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Alleviating the Suppression of Glycogen Synthase Kinase-3β by Akt Leads to the Phosphorylation of cAMP-response Element-binding Protein and Its Transactivation in Intact Cell Nuclei

2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês

10.1074/jbc.m302972200

ISSN

1083-351X

Autores

Thomas R. Salas, Shrikanth A.G. Reddy, John L. Clifford, Roger J. Davis, Akira Kikuchi, Scott M. Lippman, David G. Menter,

Tópico(s)

PI3K/AKT/mTOR signaling in cancer

Resumo

Glycogen synthase kinase-3β (GSK-3β) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3β activity opposes Akt function we used various methods to alleviate GSK-3β suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-α (TNF-α) were used to activate GSK-3β. In other experiments mutant forms of GSK-3β, GSK-3βΔ9 (a constitutively active deletion mutant of GSK-3β) and GSK-3βY216F (an inactive point mutant of GSK-3β) were used to alter GSK-3β activity. LY294002, TNF-α, and overexpression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, alleviated the suppression of GSK-3β activity in prostate carcinoma cells and enhanced the turnover of β-catenin. Forced expression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, suppressed cell growth and showed that the phosphorylation status of GSK-3β can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3β activity, overexpression of wild-type GSK-3β suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3βΔ9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3βΔ9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3βΔ9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei. Glycogen synthase kinase-3β (GSK-3β) activity is suppressed when it becomes phosphorylated on serine 9 by protein kinase B (Akt). To determine how GSK-3β activity opposes Akt function we used various methods to alleviate GSK-3β suppression in prostate carcinoma cells. In some experiments, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (a kinase involved in activating Akt) and tumor necrosis factor-α (TNF-α) were used to activate GSK-3β. In other experiments mutant forms of GSK-3β, GSK-3βΔ9 (a constitutively active deletion mutant of GSK-3β) and GSK-3βY216F (an inactive point mutant of GSK-3β) were used to alter GSK-3β activity. LY294002, TNF-α, and overexpression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, alleviated the suppression of GSK-3β activity in prostate carcinoma cells and enhanced the turnover of β-catenin. Forced expression of wild-type GSK-3β or of GSK-3βΔ9, but not GSK-3βY216F, suppressed cell growth and showed that the phosphorylation status of GSK-3β can affect its intracellular distribution. When transcription factors activator protein-1 and cyclic AMP-response element (CRE)-binding protein were analyzed as targets of GSK-3β activity, overexpression of wild-type GSK-3β suppressed AP1-mediated transcription and activated CRE-mediated transcription. Overexpression of GSK-3βΔ9 caused an (80-fold) increase in CRE-mediated transcription, which was further amplified (up to 130-fold) by combining GSK-3βΔ9 overexpression with the suppression of Jun activity. This study also demonstrated for the first time that expression of constitutively active GSK-3βΔ9 results in the phosphorylation of CRE-binding protein on serine 129 and enhancement of CRE-mediated transcription in intact cell nuclei. Glycogen synthase kinase-3β (GSK-3β) 1The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; Akt, protein kinase B; CRE, cAMP response element; CREB, CRE-binding protein; AP1, activator protein-1; PI3K, phosphatidylinositol 3-kinase; IGF, insulin-like growth factor; TNF, tumor necrosis factor; NFκB, nuclear factor κB; JNK, Jun N-terminal kinase; JBD, JNK-binding domain; TBS, triethanolamine-buffered saline; TBST, TBS containing 0.1% Tween 20; EGF, epidermal growth factor; CBP, CREB-binding protein; KID, kinase-inducible domain; PC, prostate carcinoma; LUC, luciferase; wt, wild type; CAM, calcein AM. is a proline-directed serine (Ser)/threonine (Thr) kinase (1Harwood A.J. Cell. 2001; 105: 821-824Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 2Woodgett, J. R. (2001) Science's STKE http://stke.sciencemag.org/cgi/content/full/oc_sigtrans%3b2001/100/rel2.Google Scholar, 3Frame S. Cohen P. Biochem. 