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Cell and Tumor Biology |
B Kinase
Departments of 1 Internal Medicine, 2 Pharmacology and Toxicology, and 3 Human Biological Chemistry & Genetics, University of Texas Medical Branch, Galveston, Texas
Requests for reprints: Jie Du, Department of Internal Medicine, 9.138 Medical Research Building, University of Texas Medical Branch, Galveston, TX 77555-1064. Phone: 409-772-3362; Fax: 409-747-0692; E-mail: jidu{at}utmb.edu.
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Abstract |
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B (NF-B). High levels of SGK expression were observed in human breast cancer samples. When SGK was reduced the apoptotic rate increased, and increased SGK activity prevents serum withdrawal–induced apoptosis. SGK-induced cell survival was abolished by a dominant-negative form of IB kinase ß (IKKß, K44A) or a null mutation of IKKß in mouse embryonic fibroblast cells indicating involvement of the NF-B pathway. Serum-induced SGK or increased expression of SGK activated NF-B transcriptional activity, whereas small interference RNA to SGK blocked NF-B activity. Coexpression of SGK and IKKß significantly increased the activation of NF-B (versus expression of IKKß alone). Expression of dominant-negative IKKß K44A, IB
AA, and kinase-dead SGK (127KM) blocked the ability of SGK to stimulate NF-B activity, suggesting that IKKß is a target of SGK. We also show that SGK enhances the ability of IKKß to phosphorylate endogenous IB in cells or recombinant glutathione S-transferase-IB in vitro and increases IB degradation; SGK physically associates with and activates IKKß in MDA231 cells via phosphorylation of Ser181 in IKKß. Taken together, we conclude that SGK acts as an oncogene in breast cancer cells through activation of the IKK-NF-B pathway, thereby preventing apoptosis. Blocking SGK expression/activity represents a potential therapeutic approach for breast cancer treatment.
Key Words: Breast cancer • Signal transduction • Survival factors • Transcriptional control of apoptosis
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Introduction |
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Serum and glucocorticoid inducible protein kinase (SGK) has been implicated in regulation of apoptosis. For example, a newly discovered member of the SGK family, cytokine-independent survival kinase, can phosphorylate and negatively regulate proapoptotic BAD to prevent interleukin-3 withdrawal–induced cell death (3). SGK has also been shown to protect mammary epithelial cells from undergoing apoptosis following withdrawal of glucocorticoids (4). The potential for SGK in promoting growth of cancer cells is supported by reports that SGK expression is increased in primary liver cancer or ductal breast carcinoma in situ (5, 6) . However, the mechanism by which SGK inhibits apoptosis in cancer is not fully explored.
Activation of NF-B has been shown to prevent apoptosis in cancer and other cells (7, 8). The activation of NF-B requires activation of IKK. In vivo IKK activation is preceded by phosphorylation by certain upstream kinases such as mitogen-activated protein kinase kinase kinase-1 (9–11), NF-B–inducing kinase (12), protein kinase C (13, 14), and NF-B–activating kinase (15). Another major signaling pathway that affects IKK complex and NF-B activity is the PI-3 kinase pathway (16–18). For example, platelet-derived growth factor can cause transient association between Akt and IKK in vivo plus increased IKK activity with NF-B activation and protection from apoptosis (18).
SGK belongs to the A/G protein kinase subfamily including Akt, protein kinase A, p70S6 kinase, and protein kinase C, which is activated by phosphorylation through PDK, a PI-3 kinase downstream kinase (16–18). SGK and Akt share 45% to 55% sequence identity throughout their catalytic domain (19); both kinases share a similar consensus phosphorylation site RXRXXS/T (20, 21) . However, SGK has activation and expression properties that are distinct from Akt. When epithelial cells are exposed to hyperosmotic stress, heat shock, UV irradiation, oxidative stress, or steroid hormones, SGK expression and activation increase, whereas Akt remains in a nonphosphorylated/activated state (22). Activation of the glucocorticoid receptor rapidly induces SGK expression, and activated SGK, but not Akt, protects cells from apoptosis in the mammary epithelial cell line MCF 10A (4). The mechanism by which SGK protects cells from apoptosis is not clear at present. We examined if the antiapoptotic effect of SGK involves activation of NF-B. We found that SGK physically associates with IKKß and phosphorylates IKKß, leading to release of NF-B. The results identified a growth factor–responsive SGK-IKK-NF-B survival pathway and suggest a mechanism by which SGK can suppress apoptosis of cancer cells.
