Deanna R. Brickley

Activation of the glucocorticoid receptor (GR) results in diverse physiological effects depending on cell type. For example, glucocorticoids (GC) cause apoptosis in lymphocytes but can rescue mammary epithelial cells from growth factor withdrawal-induced death. However, the molecular mechanisms of GR-mediated survival remain poorly understood. In this study, a large-scale oligonucleotide screen of GR-regulated genes was performed. Several of the genes that were found to be induced 30 min after GR activation encode proteins that function in cell survival signaling pathways. We also demonstrate that dexamethasone pretreatment of breast cancer cell lines inhibits chemotherapy-induced apoptosis in a GR-dependent manner and is associated with the transcriptional induction of at least two genes identified in our screen, serum and GC-inducible protein kinase-1 (SGK-1) and mitogen-activated protein kinase phosphatase-1 (MKP-1). Furthermore, GC treatment alone or GC treatment followed by chemotherapy increases both SGK-1 and MKP-1 steady-state protein levels. In the absence of GC treatment, ectopic expression of SGK-1 or MKP-1 inhibits chemotherapy-induced apoptosis, suggesting a possible role for these proteins in GR-mediated survival. Moreover, specific inhibition of SGK-1 or MKP-1 induction by the introduction of SGK-1- or MKP-1-small interfering RNA reversed the anti-apoptotic effects of GC treatment. Taken together, these data suggest that GR activation in breast cancer cells regulates survival signaling through direct transactivation of genes that encode proteins that decrease susceptibility to apoptosis. Given the widespread clinical administration of dexamethasone before chemotherapy, understanding GR-induced survival mechanisms is essential for achieving optimal therapeutic responses.

We previously demonstrated that activation of the glucocorticoid receptor (GR) initiates an antiapoptotic signal in the immortalized human mammary epithelial cell line MCF10A that is dependent on the GR's transcriptional activity. In this study, we show that the survival role of GR activation extends to protecting human breast cancer cells undergoing apoptosis after growth factor deprivation. Serum and glucocorticoid-regulated kinase-1 (sgk), a gene previously identified as a direct transcriptional target of the activated GR in a rat mammary tumor cell line, was rapidly induced after GR activation in human mammary epithelial cells. Furthermore, in the absence of all growth factors, ectopic sgk expression inhibited apoptosis, suggesting that SGK is a survival kinase. Finally, kinase-dead SGK expression inhibited the protection from apoptosis usually seen after GR activation. These findings suggest that SGK is an important downstream target of GR-mediated survival signaling and that it is distinct from other survival kinases because it can be primarily regulated at the level of transcription. We previously demonstrated that activation of the glucocorticoid receptor (GR) initiates an antiapoptotic signal in the immortalized human mammary epithelial cell line MCF10A that is dependent on the GR's transcriptional activity. In this study, we show that the survival role of GR activation extends to protecting human breast cancer cells undergoing apoptosis after growth factor deprivation. Serum and glucocorticoid-regulated kinase-1 (sgk), a gene previously identified as a direct transcriptional target of the activated GR in a rat mammary tumor cell line, was rapidly induced after GR activation in human mammary epithelial cells. Furthermore, in the absence of all growth factors, ectopic sgk expression inhibited apoptosis, suggesting that SGK is a survival kinase. Finally, kinase-dead SGK expression inhibited the protection from apoptosis usually seen after GR activation. These findings suggest that SGK is an important downstream target of GR-mediated survival signaling and that it is distinct from other survival kinases because it can be primarily regulated at the level of transcription. glucocorticoid receptor mammary epithelial cell hemagglutinin epidermal growth factor fetal calf serum phosphate-buffered saline dexamethasone 21-mesylate glyceraldehyde-3-phosphate dehydrogenase phosphatidylinositol 3-kinase The specificity of the glucocorticoid receptor's (GR)1 transcriptional activity varies widely between cell types, thus accounting for the diverse and sometimes opposite physiological effects of glucocorticoid in different tissues. For example, glucocorticoids have been shown to promote apoptosis in lymphocytes (1Wyllie A. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4153) Google Scholar, 2Schwartzman R. Cidlowski J. Int. Arch. Allergy Immunol. 1994; 105: 347-354Crossref PubMed Scopus (114) Google Scholar), whereas human mammary epithelial cells (MECs) (3Moran T. Gray S. Mikosz C. Conzen S. Cancer Research. 2000; 60: 867-872PubMed Google Scholar) and rat hepatoma epithelial cells (4Evans-Storms R. Cidlowski J. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (84) Google Scholar) are protected from apoptosis after GR activation. Furthermore, studies in both breast and liver epithelial cells have demonstrated that RU486, a potent GR antagonist that inhibits GR-mediated transcriptional activation, reverses the survival effect of glucocorticoids (3Moran T. Gray S. Mikosz C. Conzen S. Cancer Research. 2000; 60: 867-872PubMed Google Scholar, 4Evans-Storms R. Cidlowski J. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (84) Google Scholar). The antagonistic effects of RU486 on cell survival suggest that glucocorticoid-mediated survival is regulated specifically through GR-induced transactivation of downstream genes. Previous studies using glucocorticoid concomitantly with or without the GR antagonist RU486 have suggested that the identification of genes directly induced or repressed by GR activation might reveal important pathways relevant to epithelial cell survival signaling. One such GR-inducible gene, serum and glucocorticoid-regulated kinase-1 (sgk), encodes a serine/threonine kinase with 54% homology in its catalytic domain to the well described antiapoptotic kinase AKT.sgk was originally identified through subtractive cloning of a serum and glucocorticoid-induced mammary tumor cell cDNA library (5Webster M. Goya L. Ge Y. Maiyar A. Firestone G. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (497) Google Scholar). More recently, sgk was found to be part of a larger gene family and was designated sgk-1 (6Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (529) Google Scholar). Interestingly,sgk has also been shown to be transcriptionally induced after activation of a variety of steroid receptors including the mineralocorticoid receptor in kidney epithelial cells (7Naray-Fejes-Toth A. Canessa C. Cleaveland E. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar) and the follicle-stimulating hormone receptor in ovarian granulosa cells (8Alliston T. Maiyar A. Buse P. Firestone G. Richards J. Mol. Endocrinol. 1997; 11: 1934-1949Crossref PubMed Google Scholar).sgk transcripts have also been shown to be induced after changes in cell volume (9Waldegger S. Barth P. Raber G. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4440-4445Crossref PubMed Scopus (331) Google Scholar) and under conditions of extracellular hyperosmotic stress (10Bell L. Leong M. Kim B. Wang E. Park J. Hemmings B. Firestone G. J. Biol. Chem. 2000; 275: 25262-25272Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In this report, we have extended our original investigation of GR-mediated survival signaling from the nontumorigenic MEC cell line MCF10A to breast cancer cell lines. Although only a subset of commonly studied human breast tumor cell lines undergo significant apoptosis after growth factor deprivation, most of these growth factor-dependent cell lines were protected from apoptosis after treatment with physiological concentrations of glucocorticoid. Furthermore, the effect of glucocorticoid appears to be GR-mediated because it can be out-competed by high affinity GR antagonists. We also demonstrate that the GR survival signal is likely to be transmitted at least in part as a consequence of the transcriptional activation ofsgk since (i) sgk mRNA is induced in a GR-dependent fashion immediately after glucocorticoid treatment of human MECs, (ii) ectopic SGK expression protects MECs from apoptosis induced by growth factor withdrawal, and (iii) expression of a kinase-dead SGK inhibits protection from apoptosis. Taken together, these results suggest the existence of a novel GR-initiated antiapoptotic pathway that operates, at least in part, through transcriptional induction of the survival kinase gene,sgk. pOG-hSGK was obtained as a generous gift from Dr. Siegfried Waldegger (University of Hamburg, Germany). The human sgk cDNA fragment was amplified from pOG and cloned into the EcoRI and XhoI sites of the retroviral vector pLPCX (CLONTECH, Palo Alto, CA) by applying a polymerase chain reaction-based strategy that incorporated a hemagglutinin (HA) tag in-frame in the amino terminus of wild type SGK using the following primer (Life Technologies, Inc.): wild type HA-SGK, 5′-TAATACTCGAGGCTCCATCATGTACCCATATGACGTTCCAGACTACGCTACGGTGAAAACT-3′. ΔN60 HA-SGK was similarly constructed by inserting an HA tag in-frame 5′ to the coding sequence for amino acid 61 of SGK: ΔN60 HA-SGK, 5′-GGAATTCCTAAGCGTAGTCTGGAACGTCATATGGGTATACCCCTGCATCAGT-3′. The carboxyl-terminal primer for both constructs was 5′-GGAATTCCTCAGAGGAAAGAGTC-3′. The polymerase chain reaction mixtures were supplemented with 10% Me2SO and run withPfu DNA polymerase (Promega, Madison, WI) with an initial 95 °C, 5 min denaturation followed by 35 cycles at 95 °C for 1 min, 55 °C for 1 min, 72 °C for 3 min, and a final 72 °C for 5 min elongation. Mutations were confirmed using primers provided with the pLPCX vector by overlapping bidirectional sequencing using an ABI776 sequencer (PerkinElmer Life Sciences). All parental cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA). MCF10A and MCF10A-Myc cells (as described in Moran et al. (3Moran T. Gray S. Mikosz C. Conzen S. Cancer Research. 2000; 60: 867-872PubMed Google Scholar)) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (BioWhittaker, Walkersville, MD) supplemented with hydrocortisone (0.5 μg/ml), human recombinant EGF (10 ng/ml), and bovine insulin (5 μg/ml; Sigma). BT-20, Hs578T, MDA-MB-231, MDA-MB-468, SK-BR-3, and T-47D cells were cultured in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% heat-inactivated fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA). MCF7 cells were cultured in minimal essential medium (ATCC) supplemented with 10% heat-inactivated FCS, and HCC1937 cells were cultured in RPMI 1640 (BioWhittaker) supplemented with 10% heat-inactivated FCS. Retroviruses were made by transient transfection of retroviral vectors into amphotropic Phoenix cells (a gift of Dr. Gary Nolan, Stanford University, Palo Alto, CA) using either standard calcium phosphate precipitation or Effectene transfection reagent per manufacturer's instructions (Qiagen, Santa Clarita, CA). MECs were infected as described previously (3Moran T. Gray S. Mikosz C. Conzen S. Cancer Research. 