Glycogen Synthase Kinase-3β Is Involved in the Phosphorylation and Suppression of Androgen Receptor Activity
2004; Elsevier BV; Volume: 279; Issue: 18 Linguagem: Inglês
10.1074/jbc.m309560200
ISSN1083-351X
AutoresThomas R. Salas, Jeri Kim, Funda Vakar‐Lopez, Anita L. Sabichi, Patricia Troncoso, Guido Jenster, Akira Kikuchi, Shao-Yong Chen, Lirim Shemshedini, Milind Suraokar, Christopher J. Logothetis, John DiGiovanni, Scott M. Lippman, David G. Menter,
Tópico(s)Cancer-related gene regulation
ResumoKinases can phosphorylate and regulate androgen receptor activity during prostate cancer progression. In particular, we showed that glycogen synthase kinase-3β phosphorylates the androgen receptor, thereby inhibiting androgen receptor-driven transcription. Conversely, the glycogen synthase kinase-3β inhibitor lithium chloride suppressed the glycogen synthase kinase-3β-mediated phosphorylation of the androgen receptor, thereby enabling androgen receptor-driven transcription to occur. The androgen receptor hinge and ligand-binding domains were important for both the phosphorylation and the inhibition of transcriptional activity of the receptor by glycogen synthase kinase-3β. Furthermore, androgen receptor phosphorylation was augmented by LY294002, an indirect inhibitor of protein kinase B/Akt that inhibits glycogen synthase kinase-3β. We also showed that the mutation of various phosphorylation sites on glycogen synthase kinase-3β affected the ability of these mutants to co-distribute with the androgen receptor in the cell nucleus, also that both glycogen synthase kinase-3β and androgen receptor proteins can be found in cell nuclei of prostate cancer tissue samples. Because glycogen synthase kinase-3β activity is suppressed after the enzyme is phosphorylated by protein kinase B/Akt and Akt activity frequently increases during the progression of prostate cancer, nullification of the glycogen synthase kinase-3β-mediated suppression of androgen receptor activity by Akt likely contributes to prostate cancer progression. Kinases can phosphorylate and regulate androgen receptor activity during prostate cancer progression. In particular, we showed that glycogen synthase kinase-3β phosphorylates the androgen receptor, thereby inhibiting androgen receptor-driven transcription. Conversely, the glycogen synthase kinase-3β inhibitor lithium chloride suppressed the glycogen synthase kinase-3β-mediated phosphorylation of the androgen receptor, thereby enabling androgen receptor-driven transcription to occur. The androgen receptor hinge and ligand-binding domains were important for both the phosphorylation and the inhibition of transcriptional activity of the receptor by glycogen synthase kinase-3β. Furthermore, androgen receptor phosphorylation was augmented by LY294002, an indirect inhibitor of protein kinase B/Akt that inhibits glycogen synthase kinase-3β. We also showed that the mutation of various phosphorylation sites on glycogen synthase kinase-3β affected the ability of these mutants to co-distribute with the androgen receptor in the cell nucleus, also that both glycogen synthase kinase-3β and androgen receptor proteins can be found in cell nuclei of prostate cancer tissue samples. Because glycogen synthase kinase-3β activity is suppressed after the enzyme is phosphorylated by protein kinase B/Akt and Akt activity frequently increases during the progression of prostate cancer, nullification of the glycogen synthase kinase-3β-mediated suppression of androgen receptor activity by Akt likely contributes to prostate cancer progression. Androgens and androgen receptors (ARs), 1The abbreviations used are: AR, androgen receptor; ARE, androgen response element; GSK-3β, glycogen synthase kinase-3β; Akt, protein kinase B; R1881, 17α-methyl-17β-hydroxyestra-4,9,11-trien-3-one or metribolone; DH2O, distilled H2O; C3, pcDNA 3.1 empty vector expressing PC-3 cells; A103, hAR/pcDNA 3.1 plasmid expressing PC-3 cells; V28, VP16-AR expressing PC-3 cells; PC-3, prostate carcinoma cells; COS-1, African green monkey kidney fibroblast cells; LUC, luciferase; PB, probasin; wt, wild type; HA, hemagglutinin. 