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Materials and Methods |
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/ß H-470), IKK, p65, ß-tubulin, protein A/G agarose, and recombinant GST-IB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against phospho-IB (Ser32), phospho-IKKß (Ser177/181) was obtained from Cell Signaling Technology (Beverly, MA). Active SGK was bought from Upstate Biotechnology Inc. (Waltham, MA). Immunoaffinity purified SGK antibody was a gift from Drs. Bela Malik and D. Eaton of Emory University (Atlanta, GA). Small interference RNA (siRNA) control and siRNA to SGK were synthesized by Dharmacon, Inc. (Lafayette, CO). Insulin-like growth factor (IGF)-I was bought from Roche Applied Science (Indianapolis, IN). Vectastain Universal Quick Kit was bought from Vector Laboratory (Burlingame, CA). Luciferase assay system was purchased from Promega (Madison WI).
Cell Culture. MDA231cells derived from human breast cancer and HEK293 cells derived from human embryonic kidney were purchased from American Type Culture Collection (Rockville, MD). MDA231/SGK cell line was created by puromycin selection after retrovirus SGK infection in MDA231 cells. Retrovirus SGK was produced by the MSCV retroviral expression system (BD Biosciences Clontech, Palo Alto, CA). Briefly, SGK cDNA was cloned in pMSCV puro vector and resultant viral DNA was transfected into PT67 packaging cell line; virus-producing cell colonies were selected by puromycin. The high-titer supernatant from puromycin-selected PT67 was used to infect MDA231 cells in the presence of 8 µg/mL polybrene. Mouse embryonic fibroblast (MEF) wild-type, IKK–/– and IKKß–/– cells were kindly provided by Dr. I.M. Verma (Salk Institute, La Jolla, CA). All cells were cultured in DMEM supplemented with 10% heat-inactive fetal bovine serum (FBS; Sigma-Aldrich), 100 unit/mL penicillin, and 100 unit/mL streptomycin (Invitrogen, Grand Island, NY). Cells were propagated at 37°C in humidified air containing 5% CO2 incubator.
Expression Vectors. pCMV4-HA-SGK was a generous gift from Dr. Hemmings A. Brian (Friedrich Miescher-Institut, Basel, Switzerland); pRK-Flag-IKKß and dominant-negative pRK-Flag-IKKß K44A were generously provided by Dr. J. D. Woronicz (Department of Musculoskeletal Science, Genetics Institute, Cambridge, MA). pHIV-NF-B-luc was generated by subcloning of four tandem repeats of HIV long terminal NF-B binding consensus site from p(HIV B) 4-cat (23) into pGL2 basic vector. Adenovirus NF-B-luc was from Dr. Engelhardt (University of Iowa College of Medicine, Iowa City, IA). The vectors pCMV-EGFP, pNF-B-luc, pMyc-luc, p53-luc, pE2F-luc, and pRb-luc were bought from BD Biosciences Clontech. The expression vector pAd-CMV-track-SGK was constructed by clipping SGK from pCMV4-HA-SGK and inserting it into the KpnI and EcoRV sites in the pAd-CMV-track vector. pcDNA3 was obtained from Invitrogen. The IKKß(132-206AA) fragment was amplified from pRK-Flag-IKKß by PCR and cloned into the BamH1 and HindIII sites of pGEX-KG-GST plasmid. The IKKß mutant, GST-IKKß (177A/181A), was generously provided by Prof. Makoto Nakanishi (Nagoya City University Medical School, Nagoya, Japan).