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Additionally, certain amino acids (Arg96, Arg180, and Lys205) in the substrate binding pocket of GSK-3β determine the specificity of "primed substrate" binding and enzyme inhibition characteristics (1Harwood A.J. Cell. 2001; 105: 821-824Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 6Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar, 7Dajani R. Fraser E. Roe S.M. Young N. Good V. Dale T.C. Pearl L.H. Cell. 2001; 105: 721-732Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 8ter Haar E. Coll J.T. Austen D.A. Hsiao H.M. Swenson L. Jain J. Nat. Struct. Biol. 2001; 8: 593-596Crossref PubMed Scopus (335) Google Scholar). Several transcription factors are directly phosphorylated by GSK-3β (3Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1303) Google Scholar, 4Cohen P. Frame S. Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1325) Google Scholar, 9Scheid M.P. Woodgett J.R. J. Mammary Gland Biol. Neoplasia. 2001; 6: 83-99Crossref PubMed Scopus (31) Google Scholar, 10Kim L. Kimmel A.R. Curr. Opin. Genet. Dev. 2000; 10: 508-514Crossref PubMed Scopus (213) Google Scholar, 11Ding V.W. Chen R.H. McCormick F. J. Biol. Chem. 2000; 275: 32475-32481Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). The majority of these transcription factors are inhibited after being phosphorylated by GSK-3β; for example, GSK-3β blocks Jun-DNA binding by phosphorylating the Jun DNA binding domain. Only two transcription factors, cyclic AMP response element (CRE) binding (CREB) protein and possibly microphthalmia-associated transcription factor are stimulated after phosphorylation by GSK-3β (3Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1303) Google Scholar). In vitro studies have shown that CREB proteins and peptides are primed by phosphorylation at Ser133 (12Deak M. Clifton A.D. Lucocq L.M. Alessi D.R. EMBO J. 1998; 17: 4426-4441Crossref PubMed Scopus (855) Google Scholar) followed by progressive phosphorylation by GSK-3β at Ser129, which fully activates CRE transcription (13Fiol C.J. Williams J.S. Chou C.H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar, 14Wang Q.M. Roach P.J. Fiol C.J. Anal. Biochem. 1994; 220: 397-402Crossref PubMed Scopus (39) Google Scholar). No studies to date have demonstrated that the phosphorylation of CREB at Ser129 occurs in vivo or in intact cell nuclei (3Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1303) Google Scholar). Several signaling pathways inhibit GSK-3β activity (3Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1303) Google Scholar, 4Cohen P. Frame S. Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1325) Google Scholar). One of these signaling pathways, protein kinase B (or Akt), is often constitutively active in many types of human cancer (15Nicholson K.M. Anderson N.G. Cell. Signal. 2002; 14: 381-395Crossref PubMed Scopus (1412) Google Scholar) and phosphorylates GSK-3β on Ser9 to inactivate its kinase activity (16Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4471) Google Scholar, 17Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar, 18Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3754) Google Scholar, 19Cantley L. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4770) Google Scholar). Akt is itself activated upon phosphorylation of Ser473 and Thr308 by a complicated mechanism involving 3-phosphoinositide-dependent kinase 1 (20Scheid M.P. Marignani P.A. Woodgett J.R. Mol. Cell. Biol. 2002; 22: 6247-6260Crossref PubMed Scopus (275) Google Scholar, 21Scheid M.P. Woodgett J.R. Nat. Rev. Mol. Cell Biol. 2001; 2: 760-768Crossref PubMed Scopus (548) Google Scholar). Upstream in this pathway phosphatidylinositol 3-kinase (PI3K) commonly binds to receptors that are activated in response to insulin or growth factors, e.g. insulin-like growth factor (IGF) (18Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3754) Google Scholar, 22Angelloz-Nicoud P. Lalou C. Binoux M. Growth Horm. IGF Res. 1998; 8: 71-75Crossref PubMed Scopus (24) Google Scholar, 23Dupont J. Le Roith D. Mol. Pathol. 