The serum- and glucocorticoid-induced protein kinase-1 (Sgk-1) mitochondria connection: identification of the IF-1 inhibitor of the F(1)F(0)-ATPase as a mitochondria-specific binding target and the stress-induced mitochondrial localization of endogenous Sgk-1.

The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F1F0-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F1F0-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F1F0-ATPase, as part of the cellular stressed induced program.

Stimulus-dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity.

We originally discovered the serum and glucocorticoid inducible protein kinase, SGK, as a novel protein kinase that is under acute transcriptional control by serum and glucocorticoids. An expanding set of cell surface receptor, nuclear receptor, and cellular stress pathways has been shown to target SGK, which has implicated this regulated signaling molecule in a variety of biological functions. Compared to most other protein kinases, a distinguishing feature of SGK is the stringent stimulus-dependent regulation of its transcription, subcellular localization and enzymatic activity. In addition, SGK expression is regulated during discrete developmental stages, and during normal and abnormal physiological function. An analysis of the SGK promoter reveals many potential transcription factor sites that potentially account for the stimulus-dependent changes in SGK transcript expression observed in a variety of cell systems, although, the direct stimulus regulation of SGK promoter activity has been established only for glucocorticoids, p53 tumor suppressor protein, hyperosmotic stress and follicle stimulating hormone. In the systems tested to date, hormones, growth factors and environmental cues induce expression of a catalytically active SGK. It is now well established that the enzymatic activity of SGK is controlled by the PI 3-kinase cascade which produces a hyperphosphorylated active SGK. A critical third level of regulation is the stimulus-dependent control of SGK subcellular localization. The nuclear-cytoplasmic shuttling of SGK is regulated by a nuclear localization signal (NLS) that binds to the importin-alpha nuclear import receptor. Modeling of the 3-D structure of the central region of SGK that includes the kinase domain predicts that the NLS is located at an external surface of the molecule. Thus, multiple signal transduction pathways converge on SGK to control its availability, function and access to its substrates and non-substrate targets.

Expression of the serum- and glucocorticoid-inducible protein kinase, Sgk, is a cell survival response to multiple types of environmental stress stimuli in mammary epithelial cells.

The effects of multiple stress stimuli on the cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) were examined in NMuMg mammary epithelial cells exposed to hyperosmotic stress induced by the organic osmolyte sorbitol, heat shock, ultraviolet irradiation, oxidative stress induced by hydrogen peroxide, or to dexamethasone, a synthetic glucocorticoid that represents a general class of physiological stress hormones. Each of the stress stimuli induced Sgk protein expression with differences in the kinetics and duration of induction and in subcellular localization. The environmental stresses, but not dexamethasone, stimulated Sgk expression through a p38/MAPK-dependent pathway. In each case, a hyperphosphorylated active Sgk protein was produced under conditions in which Akt, the close homolog of Sgk, remained in its non-phosphorylated state. Ectopic expression of wild type Sgk or of the T256D/S422D mutant Sgk that mimics phosphorylation conferred protection against stress-induced cell death in NMuMg cells. In contrast, expression of the T256A/S422A Sgk phosphorylation site mutant has no effect on cell survival. Sgk is known to phosphorylate and negatively regulate pro-apoptotic forkhead transcription factor FKHRL1. The environmental stress stimuli that induce Sgk, but not dexamethasone, strongly inhibited the nuclear transcriptional activity and increased the cytoplasmic retention of FKHRL1. Also, the conditional IPTG inducible expression of wild type Sgk, but not of the kinase dead T256A mutant Sgk, protected Con8 mammary epithelial tumor cells from serum starvation-induced apoptosis. Taken together, our study establishes that induction of enzymatically active Sgk functions as a key cell survival component in response to different environmental stress stimuli.