NF-B Reporter Gene Assay. Transfected or NF-B-luc virus–infected cells were cultured in a complete growth medium for 36 hours and harvested for luciferase assays, done according to available protocols (Promega). The concentration of protein in the cell lysate was measured using the Bradford method and an equal amount of protein from each cell lysate was assayed for luciferase activity.
Apoptosis Assay. Two methods were used to detect cell apoptosis. The first was done with the Annexin V-PE apoptosis detection kit 1 (BD Biosciences) according to the manufacturer's instructions. Briefly, cells were counted, resuspended in 100 µL of binding buffer, stained with Annexin V-PE plus 7-amino-actinomycin D for 15 minutes, and analyzed by flow cytometry (Becton Dickinson, San Jose, CA). The second protocol for detecting apoptosis involved cell nuclear staining: HEK293 cells or MEF cells were transfected with 0.5 µg pCMV-EGFP or pCMV-track-HA-SGK, pRK-Flag-IKKß, pRK-Flag-IKKß K44A, or siRNA, siRNA to SGK. After 24 hours, the medium was changed to serum-free medium for another 24 (HEK293 cells) or 48 hours (MEF cells); after gently washing with PBS, the cells were immediately fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 10 minutes before staining with 1 µg/mL Hoechst 33258 (Molecular Probes, Eugene, OR) in PBS for 10 minutes as described (24). A Zeiss LSM 510 UV Meta laser-scanning confocal microscope was used at x60 magnification to count at least 500 Hoechst 33258–stained cells (blue) of transfected cells per well (GFP positive, green cells) to determine the percentage of apoptotic cells per experimental condition.
Immunoprecipitation. Cells were lysed in a lysis buffer [5 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 1% Triton X-100, 10 mmol/L glycerol, 1 mmol/L EDTA, 1 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 5 µg/mL leupeptine, and 5 µg/mL pepstatin]. Lysates were precleared for 30 minutes at 4°C with 20 µL of protein A/G agarose and 0.25 µg normal mouse IgG (Sigma). HA-SGK, Flag-IB, or Flag-IKKß was immunoprecipitated by incubating with agarose-conjugated anti-HA or anti-Flag antibody for 2 hours at 4°C. SGK or IKKß was immunoprecipitated by anti-SGK or anti-IKKß at 4°C overnight and then incubated with protein A/G agarose for 2 hours at 4°C. Immunocomplexes were washed five times with lysis buffer, boiled, subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the primary antibody was added and the mixture incubated and extensively washed with 0.1% Tris-buffered salt solution with Tween 20. The appropriate secondary antibody was added for 1 hour. Enhanced chemiluminescence detection was done using an ECL kit (Amersham).
IB Kinase Assay. HEK293 cells were transiently transfected with pCMV4-HA-SGK and pRK-Flag-IKKß in 10% FBS for 24 hours. Cells were serum starved for 24 hours before adding IGF-I (10 ng/mL) for 15 minutes and then IKKß was immunoprecipitated using anti-Flag agarose–conjugated antibody. IKK activity was measured as described (25–27) by using this immunocomplex incubated with the purified recombinant GST-IB (1-54). Briefly, the kinase activity was determined at 30°C for 30 minutes in a 30-µL reaction mixture containing 50 mmol/L HEPES (pH 8.0), 10 mmol/L MgCl2, 2.5 mmol/L EGTA, 1 mmol/L DTT, 10 µmol/L ß-glycerophosphate, 1mmol/L NaF, 0.1 mmol/L Na3VO4, 0.1 mmol/L phenylmethylsulfonyl fluoride, 10 µmol/L ATP, and 10µmol/L [
-32P]ATP. Reaction products were separated by 10% SDS-PAGE, transferred to nitrocellulose, and visualized with a phosphoimager.