2001; 54: 149-154Crossref PubMed Scopus (122) Google Scholar, 24Bevan P. J. Cell Sci. 2001; 114: 1429-1430Crossref PubMed Google Scholar). The prostate carcinoma (PC)-3 cells used in the present study exhibit growth factor-independent proliferation that involves an IGF autocrine loop (22Angelloz-Nicoud P. Lalou C. Binoux M. Growth Horm. IGF Res. 1998; 8: 71-75Crossref PubMed Scopus (24) Google Scholar, 25Huynh H. Pollak M. Zhang J.C. Int. J. Oncol. 1998; 13: 137-143PubMed Google Scholar, 26Kimura G. Kasuya J. Giannini S. Honda Y. Mohan S. Kawachi M. Akimoto M. Fujita-Yamaguchi Y. Int. J. Urol. 1996; 3: 39-46Crossref PubMed Scopus (23) Google Scholar). ThisautocrineloopcanstimulatethePI3K/3-phosphoinositide-dependent kinase 1/Akt pathway, which can phosphorylate GSK-3β on Ser9 and, thus, suppress its enzymatic activity. In addition to positively regulating cell proliferation, the PI3K/ 3-phosphoinositide-dependent kinase 1/Akt activation has been implicated in regulating sensitivity to cytokines, such as tumor necrosis factor (TNF)-α, which induce apoptosis (27Chen X. Thakkar H. Tyan F. Gim S. Robinson H. Lee C. Pandey S.K. Nwokorie C. Onwudiwe N. Srivastava R.K. Oncogene. 2001; 20: 6073-6083Crossref PubMed Scopus (265) Google Scholar, 28Voelkel-Johnson C. King D.L. Norris J.S. Cancer Gene Ther. 2002; 9: 164-172Crossref PubMed Scopus (94) Google Scholar, 29Beresford S.A. Davies M.A. Gallick G.E. Donato N.J. J. Interferon Cytokine Res. 2001; 21: 313-322Crossref PubMed Scopus (24) Google Scholar). Although the transcription factor nuclear factor κB (NFκB) is partly responsible for cellular responses to TNF-α through NFκB protein interactions with GSK-3β (30Hoeflich K.P. Luo J. Rubie E.A. Tsao M.S. Jin O. Woodgett J.R. Nature. 2000; 406: 86-90Crossref PubMed Scopus (1242) Google Scholar), other transcription factors also may be affected by TNF-α, particularly those influenced by Akt suppression of GSK-3β activity. We conducted the present study to 1) determine the effects of alleviating GSK-3β suppression by Akt and assess GSK-3β function in prostate cancer cells, 2) examine the phosphorylation of CREB on Ser129 by GSK-3β in intact cell nuclei and its effects on CRE-mediated transcription, and 3) examine the intracellular distribution of GSK-3β mutants that were altered at critical phosphorylation sites. Cells and Culture Conditions—PC-3 human prostate adenocarcinoma cells were obtained from the American Type Culture Collection (Manassas, VA). These cells were maintained in a 1:1 (vol:vol) mixture of Dulbecco's minimal essential medium and F-12 medium supplemented with 10% fetal bovine serum. Reagents—Recombinant TNF-α, IGF-1, and epidermal growth factor (EGF) were purchased from R&D Systems Inc. (Minneapolis, MN). Anti-Tyr(P)216-GSK-3 anti-β-catenin (Upstate Biotechnology, Inc., Lake Placid, NY), anti-GSK-3β-specific, anti-Ser(P)9-GSK-3, and anti-Ser(P)63-Jun (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were purchased from the respective manufacturers. Rabbit anti-Ser(P)133-CREB antibody was purchased from Upstate Biotechnology. Anti-Ser(P)9-GSK-3 and anti-Jun antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Goat anti-mouse and goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce. Goat anti-mouse and goat anti-rabbit Alexa-488-conjugated secondary antibodies were purchased from Molecular Probes (Eugene, OR). Preparation of Anti-CKRREILS129RRPS133YR-CREB Antibody— Rabbit polyclonal antibodies were raised against a diphosphorylated CREB peptide CKRREILS129RRPS133YR. The diphospho peptide CKRREILS129RRPS133YR was conjugated with a keyhole limpet hemocyanin carrier by generating