2000; 60: 867-872PubMed Google Scholar) with HA-SGK-expressing retroviruses, and clones were selected with puromycin (400 ng/ml). Individual colonies were tested for HA-SGK expression by Western analysis using an anti-HA monoclonal antibody. Cells were trypsinized and seeded subconfluently at 1 × 105 cells/3-cm well in the appropriate media. Cells were allowed to adhere overnight, rinsed twice with PBS, and subsequently cultured for 72 h in serum-free media containing insulin, EGF, hydrocortisone, dexamethasone (10−6m), or combinations of these purified growth factors in the concentrations listed above. In GR antagonist assays, RU486 (5 × 10−7m; Sigma), dexamethasone 21-mesylate (DM, 10−7m; Steraloids Inc, Newport, RI), and dexamethasone oxetanone (10−5m; a gift of Dr. Stoney Simons, National Institutes of Health) were added to media containing glucocorticoid. In some experiments, cells were pre-treated for 30 min with serum-free media and 50 μm LY294002 (Calbiochem) or vehicle alone (0.01% Me2SO in PBS) followed by the addition of appropriate growth factors. After a 72-h incubation, media and floating debris were gently aspirated from wells and replaced with 2 ml of fresh serum-free media; cells were then immediately fixed by adding 500 μl of 37% formaldehyde to each well in a dropwise fashion and incubating at room temperature for 30 min. The fixative solution was subsequently aspirated, and fixed cells were allowed to dry overnight. To score for apoptosis, cells were then stained with a 1 μm4,6-diamidino-2-phenylindole/PBS solution as described previously (11Kennedy S. Wagner A. Conzen S. Jordan J. Bellacosa A. Tsichlis P. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (980) Google Scholar). A Nikon Eclipse E800 microscope with UV illumination at 600× magnification was used to count at least 200 4,6-diamidino-2-phenylindol -stained cells per well to determine the percentage of apoptotic cells per experimental condition. All apoptosis assays were repeated a minimum of three separate times to calculate averages and S.E. Equal numbers of cells (1 × 105) were cultured in 3-cm dishes overnight. The following morning, cells were washed twice on ice with ice-cold PBS and lysed directly in 40 μl of 2× Laemmli buffer. Whole cell lysates were prepared by scraping cells with a rubber spatula, passing lysates 10 times through an 18-gauge needle, and then boiling for 5 min. Samples were then electrophoresed in 8 or 9% SDS-polyacrylamide electrophoresis gels and transferred to nitrocellulose (Osmonics, Minnetonka, MN). Equal protein loading was confirmed by visual inspection of Ponceau S staining of the nitrocellulose membrane. Nitrocellulose was then rinsed with Tris-buffered saline, 0.1% Tween and incubated with one of the following antibodies: rabbit polyclonal anti-GR (Affinity Bioreagents, Golden, CO) or rat monoclonal anti-HA (Roche Molecular Biochemicals, Indianapolis, IN). After primary antibody incubation and extensive washing with Tris-buffered saline, 0.1% Tween, the appropriate secondary antibody, either anti-rabbit IgG-horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) or IgG-horseradish peroxidase was added at a The nitrocellulose was washed with Tris-buffered saline, 0.1% Tween, incubated in to manufacturer's instructions to and were subsequently with a antibody or a antibody CA) as a loading MCF10A-Myc cells SGK constructs were lysed as described in Park et al. J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar). of by were then used to lysates were incubated with rat monoclonal HA protein for min at SGK was then used in a kinase as described in Park et al. J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar). of into was on a and the was repeated to MCF10A-Myc and cells were seeded in numbers in and allowed to in the appropriate media. cells media was aspirated, and cells were washed twice with PBS and incubated in media for 72 or After serum deprivation, cells were for 30 min with either vehicle alone or FCS. was using the per manufacturer's instructions was by and of per experimental was electrophoresed in a formaldehyde and then transferred were then with a human sgk cDNA and a rat cDNA with using the primer were then washed and to were using a that the signal be were repeated at least times to calculate mRNA and S.E. The kinase-dead and the and of were made using the per the manufacturer's were to either the to or the sites or of to (Life Technologies, of the following HA-SGK, HA-SGK, HA-SGK, are The DNA used was Mutations were confirmed using primers provided with the pLPCX vector by bidirectional sequencing using an ABI776 sequencer (PerkinElmer Life Sciences). vectors were then used to and MECs as described above. We previously demonstrated that GR activation initiates a potent antiapoptotic signal in the nontumorigenic MEC line MCF10A and its line In the study, we to determine GR activation might also apoptosis in breast cancer cell lines. We selected commonly studied breast cancer cell lines Hs578T, MDA-MB-231, MDA-MB-468, SK-BR-3, and and for GR Western analysis of cell using GR that all cell lines the GR protein as previously the T-47D line GR expression A. E. Cancer Google Scholar, M. Mol. Endocrinol. PubMed Scopus Google Scholar, S. B. J. Mol. Endocrinol. PubMed Scopus Google Scholar). To glucocorticoid treatment can a survival pathway in these tumor cell apoptosis was after 72 h in the absence of all growth factors and in the of a physiological of the glucocorticoid In the absence of all growth factors, the percentage