Phosphorylation of IKKß by SGK. The ability of SGK to phosphorylate GST-IKKß was examined at 30°C for 30 minutes in 50 µL mixture plus active SGK with GST-IKKß (132-206AA) or GST-IKKß (177A/181A). The mixture contained 20 mmol/L 4-morpholinepropanesulfonic acid (pH 7.2), 25 mmol/L ß-glycerol phosphate, 5 mmol/L EGTA, 1 mmol/L Na3VO4, 1 mmol/L DTT, 2 µmol/L protein kinase A inhibitor peptide and 1 µmol/L microcystin-LR, 15 mmol/L MgCl2, 100µmol/L cold ATP, and 10 µmol/L [-32P]ATP. Reaction products were separated by 10% SDS-PAGE, transferred to nitrocellulose, and visualized with a phosphoimager.
Immunohistochemistry Staining. Second-generation (TARP-2) multitumor tissue microarray slides were obtained from the Cooperative Human Tissue Network under the Tissue Array Research Program of the National Cancer Institute (Bethesda, MD). Briefly, this multitumor tissue microarray is composed of 0.6-mm samples of 5 normal and 75 cancer of human breast tissue and some other tissues. Tissue microarray sections were dried under a low-power UV light for 2hours. All sections were deparaffinized, dehydrated, and stained by using Vectastain Universal Quick Kit (Vector Laboratories, Burlingame, CA). Slides were incubated with purified anti-SGK (1:100) for 20 minutes followed by biotinylated universal second antibody for 10 minutes and then streptavidin/peroxidase for 5 minutes before adding the peroxidase substrate for 5 seconds. Negative (normal IgG) controls were always included.
Nuclear Extract Preparation and Gel Mobility Shift Analysis of NF-B Binding. Nuclear extracts of MDA231and MDA231/SGK cells were prepared as described previously (28) and directly used for Western blotting detection of p65 nuclear translocation, IB degradation, and NF-B binding activity assay. NF-B binding activity was determined by gel mobility shift analysis. The oligonucleotide containing the NF-B binding sites was bought from Promega. The DNA-binding reaction was done at 30°C for 15 minutes in a volume of 20 µL containing 5 µg of nuclear extract, 225 µg/mL bovine serum albumin, 1.0 x 105 counts/min of 32P-labeled probe, 0.1 µg/mL poly(deoxyinosinic-deoxycytidylic acid), and 15 µL of binding buffer [12 mmol/L HEPES (pH 7.9), 4 mmol/L Tris, 60 mmol/L KCl, 1 mmol/L EDTA, 12% glycerol, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride]. After the binding reaction, the samples were subjected to electrophoresis in 1x Tris-borate EDTA buffer using 6% native polyacrylamide gels.
Statistics. Values were considered statistically significant at P < 0.05.
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B pathway.
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–/– or IKKß–/– cells (Fig. 3C) were transfected with pCMV-track-HA-SGK or control pCMV-track. Subsequently, the cells were subjected to serum-free media for 48 hours and then fixed and stained with Hoechst 33258. The apoptotic cells were measured by the change in nuclear morphology. Expression of wild-type SGK was associated with a significant reduction in the number of apoptotic cells in MEF cells. However, expression of SGK in either MEF cells that are IKK or IKKß null (Fig. 3E) does not provide protection from apoptosis when serum is withdrawn (Fig. 3C and D). Thus, the antiapoptotic effect of SGK involves IKK, suggesting a potential role for antiapoptotic NF-B pathway.