a sulfide linkage using maleimide chemistry, and a diphospho peptide antibody was purified on a CKRREILS129RRPS133YR affinity column. A second affinity purification step was carried out using a monophospho peptide CKRREILSRRPS133YR affinity column to eliminate antibodies that interacted with the monophosphorylated peptide. The resulting pass-through fraction produced an anti-diphospho peptide antibody that was high titer (1:173,000) based on a peptide linked-enzyme immunosorbent assay that failed to bind CKRREILSRRPS133YR but could specifically recognize both CKRREILS129RRPSYR and CKRREILS129RRPS133YR. GSK-3β Assays—PC-3 cells were incubated overnight in serum-free Dulbecco's minimal essential medium/F-12 medium and then incubated with or without 10 ng/ml TNF-α for 20 min. The cells were harvested, and GSK-3β was immunoprecipitated from detergent lysates using an anti-GSK-3β antibody (Transduction Laboratories, Los Angeles, CA). GSK-3β kinase assays were then carried out as described by Pap and Cooper (17Pap M. Cooper G.M. J. Biol. Chem. 1998; 273: 19929-19932Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar) using a Ser(P)133-CREB peptide (amino acids 123–135; New England Biolabs, Beverly, MA) in the presence of [γ-32P]ATP (specific activity, 3000 Ci/mm; Amersham Biosciences). Cells were treated with 20 μm LY294002 (Sigma), a PI3K-specific inhibitor, to inhibit Akt and isolate GSK-3β in an active form. The results were normalized as a relative cpm incorporated into a CREB peptide, compared with untreated control samples, and analyzed for statistical significance using the StatView software program (SAS Institute, Inc., Cary, NC). Nuclear Extracts—Nuclear extracts were prepared as described by Schaefer et al. (31Schaefer T.S. Sanders L.K. Park O.K. Nathans D. Mol. Cell. Biol. 1997; 17: 5307-5316Crossref PubMed Google Scholar). Briefly, after cells were washed with cold phosphate-buffered saline wash solution (phosphate-buffered saline containing 0.1× BM complete protease mixture tablet (Roche Molecular Biochemicals) and 10 μm Na2VO4), they were scrape-harvested into 1.5-ml conical tubes. Cell suspensions were centrifuged at 3000 revolutions per min (rpm) at 4 °C for 5 min, and pelleted cells were resuspended in 100 μl of hypotonic lysis buffer (10 mm Hepes, pH 8.0, 1.5 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, 25 mm NaF, 1 mm Na2VO4, and 1× BM complete protease mixture inhibitors) and incubated on wet ice for 10 min. The resuspended lysate was centrifuged at 7500 rpm at 4 °C for 5 min. The nuclear suspension as washed 2× and centrifuged at 12,000 rpm for 5 min. Nuclear preparations were examined by light microscopy for the presence of cytosolic components, which were not present. The nuclear pellets were lysed with sodium dodecyl sulfate sample buffer and DNA-sheared through a 20-gauge needle. These lysates were then compared with total cell lysates for the presence of actin. Only trace amounts of actin were present in the nuclear lysate preparations, whereas the actin content was very high in the total cell lysate (data not shown). Western Blot Analysis—PC-3 cells were left untreated or treated with 10 ng/ml TNF-α, 1 ng/ml IGF-1, 10 ng/ml EGF, or 1 μm insulin for 16 h at 37 °C and subsequently analyzed for expression and activation of various proteins. Whole-cell lysates were made from various PC-3 cell samples according to a previously described method (31Schaefer T.S. Sanders L.K. Park O.K. Nathans D. Mol. Cell. Biol. 1997; 17: 5307-5316Crossref PubMed Google Scholar). Proteins were separated on a NuPAGE 6–12% gradient gel (Novex, Carlsbad, CA), electrotransferred to a nitrocellulose membrane, and immunoblotted with a primary antibody overnight at 4 °C. Immune complexes were visualized via Super Signal chemiluminescence (Pierce). Transfection and Luciferase (LUC) Assays—The commercially available plasmids used were pCRE-LUC, pCISCK (an inactive LUC control) (Stratagene, La Jolla, CA), and pAP1-LUC, (BD Biosciences Clontech, PaloAlto, CA). The pCMV-4 control plasmid was a gift from D. Russell. The plasmids were generated in the authors laboratories and included pCDNA3-Flag-JBD, pCGN/GSK-3wt, pCGN/GSK-3Δ9, and pCGN/GSK-3Y216F (32Dickens M. Rogers J.S. Cavanagh J. Raitano A. Xia Z. Halpern J.R. Greenberg M.E. Sawyers C.L. Davis R.J. Science. 