1The abbreviations used are: AR, androgen receptor; ARE, androgen response element; GSK-3β, glycogen synthase kinase-3β; Akt, protein kinase B; R1881, 17α-methyl-17β-hydroxyestra-4,9,11-trien-3-one or metribolone; DH2O, distilled H2O; C3, pcDNA 3.1 empty vector expressing PC-3 cells; A103, hAR/pcDNA 3.1 plasmid expressing PC-3 cells; V28, VP16-AR expressing PC-3 cells; PC-3, prostate carcinoma cells; COS-1, African green monkey kidney fibroblast cells; LUC, luciferase; PB, probasin; wt, wild type; HA, hemagglutinin. which like other steroid nuclear receptors, can be regulated by kinases (1Brinkmann A.O. Blok L.J. de Ruiter P.E. Doesburg P. Steketee K. Berrevoets C.A. Trapman J. J. Steroid Biochem. Mol. Biol. 1999; 69: 307-313Google Scholar, 2Jenster G. J. Pathol. 2000; 191: 227-228Google Scholar, 3Jenster G. Semin. Oncol. 1999; 26: 407-421Google Scholar, 4Feldman B.J. Feldman D. Nature Rev. Cancer. 2001; 1: 34-45Google Scholar, 5Nelson W.G. De Marzo A.M. Isaacs W.B. N. Engl. J. Med. 2003; 349: 366-381Google Scholar). In the prostate, AR helps maintain the balance between the growth and death of prostate cells (4Feldman B.J. Feldman D. Nature Rev. Cancer. 2001; 1: 34-45Google Scholar). Structurally, the AR has a transactivation domain (amino acids 1-556), a DNA-binding domain (amino acids 559-627), a hinge region (amino acids 622-670), and a ligand-binding domain (amino acids 712-919) (3Jenster G. Semin. Oncol. 1999; 26: 407-421Google Scholar). During the progression of prostate cancer, however, ARs can become mutated or their regulation compromised, which often leads to the development of androgen-independent prostate cancer (4Feldman B.J. Feldman D. Nature Rev. Cancer. 2001; 1: 34-45Google Scholar, 6Logothetis C.J. Hoosein N.M. Hsieh J.T. Semin. Oncol. 1994; 21: 620-629Google Scholar). Androgen-independent prostate cancer is a lethal recurring tumor that often develops in patients who have undergone androgen ablation therapy. Multiple factors can cause androgen-independent prostate cancer (4Feldman B.J. Feldman D. Nature Rev. Cancer. 2001; 1: 34-45Google Scholar), but the most common factor, and the one that helps drive both normal and dysfunctional receptor activity, is the phosphorylation of AR.Data suggest that kinases regulate AR function, but which kinases are involved remains unresolved. Rigorous phosphoamino acid analyses of the AR have shown that serine phosphorylation events are more prevalent than either threonine or tyrosine suggesting that serine kinases may exert most of the influence on the AR (1Brinkmann A.O. Blok L.J. de Ruiter P.E. Doesburg P. Steketee K. Berrevoets C.A. Trapman J. J. Steroid Biochem. Mol. Biol. 1999; 69: 307-313Google Scholar, 7Jenster G. de Ruiter P.E. van der Korput H.A. Kuiper G.G. Trapman J. Brinkmann A.O. Biochemistry. 1994; 33: 14064-14072Google Scholar). Many consensus sites are present in the AR for serine kinases including mitogen-activated protein kinases, calmodulin-dependent protein kinases, and glycogen synthase kinase (GSK)-3 (8Kennelly P.J. Krebs E.G. J. Biol. Chem. 1991; 266: 15555-15558Google Scholar). In one instance, Ser308 in the AR was identified as a phosphorylation site but the kinase that phosphorylates it remains to be identified (9Zhu Z. Becklin R.R. Desiderio D.M. Dalton J.T. Biochem. Biophys. Res. Commun. 2001; 284: 836-844Google Scholar). Others have shown that serines 81 and 94 become phosphorylated soon after translation and when these sites are mutated this limits ligand-induced phosphorylation but not transactivation (7Jenster G. de Ruiter P.E. van der Korput H.A. Kuiper G.G. Trapman J. Brinkmann A.O. Biochemistry. 1994; 33: 14064-14072Google Scholar, 10Zhou Z.X. Kemppainen J.A. Wilson E.M. Mol. Endocrinol. 1995; 9: 605-615Google Scholar). Also, the mutation of serine 650 reduces transcriptional regulation of an androgen response element (ARE) containing promoter construct by 30% (10Zhou Z.X. Kemppainen J.A. Wilson E.M. Mol. Endocrinol. 1995; 9: 605-615Google Scholar). The importance of Akt kinase activity in prostate cancer has been well documented (11Davies M.A. Koul D. Dhesi H. Berman R. McDonnell T.J. McConkey D. Yung W.K. Steck P.A. Cancer Res. 1999; 59: 2551-2556Google Scholar, 12Manin M. Baron S. Goossens K. Beaudoin C. Jean C. Veyssiere G. Verhoeven G. Morel L. Biochem. J. 2002; 366: 729-736Google Scholar, 13Davies M.A. Kim S.J. Parikh N.U. Dong Z. Bucana C.D. Gallick G.E. Clin. Cancer Res. 2002; 8: 1904-1914Google Scholar, 14Malik S.N. Brattain M. Ghosh P.M. Troyer D.A. Prihoda T. Bedolla R. Kreisberg J.I. Clin. Cancer Res. 2002; 8: 1168-1171Google Scholar, 15Paweletz C.P. Charboneau L. Bichsel V.E. Simone N.L. Chen T. Gillespie J.W. Emmert-Buck M.R. Roth M.J. Petricoin I.E. Liotta L.A. Oncogene. 