SGK Increases NF-B Activity through an IB Kinase. Next, we evaluated the effect of SGK expression on NF-B activation using a luciferase-promoter assay. When HEK293 cells were cotransfected with wild-type SGK and pNF-B-luc or with four other report vectors (pMyc-luc, p53-luc, pE2F-luc, and pRb-luc), we found that SGK expression dramatically increased the NF-B promoter activity. In sharp contrast, SGK had little or no influence on the luciferase activity driven by promoters for Myc, p53, E2F, or Rb (Fig. 4A). The selective enhancement of the NF-B–responsive promoter suggests that SGK specifically induces NF-B activity. As a control, we found that SGK does not increase mutant NF-B activity (Fig. 4B). In this experiment, MDA231 cells were transfected with SGK and wild-type or mutant HIV-NFB-luc, a construct consisting of four HIV-NF-B binding sites (23), then treated with 10% serum for 24 hours. FBS induced a 5-fold increase in HIV-NFB-luc promoter activity in the presence of SGK (Fig. 4B) but failed to increase mutant HIV-NFB activity with SGK expression (Fig. 4B).
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B under physiologic conditions, we infected MDA231 cells with adenovirus NF-B-luc. After serum starvation, the cells were treated with 10% FBS to increase the SGK level. The luciferase assay indicated that induction of SGK with serum resulted in a higher level of NF-B activity (Fig. 4C). To examine the specificity of SGK-increased NF-B, we blocked endogenous SGK by transfection of siRNA of SGK into MDA231 cells and used a nonspecific siRNA as control. The results indicate that expression of SGK increased NF-B activity 82% over the expression of siRNA control (control), whereas transfection of siRNA against SGK blocked 90% of NF-B activity compared with siRNA control (Fig. 4D). Moreover, serum failed to activate NF-B when SGK is blocked by the siRNA SGK (Fig. 4E). NF-B is normally sequestered in the cytoplasm by its interaction with the inhibitory protein, IB (29). Phosphorylation of IB by IKK leads to IB degradation with release of NF-B, which translocates to the nucleus to initiate gene transcription. To test if the activation of NF-B transcriptional activity by SGK involves IKK, we found that HEK293 cells expressing SGK or IKKß alone yielded a 6- to 8-fold increase in NF-B activity, respectively (Fig. 4F). However, the combination of SGK and IKKß expression produced an additive effect (i.e., a 12-fold increase over control values; Fig. 4F). In contrast, kinase-dead SGK or dominant-negative IKKß K44A inhibited SGK-induced NF-B activity (Fig. 4F). The nondegradable serine-to-alanine mutant IB (IB-AA) also inhibited the activation of NF-B (Fig. 4G). These results suggest IKK is acting downstream of SGK to mediate NF-B activation.
SGK Increases IB Degradation, p65 Nuclear Translocation, and NF-B DNA-Binding Activity. To provide additional evidence for SGK increased NF-B transcriptional activity, IB degradation, p65 nuclear translocation, and NF-B binding were analyzed. We compared MDA231 cells with the MDA231/SGK cell line treated with or without serum. Serum treatment for 30 minutes increased NF-B binding activity in MDA231/SGK cells compared with MDA231 cells (Fig. 4H). Note that in serum-free condition, there is also a higher NF-B binding activity in SGK cells. Consistent with the gel shift results, we found higher p65 nuclear translocation and IB degradation in MDA231/SGK cells than MDA231 cell (Fig. 4I and J).
SGK Enhances IKK Activity in Cultured Cells. To examine if SGK regulates IKK activity and phosphorylation of IB at Ser32 and Ser36 (30), we overexpressed wild-type IB tagged with Flag in HEK293 cells that were transfected with or without the wild-type SGK and with or without wild-type Flag-IKKß. IB was immunoprecipated by anti-Flag and detected by Western blotting with a phospho-IB antibody (phospho-Ser32). As shown in Fig. 5A, SGK expression alone induced minimal phosphorylation of IB at Ser32 (Fig. 5A, lane 3), whereas modest expression of IKKß alone induced a limited degree of Ser32 phosphorylation (Fig. 5A, lane 2). In contrast, expression of SGK and IKKß together dramatically increased IB phosphorylation (Fig. 5A, lane 4) indicating that a high SGK can increase IKK activity in HEK293 cells. There also was phosphorylation of IB in response to SGK occurring in a SGK dose-dependent manner (Fig. 5B). Next, we activated SGK by 10 ng/mL IGF-I and, as shown in Fig. 5C, IB phosphorylation was significantly increased when SGK was activated.