of apoptosis in breast cancer cell lines from in cells to only in MCF7 cells In of the tumor cell lines MDA-MB-231, BT-20, Hs578T, and MDA-MB-468, treatment with dexamethasone inhibited apoptosis with growth factor To determine glucocorticoid-mediated survival was specifically through activation of the MCF10A-Myc and cells were concomitantly with dexamethasone and one of three of GR activation and its transcriptional RU486 (5 × C. S. DNA Biol. 1993; PubMed Scopus Google Scholar), dexamethasone oxetanone N. L. M. E. Endocrinology. PubMed Scopus Google Scholar), or dexamethasone 21-mesylate F. A. G. Mol. Endocrinol. PubMed Scopus Google Scholar) All three GR the antiapoptotic effect of glucocorticoids to on the cell line 2 In MCF10A-Myc RU486 was the most potent antagonist of glucocorticoid-mediated in a in apoptosis. In was the most potent antagonist of survival signaling by in a in apoptosis at 72 h with cells protected by glucocorticoid alone 2 for a of glucocorticoid activation, the effect of all three GR be out-competed in MCF10A-Myc cells and cells with concentrations of Taken together, these results suggest that glucocorticoid-mediated survival in breast cancer cell lines is specifically through the activation of the We to the survival downstream of GR activation. a human cancer with genes we gene expression by of mRNA from MCF10A cells for 30 min with either vehicle alone dexamethasone (10−6m), or both dexamethasone and RU486 We identified genes that were induced or repressed at least by 30 min of glucocorticoid treatment expression was after treatment with the GR antagonist RU486 One of the most GR-induced genes identified from this of MEC transcripts was a serine/threonine kinase previously shown to be 54% in its catalytic domain to the well antiapoptotic serine/threonine kinase (5Webster M. Goya L. Ge Y. Maiyar A. Firestone G. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (497) Google Scholar). Although sgk been shown to be transcriptionally induced by glucocorticoid treatment in rat mammary cells (5Webster M. Goya L. Ge Y. Maiyar A. Firestone G. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (497) Google Scholar), found that were induced by glucocorticoid treatment in human kidney epithelial cells (9Waldegger S. Barth P. Raber G. Lang F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4440-4445Crossref PubMed Scopus (331) Google Scholar). the of a in sgk activation by we confirmed our using analysis of sgk mRNA transcripts 30 min after glucocorticoid treatment in human breast cell MCF10A-Myc and analysis of mRNA using an human sgk cDNA demonstrated that in MCF10A-Myc GR activation induced sgk mRNA The GR antagonist RU486 inhibited sgk mRNA whereas expression under all cells a induction of sgk transcripts after glucocorticoid treatment that was also by RU486 treatment In both cell types, serum These suggest that glucocorticoid of human MECs appears to directly through a GR-dependent that can be by We the activity of SGK in MCF10A-Myc cells to apoptotic SGK was in MCF10A-Myc cells after with either an wild type human SGK or a SGK containing amino The ΔN60 SGK protein been previously shown to be wild type SGK in human kidney cells (6Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (529) Google Scholar). Individual clones either wild type HA-SGK or ΔN60 HA-SGK were by puromycin and cell lysates were for HA-SGK protein expression by Western analysis using a monoclonal anti-HA antibody. to previously results in human kidney ΔN60 HA-SGK was the wild type HA-SGK, both were in to cells with the retroviral pLPCX vector alone To determine wild type and ΔN60 HA-SGK have kinase activity MCF10A-Myc cells these constructs were of all growth factors overnight, and SGK activity was the kinase activity of SGK from wild type HA-SGK, ΔN60 HA-SGK, and a kinase-dead HA-SGK in MCF10A-Myc cells. an for SGK kinase activity is we used the previously described as a J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar). kinase activity was seen in both wild type and ΔN60 HA-SGK from MCF10A-Myc cells with wild type and ΔN60 HA-SGK both as a in Western suggesting that of SGK in MCF10A-Myc cells the absence of growth factors in the MCF10A-Myc cell lines were then for apoptosis after conditions of growth factor deprivation. In the absence of all growth factors both wild type and ΔN60 HA-SGK-expressing cell lines an of of apoptosis with cells only the pLPCX These results suggest that in the absence of growth factors, expression of either the wild type or ΔN60 HA-SGK can MECs from apoptosis. the addition of insulin and EGF previously in MCF10A cells to the 3-kinase pathway to (3Moran T. Gray S. Mikosz C. Conzen S. Cancer Research. 2000; 60: 867-872PubMed Google Scholar)) survival in cells ectopic The antiapoptotic activity of SGK in the absence of EGF or insulin was because SGK activation has been shown previously to be dependent on the pathway J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar). To determine the survival effect after of wild type SGK in SGK kinase we the antiapoptotic activity of kinase-dead SGK HA-SGK and HA-SGK, a of the in SGK J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar). These constructs were used to MCF10A-Myc cell lines kinase-dead HA-SGK cell lines were then in apoptosis In to wild kinase-dead HA-SGK provided significant protection from apoptosis under conditions of serum 5 These results suggest that SGK kinase activity is in essential for its antiapoptotic We kinase-dead SGK as a and survival in MECs with glucocorticoid. lines HA-SGK were of all growth factors or with