Importin-alpha mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain.

The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase-dependent phosphorylation of Sgk. Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.

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.

The stimulus-dependent co-localization of serum- and glucocorticoid-regulated protein kinase (Sgk) and Erk/MAPK in mammary tumor cells involves the mutual interaction with the importin-alpha nuclear import protein.

In Con8 rat mammary epithelial tumor cells, indirect immunofluorescence revealed that Sgk (serum- and glucocorticoid-regulated kinase) and Erk/MAPK (extracellular signal-regulated protein kinase/mitogen activated protein kinase) co-localized to the nucleus in serum-treated cells and to the cytoplasmic compartment in cells treated with the synthetic glucocorticoid dexamethasone. Moreover, the subcellular distribution of the importin-alpha nuclear transport protein was similarly regulated in a signal-dependent manner. In vitro GST-pull down assays revealed the direct interaction of importin-alpha with either Sgk or Erk/MAPK, while RNA interference knockdown of importin-alpha expression disrupted the localization of both Sgk and Erk into the nucleus of serum-treated cells. Wild type or kinase dead forms of Sgk co-immunoprecipitated with Erk/MAPK from either serum- or dexamethasone-treated mammary tumor cells, suggesting the existence of a protein complex containing both kinases. In serum-treated cells, nucleus residing Sgk and Erk/MAPK were both hyperphosphorylated, indicative of their active states, whereas, in dexamethasone-treated cells Erk/MAPK, but not Sgk, was in its inactive hypophosphorylated state. Treatment with a MEK inhibitor, which inactivates Erk/MAPK, caused the relocalization of both Sgk and ERK to the cytoplasm. We therefore propose that the signal-dependent co-localization of Sgk and Erk/MAPK mediated by importin-alpha represents a new pathway of signal integration between steroid and serum/growth factor-regulated pathways.

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.