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B (1-54) in the presence of [-32P]ATP. First, IKK was immunopurified from HEK293 cells that has been transfected with SGK plus wild-type or dominant-negative IKKß. As shown in Fig. 5D, SGK increases IKK-mediated phosphorylation of IB, unless IKK was mutated. Second, we immunoprecipitated HA-SGK and Flag-IKKß from individual HEK293 cells transfected with HA-SGK or Flag-IKKß, respectively. As shown in Fig. 5E, combining the two immunocomplexes increased the phosphorylation of GST-IB (1-54; Fig. 5E). These results indicate that SGK could act upstream to activate IKK. SGK Associates with and Phosphorylates IKK. To test if SGK could increase IKK activity by forming a protein-protein interaction complex, we transfected HEK293 cells with HA-SGK and Flag-IKKß. In immunocomplexes obtained using an anti-HA antibody, we found the presence of IKKß (Fig. 5F, lane 2). Likewise, the association of SGK and IKK was also detected in HA-SGK–transfected MDA231 cells (Fig. 5F, lane 1). To correlate the association of SGK with IKKß in untransfected MDA231 cells, we treated serum-starved cells with 10% FBS to increase SGK expression. The cell lysate was immunoprecipated with anti-SGK or anti-IKKß antibody. SGK has a molecular weight close to that of the IgG heavy chain, so we labeled the SGK antibody with an antibody labeling kit (IRDye 800CW, LI-COR Biosciences, Lincoln, NE) and did not use a secondary antibody. As shown in Fig. 5G, IKKß was present in SGK complexes and SGK was present in IKKß-based complexes. These observations show that SGK and IKKß physically associate in MDA231 cancer cells.
To determine if SGK can phosphorylate the specific activation site in IKKß, we purified GST-IKKß wild-type (132 to 206AA) and serine mutant peptide (S177A/S181A) using a glutathione sepharose 4B column. Phosphorylation of these IKKß peptide fusion proteins by recombinant active SGK was measured in the presence of [-32P]ATP. As shown in Fig. 5H, SGK phosphorylated the wild-type recombinant IKKß protein but not the mutant IKKß (S177A/S181A; Fig. 5H). Finally, we expressed IKKß with or without SGK in HEK293 cells and found that expression of SGK stimulated phosphorylation of Ser177/181 of IKKß, which was measured by phosphor-IKKß 177/181 antibody (Fig. 5I). These results show that SGK can phosphorylate IKK both in vitro and in cultured cells.
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Discussion |
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B activity. A high level of expression of SGK in breast cancer cells suggests that this kinase may function to protect tumor cells from apoptosis, hence, act as an oncogene. These findings provide a potential explanation of why strategies such as antiangiogenic therapy are only partially successful in treating breast cancer. The high level of SGK in breast cancer with antiapoptotic properties would counteract the apoptotic response that should occur when blood supply is reduced. In short, an elevated SGK in advanced tumor could protect cancer cells from serum withdrawal–induced apoptosis. Several studies show that SGK is involved in cell survival. For example, gene expression analysis shows that SGK expression is increased in tumors that include liver and breast cancer (5, 6). There is also evidence that SGK protects cells during growth factor withdrawal (3, 4, 31). We found that a high level of SGK expression is present in many human breast cancer tissues, whereas down-regulation of SGK by siRNA SGK will enhance serum withdrawal–induced apoptosis of the breast cancer MDA231 cell while SGK expression inhibits apoptosis.