1997; 277: 693-696Crossref PubMed Scopus (630) Google Scholar, 33Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (64) Google Scholar, 34Rogatsky I. Waase C.L. Garabedian M.J. J. Biol. Chem. 1998; 273: 14315-14321Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Cells were plated at a density of 40% in triplicate wells, using six-well plates. The next day, FuGENE 6 (Roche Diagnostics, Indianapolis, IN) was used to transfect cells in accordance with the manufacturer's instructions. Each plasmid concentration was 1 μg/ml, unless stated otherwise. To normalize LUC activity either pCRE-LUC or pAP1-LUC was transfected in excess of 6-fold (0.86 μg/ml) greater than Renilla-LUC (0.13 μg/ml; pTK-LUC, Promega, Madison, WI). The transfected cells were harvested and analyzed using the Firelight system (a gift from Corinn Rich and Packard Instrument Co., Naperville, IL) in accordance with the manufacturer's instructions. Quantification was performed in a 96-well black plate using a TOPCOUNT multiwell plate scintillation detector (Packard Instrument Co.) in the photon-counting mode. All pCRE-LUC or pAP1-LUC data were normalized to Renilla-LUC that was co-transfected to determine the effect on the relative promoter activity. To establish if there was any change in the level of pCRE-LUC and pAP1-LUC DNA in PC-3 cells or those cotransfected with GSK-3β LUC, DNA template levels were determined by PCR using primers designed to amplify LUC. The levels of LUC DNA were the same in cells transfected with either pCRE-LUC or pAP1-LUC DNA alone or when LUC reporter constructs were cotransfected with any of the GSK-3β expression vectors (data not shown). Densitometry—These experiments were performed multiple times and quantified by densitometry, and these data were subjected to statistical analysis. All densitometric analyses were performed using a Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA) and the corresponding software program, ImageQuant (Molecular Dynamics). Images were quantified using NIH Image 1.62 (National Institutes of Health), and statistical analysis was performed using StatView 5.01 (SAS Institute Inc.). The density of the proteins analyzed was normalized to the appropriate controls (e.g. actin). Student's t test was used to determine the significant differences between the mean relative densities of the various experimental groups, represented as p values. Immunofluorescence—Immunofluorescence analysis was performed as described previously (35Menter D.G. Sabichi A.S. Lippman S.M. Cancer Epidemiol. Biomark. Prev. 2000; 9: 1171-1182PubMed Google Scholar). Briefly, cells were grown on laminin-coated coverslips, which were used for TNF-α treatment. The coverslips were fixed with 1% paraformaldehyde and permeabilized using 1% Nonidet P-40 detergent. Next, samples were blocked with 3% bovine serum albumin solution in triethanolamine-buffered saline (TBS) containing 0.1% Tween 20 (TBST). A primary rabbit polyclonal antibody recognizing GSK-3β, Ser(P)133-CREB or Ser(P)129-Ser(P)133-CREB was diluted (1:200; vol:vol) in TBST containing 1% bovine serum albumin. The samples were then rinsed with TBS and incubated with the secondary antibody Alexa 488 (Molecular Probes) in TBST. After the cells were stained with secondary antibodies, counterstaining was performed using 500 nm 4′,6-diamidino-2-phenylindole dilactate (Molecular Probes) to identify the nuclei and 1 unit/ml Alexa 594-phalloidin (Molecular Probes) in TBST at 4 °C to stain the cytoplasmic actin. The samples were incubated overnight and then washed and mounted on glass slides using Prolong antifade solution (Molecular Probes). The slides were then analyzed via epifluorescence microscopy, and