dexamethasone alone for 72 In HA-SGK-expressing cell lines with a in apoptosis was seen with the parental cell line 5 These results suggest that the survival effect seen after glucocorticoid induction of SGK can be by the of kinase-dead a for kinase-dead The absence of a for either insulin or EGF in with wild type SGK suggested that in our either an pathway to 3-kinase signaling was of SGK activation or 3-kinase activity was to To determine 3-kinase activity was in for SGK antiapoptotic we cells with LY294002 a of MEC lines with LY294002 in a significant in the percentage of apoptosis in both wild type HA-SGK and ΔN60 HA-SGK-expressing cells in cells These results suggest that 3-kinase signaling is for SGK survival activity and that 3-kinase activity in MECs appears to be to in the absence of insulin and EGF We have identified a novel pathway of mammary cell survival that GR activation in both nontumorigenic MECs and breast cancer cell lines. The between GR activation and induced and repressed genes was by One of the GR-induced encodes a serum and glucocorticoid-regulated kinase with homology in its catalytic domain to the catalytic domain of the antiapoptotic kinase (5Webster M. Goya L. Ge Y. Maiyar A. Firestone G. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (497) Google Scholar). In a of MEC cell MCF10A-Myc cells and cells were found to be most to growth factor apoptosis and also demonstrated the of SGK expression treatment of these cell lines induced sgk mRNA and in Furthermore, SGK cell after growth factor withdrawal, of a kinase-dead SGK cells from apoptosis. The induction of from glucocorticoid treatment an important transcriptional for the activity of this a novel of kinase activation that appears of cell growth factor receptor signaling. the downstream of SGK kinase activity to be SGK activity in human kidney cells has been shown to be regulated by (6Kobayashi T. Cohen P. Biochem. J. 1999; 339: 319-328Crossref PubMed Scopus (529) Google Scholar, J. Leong M. Buse P. Maiyar A. Firestone G. Hemmings B. J. 1999; PubMed Scopus Google Scholar), our results suggest that 3-kinase activity be to One is that kinase 1 a downstream target of is high in a kinase kinase be for SGK activation in these cells. A is that human MECs or activity that 3-kinase activity. The through SGK apoptosis in kidney SGK has been demonstrated to be a target of of (7Naray-Fejes-Toth A. Canessa C. Cleaveland E. Aldrich G. Fejes-Toth G. J. Biol. Chem. 1999; 274: 16973-16978Abstract Full Text Full Text PDF PubMed Scopus (394) Google S. A. L. Wang J. Buse P. Firestone G. F. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, A. C. E. J. 2000; PubMed Google Scholar), SGK in the of cell is to be an of apoptosis J. A. A. J. PubMed Scopus Google Scholar), SGK expression the changes in cell volume that apoptosis by The that SGK its antiapoptotic effects by and volume is the of In we have identified GR activation as a potent survival signal in both and human breast epithelial cells. a novel of the family of serine/threonine protein has been identified as a downstream of GR survival signaling. of antiapoptotic activity in breast epithelial cells was found to be of insulin or EGF 3-kinase activity is Furthermore, transcriptional of SGK expression appears to be the of induction of SGK suggesting a novel between steroid hormone receptor activation and serine/threonine We Dr. Stoney for dexamethasone Dr. Siegfried Waldegger for the human sgk for in cell for with and and Hay for We also the DNA of the of

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (Δ60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1. The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (Δ60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1. ubiquitin-protein isopeptide ligase green fluorescent protein hemagglutinin fetal calf serum N-acetyl-Leu-Leu-norleucinal N-acetyl-Leu-Leu-normethional horseradish peroxidase Glucocorticoid receptor activation in mammary epithelial cells (1Moran T.J. Gray S. Mikosz C.A. Conzen S.D. Cancer Res. 2000; 60: 867-872PubMed Google Scholar) and hepatocytes (2Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar) initiates a potent antiapoptotic signaling pathway. Activation of the glucocorticoid receptor by ligand binding directly regulates the transcription of several potential downstream mediators of this survival pathway, including SGK-1 (3Webster M.K. Goya L., Ge, Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (492) Google Scholar, 4Mikosz C.A. Brickley D.R. Sharkey M.S. Moran T.W. Conzen S.D. J. Biol. Chem. 2001; 276: 16649-16654Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). SGK-1 is a member of an important subfamily of protein kinases known as the “AGC” subfamily that includes protein kinase A, protein kinase B, protein kinase G, and protein kinase C isoforms. SGK-1 is 54% identical in its catalytic domain to protein kinase Bα, also known as AKT-1. Two other isoforms of sgk, sgk-2 and sgk-3 (or cytokine-independent survival kinase, cisk), have been identified, and all three products can be phosphorylated and activated following phosphatidylinositol 3-kinase pathway signaling to downstream phosphatidylinositol-dependent kinases including phosphatidylinositol-dependent kinase-1 and -2 (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Furthermore, phosphatidylinositol-dependent kinase-1 and -2 activation of AKT, SGK-1, and SGK-3/CISK (6Xu J. Liu D. Gill G. Songyang Z. J. Cell Biol. 2001; 154: 699-705Crossref PubMed Scopus (94) Google Scholar) can promote cell survival under a variety of conditions that