2001; 20: 1981-1989Google Scholar), particularly the direct involvement of Akt in phosphorylating AR. For example, one study showed that the phosphorylation of AR on Ser210 and Ser790 by Akt stimulated AR-driven transcriptional activity (16Wen Y. Hu M.C. Makino K. Spohn B. Bartholomeusz G. Yan D.H. Hung M.C. Cancer Res. 2000; 60: 6841-6845Google Scholar), whereas another study showed that Akt suppressed activity (17Lin H.K. Yeh S. Kang H.Y. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7200-7205Google Scholar). However, both of these studies using in vitro kinase assays in combination with site-directed mutagenesis, putatively showed that Ser213 and Ser791 were the amino acids on AR that were phosphorylated by Akt. Limited in vivo analysis further indicated that Akt mediated the phosphorylation of AR in COS-1 cells, but no attempt was made to determine whether Akt and AR were co-localized within cells (17Lin H.K. Yeh S. Kang H.Y. Chang C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7200-7205Google Scholar). In contrast with these findings, other studies by Gioeli et al. (18Gioeli D. Ficarro S.B. Kwiek J.J. Aaronson D. Hancock M. Catling A.D. White F.M. Christian R.E. Settlage R.E. Shabanowitz J. Hunt D.F. Weber M.J. J. Biol. Chem. 2002; 277: 29304-29314Google Scholar) who used intact cells to perform [32P]orthophosphate labeling in vivo followed by peptide mapping and mass spectroscopy, failed to confirm the phosphorylation of AR by Akt on Ser213 and Ser791. These conflicting in vitro findings with regard to the Akt phosphorylation of Ser213 and Ser791 on AR and the failure to confirm in vivo that these sites are phosphorylated led us to postulate that GSK-3β regulates AR as an indirect consequence of Akt activity. GSK-3β is a likely candidate for regulating AR as an indirect mediator of Akt activity for multiple reasons. One reason is that GSK-3β is known to phosphorylate and mediate the activity of numerous nuclear transcription factors (19Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Google Scholar); another is that GSK-3β is inactivated after being phosphorylated on Ser9 by Akt (19Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Google Scholar, 20Frame S. Cohen P. Biondi R.M. Mol. Cell. 2001; 7: 1321-1327Google Scholar).We have recently shown that GSK-3β can phosphorylate cyclic AMP response element-binding protein and activate its transcriptional activity in the nuclei of PC-3 prostate carcinoma cells (21Salas T.R. Reddy S.A. Clifford J.L. Davis R.J. Kikuchi A. Lippman S.M. Menter D.G. J. Biol. Chem. 2003; 278: 41338-41346Google Scholar). In the present study, we used prostate carcinoma cells and COS-1 cells to: 1) examine the phosphorylation of AR by GSK-3β and its effects on AR-mediated transcription; 2) determine the AR protein domain that interacts with and is phosphorylated by GSK-3β; and 3) examine the intracellular co-distribution of AR with mutated forms of GSK-3β.EXPERIMENTAL PROCEDURESCells and Culture Conditions—The human prostate adenocarcinoma cells PC-3, DU145, LNCaP, as well as COS-1 cells, were obtained from the American Tissue Type Culture Collection (Manassas, VA). PC-3 cells were selected that stably expressed empty pcDNA 3.1 vector (C3 cells), hAR/pcDNA 3.1 plasmid (A103 cells), or VP16-AR (V28 cells). All tumor cells were maintained in Dulbecco's modified Eagle's medium and F-12 medium (mixed 1:1) supplemented with 10% fetal bovine serum.Western Blot Analysis—Western blot analysis was performed to detect various proteins in human prostate cancer cells. Whole cell lysates were prepared as previously described (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar). Proteins were then separated by SDS-PAGE, electrotransferred to nitrocellulose, and analyzed by chemiluminescence procedures, as previously described (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar).GSK-3β Phosphorylation of AR in Cells—Various GSK-3β constructs were transfected into cells using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Cells were washed with phosphate free Dulbecco's modified Eagle's medium (Invitrogen) and then incubated with 0.5 mCi/ml [32P]orthophosphate (Amersham Biosciences) in the presence of 1 nm 17α-methyl-17β-hydroxyestra-4,9,11-trien-3-one (R1881, otherwise known as metribolone, a synthetic nonmetabolizable androgen) for 6 h at 37 °C (23Dube J.Y. Chapdelaine P. Dionne F.T. Cloutier D. Tremblay R.R. J. Clin. Endocrinol. Metab. 