data were acquired using digital image analysis as described previously (35Menter D.G. Sabichi A.S. Lippman S.M. Cancer Epidemiol. Biomark. Prev. 2000; 9: 1171-1182PubMed Google Scholar). Cell Growth—Cell growth was measured using the vital dye calcein AM (CAM) ester (Molecular Probes), which is membrane-permeable and nonfluorescent before activation by nonspecific esterases within viable cells. The cleavage product CAM emits green fluorescence. Cells transfected with either GSK-3β expression vectors or control vector were plated in 96-well plates and incubated with CAM ester in HEPES-buffered saline solution for 15 min at 25 °C. Fluorescence was quantified using a Biolumin 9600 plate reader at 488 nm, and data were analyzed using the Statview software program (SAS Institute, Inc.). Immunostaining of Paraffin Sections for Ser(P)129-S(P)133-CREB— Immunohistochemistry was carried out in paraffin-embedded sections (5 μm) after deparaffinization and rehydration in xylene, graded alcohol, and phosphate-buffered saline, respectively. The endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide for 20 min at room temperature. After blocking the sections with 1.5% normal horse serum in phosphate-buffered saline for 1 h, anti-Ser(P)129-Ser(P)133-CREB was applied to the sections and incubated overnight at 4 °C. Then the sections were incubated with the appropriate biotinylated secondary antibody and ABC-AP reagent according to the manufacturer's instructions (Vectastain ABC Elite kit, Vector Laboratories, Burlingame, CA). Peroxidase activity was detected with by applying 3,3′-diaminobenzidine tetrahydrochloride containing 0.02% of hydrogen peroxide for 10 min. The sections were counter-stained with hematoxylin and mounted with coverslips. Molecular Modeling—Structural data obtained from Protein Data Bank code 1KDX (36Radhakrishnan I. Perez-Alvarado G.C. Parker D. Dyson H.J. Montminy M.R. Wright P.E. Cell. 1997; 91: 741-752Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar) were used to generate a model of CREB-kinase inducible domain (KID) and CBP-KID interaction domain interactions. BiotechniX 3d software (Gentech, Parc Sophia Antipolis, France) was used for three-dimensional rendering of CREB on a G4 Titanium Powerbook Computer (Apple Computer Corp., Cupertino, CA). GSK-3β Activity Is Suppressed in PC-3 Cells—Endogenous GSK-3β enzymatic activity was low in PC-3 cells (Fig. 1A, bar 1) as determined by the immune complex kinase assay using CREB peptide as the substrate. This low GSK-3β activity in untreated cells correlated with a relatively high level of phosphorylation on Ser9 (Fig. 1B, lane 1). Because PI3K is involved in the activation of Akt, which in turn phosphorylates GSK-3β at Ser9 and, thus, inactivates it, we treated cells with LY294002, a PI3K inhibitor, to determine whether GSK-3β is suppressed through this pathway. LY294002 increased GSK-3β activity ∼2-fold (Fig. 1A, bar 2), and this effect was enhanced by combining LY294002 with TNF-α (Fig. 1A, bar 4). This increase in enzyme activity correlated with a decrease in the phosphorylation of Ser9 with LY294002 alone (Fig. 1B, lane 2), which decreased further when LY294002 treatment was combined with TNF-α (Fig 1B, lane 8). Furthermore, PC-3 cells were treated with increasing concentrations of LY294002, and then enzymatic activity was determined by CREB peptide phosphorylation along with measuring the phosphorylation of Ser9 by Western analysis. After densitometric quantification, regression analysis was performed using the Statview program to compare the relative GSK-3β enzymatic activity in relation to the inverse value of the mean phospho-Ser9 density. An R 2 value approximately equal to 0.9 indicated that the relative activity of GSK-3β decreased in conjunction with increasing the phosphorylation of GSK-3β on Ser9. In addition, two PI3K stimulators, EGF and IGF-1(Fig. 1B, lanes 3 and 4, respectively), did not further suppress GSK-3β activity by increasing the phosphorylation of GSK-3β at