normally favor apoptosis (for review, see Ref. 7Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (788) Google Scholar).In addition to SGK-1 regulation via reversible phosphorylation,sgk-1 transcription is acutely up-regulated after glucocorticoid receptor activation, serum stimulation, and cell stress (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). The role of transcriptional activation in modulating a serine-threonine kinase is unusual and to date has not been implicated in the regulation of either sgk-2 or sgk-3expression (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar).Another increasingly recognized mechanism of regulation of signaling proteins is through posttranslational modification via covalent addition of one or more ubiquitin molecules (reviewed in Ref. 9Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Covalent attachment of ubiquitin, either as a single molecule or as a polyubiquitin chain, can regulate a variety of processes including protein degradation and subcellular trafficking of the substrate protein. For example, activity of the non-receptor tyrosine kinase c-Abl has been recently shown to be down-regulated by a ubiquitin-dependent degradation pathway (10Echarri A. Pendergast A.M. Curr. Biol. 2001; 11: 1759-1765Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Similarly, protein kinase C-λ is ubiquitinated and down-regulated by the Von Hippel Lindau-Cul 2 E31ubiquitin ligase (11Okuda H. Saitoh K. Hirai S. Iwai K. Takaki Y. Baba M. Minato N. Ohno S. Shuin T. J. Biol. Chem. 2001; 276: 43611-43617Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Polyubiquitination also directly regulates the ability of TAK1 to phosphorylate MKK6, which in turn activates the c-Jun NH2-terminal kinase-p38 kinase pathway (12Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). Finally, the PHD domain of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 was recently found to act as an E3 ubiquitin ligase and to thereby mediate ubiquitination and degradation of extracellular signal-regulated kinase 1/2 (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar).In the present study we demonstrate that the serine-threonine kinase SGK-1 is modified by polyubiquitination and ultimately degraded by the 26S proteasome. The rapid degradation of SGK-1 suggests that in addition to transcriptional up-regulation and reversible phosphorylation, ubiquitin modification plays an important role in determining the availability of SGK-1 as a kinase. We also demonstrate that the amino terminus of SGK-1 contains a domain that regulates both ubiquitin/proteasome-mediated degradation and efficient association of SGK-1 with the plasma membrane. Furthermore, fractionation studies reveal that although SGK-1 is found in both membrane and cytosolic fractions, the ubiquitin-modified SGK-1 is predominantly membrane-associated. Taken together, these findings suggest that SGK-1, a serine-threonine kinase that phosphorylates Nedd4-2 (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) and the Na+/H+ exchanger 3 (NHE3) (16Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), is negatively regulated by ubiquitin modification and proteasome degradation.DISCUSSIONAlthough it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction.While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6).Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane.In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. Glucocorticoid receptor activation in mammary epithelial cells (1Moran T.J. Gray S. Mikosz C.A. Conzen S.D. Cancer Res. 2000; 60: 867-872PubMed Google Scholar) and hepatocytes (2Evans-Storms R.B. Cidlowski J.A. Endocrinology. 2000; 141: 1854-1862Crossref PubMed Scopus (83) Google Scholar) initiates a potent antiapoptotic signaling pathway. Activation of the glucocorticoid receptor by ligand binding directly regulates the transcription of several potential downstream mediators of this survival pathway, including SGK-1 (3Webster M.K. Goya L., Ge, Y. Maiyar A.C. Firestone G.L. Mol. Cell. Biol. 1993; 13: 2031-2040Crossref PubMed Scopus (492) Google Scholar, 4Mikosz C.A. Brickley D.R. Sharkey M.S. Moran T.W. Conzen S.D. J. Biol. Chem. 2001; 276: 16649-16654Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). SGK-1 is a member of an important subfamily of protein kinases known as the “AGC” subfamily that includes protein kinase A, protein kinase B, protein kinase G, and protein kinase C isoforms. SGK-1 is 54% identical in its catalytic domain to protein kinase Bα, also known as AKT-1. Two other isoforms of sgk, sgk-2 and sgk-3 (or cytokine-independent survival kinase, cisk), have been identified, and all three products can be phosphorylated and activated following phosphatidylinositol 3-kinase pathway signaling to downstream phosphatidylinositol-dependent kinases including phosphatidylinositol-dependent kinase-1 and -2 (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Furthermore, phosphatidylinositol-dependent kinase-1 and -2 activation of AKT, SGK-1, and SGK-3/CISK (6Xu J. Liu D. Gill G. Songyang Z. J. Cell Biol. 2001; 154: 699-705Crossref PubMed Scopus (94) Google Scholar) can promote cell survival under a variety of conditions that normally favor apoptosis (for review, see Ref. 7Kandel E.S. Hay N. Exp. Cell Res. 1999; 253: 210-229Crossref PubMed Scopus (788) Google Scholar). In addition to SGK-1 regulation via reversible phosphorylation,sgk-1 transcription is acutely up-regulated after glucocorticoid receptor activation, serum stimulation, and cell stress (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). The