1978; 47: 41-45Google Scholar, 24Kaufman M. Pinsky L. Hollander R. Bailey J.D. J. Steroid Biochem. 1983; 18: 383-390Google Scholar, 25Traish A.M. Muller R.E. Wotiz H.H. J. Biol. Chem. 1981; 256: 12028-12033Google Scholar, 26Schilling K. Liao S. Prostate. 1984; 5: 581-588Google Scholar). Radiolabeled medium was removed and cells were washed 3 times with cold phosphate-buffered saline and then scrape-harvested as previously described (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar). Immunoprecipitation studies were performed on 200 μg of total protein using rabbit anti-AR antibody (Santa Cruz Biotechnology, Santa Cruz, CA), after which the proteins were examined by SDS-PAGE, electrotransferred to nitrocellulose, and subjected to autoradiography performed to detect 32P-labeled AR. Electrotransferred proteins were probed with rabbit anti-AR polyclonal or mouse anti-AR monoclonal primary antibody (Santa Cruz Biotechnology) and visualized using the Super-Signal chemiluminescence substrate (Pierce).Co-immunoprecipitation Assays—After starvation prostate cells were incubated with 1 nm R1881 for 20 min. Following treatment, cells were harvested and 200 μg of total protein lysate was assayed by immunoprecipitation/Western blot analysis with either an N- or C-terminal specific AR binding antibody or GSK-3β specific antibody (Santa Cruz Biotechnology). Briefly, protein lysates were precleared by incubating with protein A/G-agarose (Pierce) for 30 min at 25 °C. Specific antibodies (10 μg/ml) were added to lysates and incubated for 16 h at 4 °C, followed by washing 3 times with HEPES-buffered saline. Proteins that were bound to protein A/G were removed using SDS sample buffer and resolved by SDS-PAGE. Samples were then electrotransferred to nitrocellulose and probed with the reciprocal antibody overnight at 4 °C. Immune complexes were visualized with West Femto chemiluminescence kit (Pierce).Kinase Assays—pRSET-GST-ARLBD protein was produced in Escherichia coli and purified using the B-PER/GST Spin Purification Kit (Pierce) in the presence of 1 nm R1881. Purified GST-ARLBD was incubated with various concentrations of GSK-3β (New England Biolabs) in the presence of [γ-32P]ATP (Amersham Biosciences) and analyzed by NuPage gradient gel electrophoresis (Invitrogen) followed by autoradiography.Immunofluorescence Analysis—Immunofluorescence analysis was performed as described previously (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar). A primary rabbit polyclonal antibody recognizing GSK-3β (Santa Cruz) or mouse monoclonal antibody recognizing AR (BD Biosciences) was diluted (1:200; v/v) in buffer as previously described (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar). The samples were then incubated with an anti-rabbit secondary antibody Alexa-488 (green), an anti-mouse secondary antibody Alexa-350 (blue), and actin probe Alexa-594 phalloidin (red) (Molecular Probes), after which they were examined as previously described (22Subbarayan V. Sabichi A.L. Llansa N. Lippman S.M. Menter D.G. Cancer Res. 2001; 61: 2720-2726Google Scholar).Luciferase Assays—Transfection experiments were performed in triplicate using FuGENE 6. In one set of experiments, various AR constructs (ARwt, AR5, AR65, AR104, AR106, and AR126), previously described (27Jenster G. Trapman J. Brinkmann A.O. Biochem. J. 1993; 293: 761-768Google Scholar, 28Jenster G. van der Korput H.A. Trapman J. Brinkmann A.O. J. Biol. Chem. 1995; 270: 7341-7346Google Scholar) were transfected in combination with wild-type GSK-3β (GSK-3βwt) (Fig. 5A). In another set of experiments, various GSK-3β constructs previously described (29Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Google Scholar) (GSK-3βwt, GSK-3βΔ9, GSK-3βK85M, and GSK-3βY216F) were transfected in combination with ARwt into COS-1 cells. Probasin-luciferase (PB-LUC) and