Ser9 beyond control levels. Similar studies showed that these treatments caused a decrease in β-catenin levels (Fig. 2). Furthermore, results similar to those observed for EGF and IGF were obtained using insulin (data not shown). These results indicate that endogenous GSK-3β activity is largely suppressed by the PI3K pathway in proliferating PC-3 cells and that activation of GSK-3β enhanced the breakdown of β-catenin.Fig. 2The activation of GSK-3β enhanced the turnover of β-catenin. PC-3 cells were left untreated or treated with TNF-α (10 ng/ml), EGF (10 ng/ml), or IGF-1 (1 ng/ml) in the absence (lanes 1–4) or presence (lanes 5–8) of PI3K-specific inhibitor LY294002 (20 μm) for 16 h, and whole-cell lysates were prepared and analyzed by Western blot. Treatment with PI3K inhibitor LY294002 caused a significant decrease in β-catenin levels. Statistical analysis showed a significant difference (a, p < 0.003) between control samples and TNF-α- or LY294002-treated samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GSK-3β Stimulated CRE-mediated Transcription and Suppressed Activator Protein-1 (AP1)-mediated Transcription— CREB and Jun family members can take part in binding to and activating CRE-containing promoters; Jun family members can also activate AP1 transcription response elements. To determine how CRE and AP1 transcriptional activities were affected by GSK-3β, PC-3 cells were cotransfected with GSK-3β, with either the CRE or AP1 response element-containing luciferase reporter constructs (CRE-LUC and AP1-LUC, respectively). The CRE-LUC activity in PC-3 cells cotransfected with GSK-3β was 6-fold higher than that in cells transfected with CRE-LUC alone (Fig. 3). The LUC vector without the CRE elements (pCISCK) showed no LUC activity, and the expression vector pCMV4 without the GSK-3 cDNA did not affect LUC activity (data not shown). We also observed that in the absence of cotransfected GSK-3β, AP1-LUC activity was about 20 times higher than for CRE-LUC. In contrast to its effect on CRE-LUC activity, GSK-3β strongly inhibited AP1-LUC activity. These data indicate that overexpression of GSK-3β can differentially regulate CREB-mediated and AP1-mediated activity in PC-3 cells. Phosphorylation of GSK-3β Variants—We next investigated the overexpression of GSK-3β proteins that contained various point mutations or deletions to determine how these molecular changes affected the protein phosphorylation patterns and the intracellular distribution of GSK-3β (Fig. 4). Deletion of the first nine amino acids of GSK-3β results in the GSK-3βΔ9 protein, which is constitutively active and cannot be inactivated by PI3K/Akt. Ser9 phosphorylation was present on all forms of GSK-3β except on the GSK-3βΔ9 transfectants (Fig. 4A, Δ9 lane lower band is absent). In contrast, GSK-3βY216F contains a mutation of tyrosine Tyr216 to a phenylalanine, resulting in a dominant-negative-acting protein that cannot activate CREB. Tyr216 was phosphorylated on endogenous GSK-3β in PC-3 cells (Fig. 4A, lane C) and on the wild-type (wt) and Δ9-transfected forms of GSK-3β. The total Tyr216 phosphorylation was reduced in the GSK-3βY216F transfectants, which included some Tyr216 phosphorylation on endogenous GSK-3β, since the phenylalanine on GSK-3βY216F cannot be phosphorylated (Fig. 4A, lane Y216F). Intracellular Distribution of GSK-3β Variants—Examination of the intracellular distribution of the different GSK-3β proteins by immunofluorescence using a GSK-3β-specific antibody showed that untransfected and empty vector-transfected control cells contained low levels of endogenous GSK-3β in both the cytoplasm and nucleus of PC-3 cells (Fig. 4B, control (C)). Exogenous, wt GSK-3β (when overexpressed) was found in both the cytoplasm and the nucleus of PC-3 cells (Fig. 4B, wt). The GSK-3βY216F protein was predominantly found in the cytoplasm, and the GSK-3βΔ9 protein was almost exclusively nuclear (Fig. 4B, Y216F and Δ9). β-Catenin Expressio

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