role of transcriptional activation in modulating a serine-threonine kinase is unusual and to date has not been implicated in the regulation of either sgk-2 or sgk-3expression (5Kobayashi T. Deak M. Morrice N. Cohen P. Biochem. J. 1999; 344: 189-197Crossref PubMed Scopus (333) Google Scholar). Another increasingly recognized mechanism of regulation of signaling proteins is through posttranslational modification via covalent addition of one or more ubiquitin molecules (reviewed in Ref. 9Pickart C.M. Mol. Cell. 2001; 8: 499-504Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Covalent attachment of ubiquitin, either as a single molecule or as a polyubiquitin chain, can regulate a variety of processes including protein degradation and subcellular trafficking of the substrate protein. For example, activity of the non-receptor tyrosine kinase c-Abl has been recently shown to be down-regulated by a ubiquitin-dependent degradation pathway (10Echarri A. Pendergast A.M. Curr. Biol. 2001; 11: 1759-1765Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Similarly, protein kinase C-λ is ubiquitinated and down-regulated by the Von Hippel Lindau-Cul 2 E31ubiquitin ligase (11Okuda H. Saitoh K. Hirai S. Iwai K. Takaki Y. Baba M. Minato N. Ohno S. Shuin T. J. Biol. Chem. 2001; 276: 43611-43617Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Polyubiquitination also directly regulates the ability of TAK1 to phosphorylate MKK6, which in turn activates the c-Jun NH2-terminal kinase-p38 kinase pathway (12Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1612) Google Scholar). Finally, the PHD domain of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase-1 was recently found to act as an E3 ubiquitin ligase and to thereby mediate ubiquitination and degradation of extracellular signal-regulated kinase 1/2 (13Lu Z., Xu, S. Joazeiro C. Cobb M.H. Hunter T. Mol. Cell. 2002; 9: 945-956Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In the present study we demonstrate that the serine-threonine kinase SGK-1 is modified by polyubiquitination and ultimately degraded by the 26S proteasome. The rapid degradation of SGK-1 suggests that in addition to transcriptional up-regulation and reversible phosphorylation, ubiquitin modification plays an important role in determining the availability of SGK-1 as a kinase. We also demonstrate that the amino terminus of SGK-1 contains a domain that regulates both ubiquitin/proteasome-mediated degradation and efficient association of SGK-1 with the plasma membrane. Furthermore, fractionation studies reveal that although SGK-1 is found in both membrane and cytosolic fractions, the ubiquitin-modified SGK-1 is predominantly membrane-associated. Taken together, these findings suggest that SGK-1, a serine-threonine kinase that phosphorylates Nedd4-2 (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) and the Na+/H+ exchanger 3 (NHE3) (16Yun C.C. Chen Y. Lang F. J. Biol. Chem. 2002; 277: 7676-7683Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), is negatively regulated by ubiquitin modification and proteasome degradation. DISCUSSIONAlthough it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction.While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6).Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane.In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. Although it is well-established that sgk-1 mRNA is up-regulated as an immediate early response to serum and glucocorticoid stimulation, the regulation of SGK-1 kinase activity is only partly understood. It has been previously established that SGK-1 is phosphorylated and activated by a phosphatidylinositol 3-kinase-dependent pathway on conserved serine and threonine residues homologous to those found in AKT-1. However, in most cell types, steady-state levels of SGK-1 are far less abundant than AKT-1 (8Lang F. Cohen P. Science's STKE. 2001; (http://www.stke.org/cgi/content/full/oc_sigtrans;2001/108/re17)PubMed Google Scholar). In this report, we demonstrate for the first time that in addition to the transcriptional regulation of sgk-1, SGK-1 protein levels are regulated by the ubiquitin-proteasome pathway, adding another dimension to the mechanism of regulation of this unusual serine-threonine kinase. We also show that polyubiquitin-modified SGK-1 is predominantly localized to the membrane-associated fraction of the cell. These observations suggest that SGK-1 is ubiquitinated at or near the membrane rather than in the cytosol. Alternatively, deubiquitinating enzymes may preferentially act on SGK-1 in the cytosolic compartment, thereby resulting in accumulation of polyubiquitinated SGK-1 in the membrane fraction. While these studies were ongoing, SGK-1 was shown to directly phosphorylate and inactivate Nedd4-2, a known membrane-associated protein that is the E3 ligase responsible for ubiquitination and degradation of the epithelial sodium channel (ENaC) (14Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Prat J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 15Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar). This raises the interesting possibility that SGK-1 may both phosphorylate and act as an E3 substrate of Nedd4-2 (or another E3 ligase), thereby achieving a tight negative feedback of SGK-1 activity. Our finding that the majority of ubiquitinated SGK-1 is associated with the plasma membrane is consistent with this proposed model (see Fig.6). Unlike SGK-3 (CISK), which contains a complete phosphatidylinositol phosphate-binding PX domain that is required to localize SGK-3 to early endosomes (24Virbasius J.V. Song X. Pomerleau D.P. Zhan Y. Zhou G.W. Czech M.