How does an actual SGK prevent apoptosis? Activated NF-B leads to transcriptional activation of genes whose products block apoptosis, including members of the Bcl-2 family, cellular inhibitors of apoptosis, and others (32). Previous studies have identified targets for this response in phosphorylation and negative regulation of the proapoptotic BAD and Forkhead transcriptional factors (3, 31). We provide evidence that IKK-NF-B is involved and that a high level of SGK can increase NF-B transcription activity by 82%, whereas knockdown of SGK in MDA231 cell by siRNA SGK will block NF-B transcriptional activity by 92%.
How does SGK stimulate NF-B transcriptional activity? We showed for the first time that SGK physically associates with IKKßin cultured cells and, more important, phosphorylates IKKßin vitro to enhance IKK activity, thus increasing IB phosphorylation to cause its degradation and increasing p65 translocation and NF-B DNA binding. Our results indicate that SGK directly activates IKKßto stimulate NF-B. However, we found that the SGK survival effect is abolished in IKK MEF cells(Fig. 3C and D), suggesting this response also requires the presence of IKK. This is not surprising because others have shown that IKK and IKKß are both necessary for the expression of NF-B–dependent, induced genes in cells (33). Moreover, several studies indicated that IKK is also essential to contribute NF-B activation by modification of chromatin, that is, phosphorylation of histone H3 to modulate chromatin accessibility at NF-B–responsive promoters (34–36). In line with these considerations, we and others find that knockout of IKK leads to a 70% reduction in basal NF-B activity compared with wild-type cells (data not shown). We conclude that both IKK and IKKß are required for maximal activation of NF-B, but our finding that there is a physical association of SGK with IKKß and phosphorylation of IKKß point to a new mechanism by which SGK promotes cell survival.
It has been reported that PDGF-induced Akt activation causes a transient association of Akt with IKK to stimulate the NF-B signaling pathway (17, 18). Because the kinase domain of SGK exhibits high (80%) homology with Akt and both are activated by phosphorylation via PDK1 and PDK2 kinases, which are downstream from PI-3 kinase (21), our data suggest an important alternative pathway for PI-3 kinase–mediated NF-B activation. This is an important finding because there are differences between SGK and Akt, including recognition sites for phosphorylation. For example, SGK phosphorylates Ser315 of the FKHRL1 transcription factor, whereas Akt favors Ser253 (31). Moreover, stimulation of SGK expression and activity is different from Akt because epithelial cells exposed to multiple types of environmental stress activated SGK, whereas Akt remained in a nonphosphorylated state (22). In short, SGK could act as an alternative signaling pathway for PI-3 kinase–mediated NF-B transcriptional factor activation.
In summary, our results point to novel mechanisms that link SGK to cell survival. SGK directly activates IKK to stimulate NF-B activity and the high level of expression of SGK in breast cancer cells suggests that this kinase is functioning to promote tumor cell survival and hence, acts as an oncogene.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6/ 3/04. Revised 11/ 5/04. Accepted 11/ 9/04.
