here the identification of sgk (serum and glucocorticoid-regulated kinase), a member of the serine-threonine kinase family, as an aldosterone-induced regulator of ENaC activity. sgk mRNA and protein were strongly and rapidly hormone stimulated both in A6 cells and in rat kidney. Furthermore, sgk stimulated ENaC activity approximately 7-fold when they were coexpressed in Xenopus laevis oocytes. These data suggest that sgk plays a central role in aldosterone regulation of Na ؉ absorption and thus in the control of extracellular f luid volume, blood pressure, and sodium homeostasis.

Serum and glucocorticoid-inducible kinase (SGK) is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated. In this study, we have investigated the regulatory mechanisms that control SGK activity. We have established a peptide kinase assay for SGK and present evidence demonstrating that SGK is a component of the phosphoinositide 3 (PI 3)-kinase signaling pathway. Treatment of human embryo kidney 293 cells with insulin, IGF-1 or pervanadate induced a 3-to 12-fold activation of ectopically expressed SGK. Activation was completely abolished by pretreatment of cells with the PI 3-kinase inhibitor, LY294002. Treatment of activated SGK with protein phosphatase 2A in vitro led to kinase inactivation. Consistent with the similarity of SGK to other secondmessenger regulated kinases, mutation of putative phosphorylation sites at Thr256 and Ser422 inhibited SGK activation. Cotransfection of PDK1 with SGK caused a 6-fold activation of SGK activity, whereas kinase-dead PDK1 caused no activation. GST-pulldown assays revealed a direct interaction between PDK1 and the catalytic domain of SGK. Treatment of rat mammary tumor cells with serum caused hyperphosphorylation of endogenous SGK, and promoted translocation to the nucleus. Both hyperphosphorylation and nuclear translocation could be inhibited by wortmannin, but not by rapamycin.

The serum-and glucocorticoid-inducible kinase (sgk) is a novel serine/threonine protein kinase that is transcriptionally regulated in rat mammary tumor cells by serum under proliferative conditions or by glucocorticoids that induce a G 1 cell cycle arrest. Our results establish that the subcellular distribution of Sgk is under stringent cell cycle and hormonal control. Sgk is localized to the perinuclear or cytoplasmic compartment as a 50-kDa hypophosphorylated protein in cells arrested in G 1 by treatment with the synthetic glucocorticoid dexamethasone. In serum-stimulated cells, Sgk was transiently hyperphosphorylated and resided in the nucleus. Laser scanning cytometry, which monitors Sgk localization and DNA content in individual mammary tumor cells of an asynchronously growing population, revealed that Sgk actively shuttles between the nucleus (in S and G 2 /M) and the cytoplasm (in G 1 ) in synchrony with the cell cycle. In cells synchronously released from the G 1 /S boundary, Sgk localized to the nucleus during progression through S phase. The forced retention of exogenous Sgk in either the cytoplasmic compartment, using a wild type sgk gene, or the nucleus, using a nuclear localization signal-containing sgk gene (NLS-Sgk), suppressed the growth and DNA synthesis of serumstimulated cells. Thus, our study implicates the nuclearcytoplasmic shuttling of sgk as a requirement for cell cycle progression and represents a novel convergence point of anti-proliferative and proliferative signaling in mammary tumor cells.A dynamic balance of steroid hormones, protein growth factors, and other environmental cues coordinately regulates an intricate network of intracellular processes that stringently control mammalian cell proliferation (1-5). A large body of literature has characterized the individual cellular events activated by either steroid hormones (6 -10) or by protein hormones and growth factors (3,(11)(12)(13)(14)(15), which trigger the two principal signal transduction pathways that eukaryotic cells employ to respond to external stimuli. To understand the functional connections between the transcriptional events regulated by nuclear steroid receptors (7,8,10,16, 17) and the cascades of phosphorylation-dephosphorylation reactions mediated by the cell-surface growth factor receptors (3,11,13,15,18), a crucial issue was to define the key steps at which these signal transduction pathways converge. There are a variety of potential mechanisms of cross-talk between growth factor and steroid-responsive pathways (5, 19). These regulatory steps include steroid-mediated changes in the expression of growth factors, their cognate receptors, and components of phosphorylation and dephosphorylation cascades (20 -29). In other cellular contexts, the phosphorylation of steroid receptors can alter their function and target gene specificity (30, 31). The regulation of cell signaling events in the nucleus for the coordinate control of target genes allows cells to respond to external stimuli in a physiologically appropriate manne...