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12908-12913Crossref PubMed Scopus (75) Google Scholar), SGK-1 contains only the α-helix-2 of this domain and does not co-localize with early endosomes. 2C. A. Mikosz and S. D. Conzen, unpublished data. Furthermore, mutation of the α-helix-2 domain arginine residues that are predicted to be required for binding phosphatidylinositols (25Bravo J. Karathanassis D. Pacold C.M. Pacold M.E. Ellson C.D. Anderson K.E. Butler P.J. Lavenir I. Perisic O. Hawkins P.T. Stephens L. Williams R.L. Mol. Cell. 2001; 8: 829-839Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) does not alter SGK-1 membrane localization or kinase activity. 3D. R. Brickley, C. A. Mikosz, and S. D. Conzen, unpublished data. Therefore, SGK-1 appears to use an independent mechanism, perhaps via a protein-protein interaction requiring the amino terminus, for translocation to the cell membrane. In summary, we have demonstrated that the steady-state protein levels and kinase activity of SGK-1 are down-regulated via polyubiquitin modification of the membrane-associated fraction of SGK-1. Regulation of serine-threonine kinases via ubiquitin modification underscores the potential importance of phosphorylation-independent mechanisms in the regulation of signaling molecules. In cell types or disease conditions in which phosphatidylinositol 3-kinase activity is constitutively high (e.g. with HER-2/neu amplification, Ras mutations, or PTEN deletions), the availability of downstream targets is likely to be of critical importance to the activity of these pathways. We thank members of our laboratory and Yair Argon, Jan Burkhardt, Geoffrey Greene, Clive Palfrey, and Marsha Rosner for reagents and useful discussions. We also thank Jerrold Turner and Vytas Bindokas for assistance with confocal microscopy.

PURPOSE: To prevent chemotherapy-related side effects, synthetic glucocorticoids, for example, dexamethasone, are routinely administered to patients with ovarian cancer. However, preclinical data implicate glucocorticoids in suppressing chemotherapy-mediated apoptosis in epithelial tumors. The anti-apoptotic mechanisms underlying this increased survival have been shown to require up-regulation of prosurvival genes, including serum and glucocorticoid-regulated kinase 1 (SGK1) and map kinase phosphatase 1 (MKP1)/dual specificity phosphatase 1 (DUSP1). Despite abundant preclinical data, there are no correlative studies in patients. We therefore evaluated anti-apoptotic gene expression in tumor samples from patients randomized to dexamethasone or normal saline. EXPERIMENTAL DESIGN: Eighteen patients were randomized before exploratory laparotomy for suspected ovarian cancer. Dexamethasone or normal saline was administered i.v. following anesthesia. Ovarian and omental tumor samples were collected intra-operatively before and after infusion. Samples were analyzed for histology and glucocorticoid receptor expression by immunohistochemistry. SGK1 and MKP1/DUSP1 mRNA levels were determined using quantitative real-time PCR. RESULTS: Ten patients were evaluable. At 30 min postinfusion, tumor samples from five patients receiving dexamethasone revealed an average SGK1 mRNA induction of 6.1-fold (SEM, +/-2.6) compared with only 1.5-fold (SEM, +/-0.4) in tumor samples from five patients receiving normal saline (P = 0.028). Average MKP1/DUSP1 mRNA expression was increased by 8.2-fold (SEM, +/-2.9) following dexamethasone versus 1.1-fold (SEM, +/-0.4) following normal saline (P = 0.009). All samples expressed glucocorticoid receptor. CONCLUSION: Glucocorticoid administration to patients is associated with rapid up-regulation of SGK1 and MKP1 expression in ovarian tumors. This finding supports the hypothesis that pharmacologic doses of glucocorticoids may decrease chemotherapy effectiveness in ovarian cancer patients through increased anti-apoptotic gene expression.

Activation of the glucocorticoid receptor (GR) plays a critical role in the stress response of virtually all cell types. Despite recent advances in large-scale genomic and proteomic data acquisition, identification of physiologically relevant molecular events downstream of nuclear hormone receptor activation remains challenging. By analyzing gene expression changes 30 min after dexamethasone (Dex) treatment, we previously found that immediate induction of serum and glucocorticoid-regulated kinase-1 (SGK-1) expression is required for GR-mediated mammary epithelial cell survival signaling. We now report that activation of the GR mediates Forkhead transcription factor 3a (FOXO3a) phosphorylation and inactivation in mammary epithelial cells. GR-mediated induction of SGK-1 expression is required for FOXO3a inactivation; additional growth factor stimulation is not required. To further explore the gene expression changes that occur downstream of GR-mediated FOXO3a inactivation, we analyzed temporal gene expression data and selected GR-down-regulated genes containing core FOXO3a binding motifs in their proximal promoters. This approach revealed several previously unrecognized transcriptional target genes of FOXO3a, including IGF binding protein-3 (IGFBP-3). Endogenous IGFBP-3 expression was confirmed to be dependent on the GR-SGK-1-FOXO3a signaling pathway. Moreover, GR activation decreased FOXO3a-induced apoptosis in SK-BR-3 breast cancer cells. Collectively, our data suggest that GR-mediated FOXO3a inactivation is an important mechanism contributing to glucocorticoid-mediated mammary epithelial cell survival.