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 V. Brune, E. Tiacci, I. Pfeil, C. Doring, S. Eckerle, C. J.M. van Noesel, W. Klapper, B. Falini, A. von Heydebreck, D. Metzler, et al. Origin and pathogenesis of nodular lymphocyte-predominant Hodgkin lymphoma as revealed by global gene expression analysis J. Exp. Med., September 29, 2008; 205(10): 2251 - 2268. [Abstract] [Full Text] [PDF]
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 A. B. Sherk, D. E. Frigo, C. G. Schnackenberg, J. D. Bray, N. J. Laping, W. Trizna, M. Hammond, J. R. Patterson, S. K. Thompson, D. Kazmin, et al. Development of a Small-Molecule Serum- and Glucocorticoid-Regulated Kinase-1 Antagonist and Its Evaluation as a Prostate Cancer Therapeutic Cancer Res., September 15, 2008; 68(18): 7475 - 7483. [Abstract] [Full Text] [PDF]
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S. de Seigneux, V. Leroy, H. Ghzili, M. Rousselot, S. Nielsen, B. C. Rossier, P.-Y. Martin, and E. Feraille NF-{kappa}B Inhibits Sodium Transport via Down-regulation of SGK1 in Renal Collecting Duct Principal Cells J. Biol. Chem., September 12, 2008; 283(37): 25671 - 25681. [Abstract] [Full Text] [PDF]
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L. Belova, D. R. Brickley, B. Ky, S. K. Sharma, and S. D. Conzen Hsp90 Regulates the Phosphorylation and Activity of Serum- and Glucocorticoid-regulated Kinase-1 J. Biol. Chem., July 4, 2008; 283(27): 18821 - 18831. [Abstract] [Full Text] [PDF]
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Y. Terada, H. Kuwana, T. Kobayashi, T. Okado, N. Suzuki, T. Yoshimoto, Y. Hirata, and S. Sasaki Aldosterone-Stimulated SGK1 Activity Mediates Profibrotic Signaling in the Mesangium J. Am. Soc. Nephrol., February 1, 2008; 19(2): 298 - 309. [Abstract] [Full Text] [PDF]
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 F. Lang, C. Bohmer, M. Palmada, G. Seebohm, N. Strutz-Seebohm, and V. Vallon (Patho)physiological Significance of the Serum- and Glucocorticoid-Inducible Kinase Isoforms. Physiol Rev, October 1, 2006; 86(4): 1151 - 1178. [Abstract] [Full Text] [PDF]
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 W. Wu, M. Zou, D. R. Brickley, T. Pew, and S. D. Conzen Glucocorticoid Receptor Activation Signals through Forkhead Transcription Factor 3a in Breast Cancer Cells Mol. Endocrinol., October 1, 2006; 20(10): 2304 - 2314. [Abstract] [Full Text] [PDF]
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M. Yu, J. Yeh, and C. Van Waes Protein Kinase Casein Kinase 2 Mediates Inhibitor-{kappa}B Kinase and Aberrant Nuclear Factor-{kappa}B Activation by Serum Factor(s) in Head and Neck Squamous Carcinoma Cells. Cancer Res., July 1, 2006; 66(13): 6722 - 6731. [Abstract] [Full Text] [PDF]
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 R. S. BelAiba, T. Djordjevic, S. Bonello, F. Artunc, F. Lang, J. Hess, and A. Gorlach The Serum- and Glucocorticoid-Inducible Kinase Sgk-1 Is Involved in Pulmonary Vascular Remodeling: Role in Redox-Sensitive Regulation of Tissue Factor by Thrombin Circ. Res., March 31, 2006; 98(6): 828 - 836. [Abstract] [Full Text] [PDF]
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 Y. Ma, L. Zhang, T. Peng, J. Cheng, S. Taneja, J. Zhang, P. Delafontaine, and J. Du Angiotensin II Stimulates Transcription of Insulin-Like Growth Factor I Receptor in Vascular Smooth Muscle Cells: Role of Nuclear Factor-{kappa}B Endocrinology, March 1, 2006; 147(3): 1256 - 1263. [Abstract] [Full Text] [PDF]
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L. Zhang, J. Cheng, Y. Ma, W. Thomas, J. Zhang, and J. Du Dual Pathways for Nuclear Factor {kappa}B Activation by Angiotensin II in Vascular Smooth Muscle: Phosphorylation of p65 by I{kappa}B Kinase and Ribosomal Kinase Circ. Res., November 11, 2005; 97(10): 975 - 982. [Abstract] [Full Text] [PDF]
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 M. N. Helms, L. Yu, B. Malik, D. J. Kleinhenz, C. M. Hart, and D. C. Eaton Role of SGK1 in nitric oxide inhibition of ENaC in Na+-transporting epithelia Am J Physiol Cell Physiol, September 1, 2005; 289(3): C717 - C726. [Abstract] [Full Text] [PDF]
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