pARE-LUC were the reporter constructs used to analyze AR-driven transcriptional activity. Cells were harvested and analyzed using the LUCLITE system (Packard Instrument Co.) and then quantitated in a 96-well black plate using a TOPCOUNT NT multiwell plate scintillation detector (Packard Instrument Co.). All results from the luciferase assays were normalized to Renilla luciferase activity, which was generated from pRLTK (Promega Corp.).Immunostaining of Paraffin Sections—Immunohistochemistry was carried out in paraffin-embedded sections (3-4 μm) following deparaffinization and rehydration in xylene, graded alcohol, and distilled H2O (DH2O), respectively. Antigen retrieval was performed in citrate buffer, pH 6.0, in steam. The endogenous peroxidase activity was quenched by incubating the sections in 3% hydrogen peroxide for 10 min at room temperature. After blocking the sections with avidin block followed by biotin block for 15 min each (Biogenex, San Ramon, CA) and then rinsing three times between treatments with phosphate-buffered saline, protein nonspecific blocking solution (Dako Corp., Carpinteria, CA) was applied and incubated for 20 min and slides were rinsed three times with phosphate-buffered saline. Rabbit anti-human GSK-3β (Cell Signaling Technology, Inc.) or AR antibodies (Santa Cruz Biotechnology, Inc.) diluted 1:50 with background reducing diluent (Dako Corp.) were applied to the sections and incubated overnight at 25 °C. Then, the sections were incubated with the appropriate biotinylated secondary antibody and horseradish peroxidase-streptavidin (Dako Corp.) and rinsed three times with phosphate-buffered saline between steps. Peroxidase activity was detected by applying 3,3′-diaminobenzidine tetrahydrochloride containing 0.02% hydrogen peroxide for 10 min. The sections were counterstained with hematoxylin, rinsed with DH2O, intensified with a saturated lithium carbonate as a bluing agent, and rinsed again with DH2O. The sections were then dehydrated in graded alcohols, cleared in xylene, and mounted in Permount (Fisher) and covered with coverslips.Image Analysis—Digital capture methods were optimized to provide the best signal-to-noise contrast for image analysis. We used a Quantix CCD camera that is driven by IP Labs (Scanalytics Inc., Fairfax, VA) image analysis software on a Power PC G4 computer with 128 megabytes of RAM. This image-acquisition equipment is attached to an IX70 inverted research light microscope (Olympus America Inc., Melville, NY).RESULTSLow GSK-3β Activity in Proliferating Prostate Cancer Cells—We used Western blots to analyze GSK-3β protein expression and phosphorylation in proliferating prostate cells. The total levels of GSK-3β protein expression were similar in all the prostate cancer cells with little variation in phosphorylation at Tyr216 (a constitutive phosphorylation event that activates this enzyme). In contrast, phosphorylation at Ser9 suppressed GSK-3β activity and was very high in the androgen-dependent LNCaP prostate cancer cells (Fig. 1A, lanes 2 and 4) but lower in the androgen-independent PC-3 and DU145 prostate cancer cells (Fig. 1A, lanes 1 and 3). Previously we verified that GSK-3β enzymatic activity inversely correlates with (P)-Ser9-GSK-3β levels in these prostate cancer cells. 2T. R. Salas, S. M. Lippman, and D. G. Menter, unpublished data. Fig. 1Changes in the phosphorylation state of GSK-3β alter its ability to phosphorylate the AR. A, Western blot analysis was performed to compare the phosphorylation status of GSK-3β in AR-deficient (AR-) (PC-3, DU145, and C3) and AR-expressing cells (AR+) (LNCaP, A103, and V28) prostatic carcinoma cells. The total levels of GSK-3β were similar in all cell types (bottom row), and there was little difference in the (P)Tyr216-GSK-3β levels (middle row). The (P)Ser9-GSK-3β level was higher in the AR+ cells, LNCaP, compared with AR- cells, PC-3, DU145, C3, or AR+ cells derived from an AR- lineage, i.e. A103 and V28 cells (top row). The relative density represents the mean pixel density of the (P)Ser9-GSK-3β normalized to that of the total GSK-3β. B, this diagram shows the structure of HA-tagged GSK-3β and mutants used in this study. 1, GSK-3βwt contains an enzymatic site and two critical regulatory phosphorylation sites Ser9 and Tyr216. 2, GSK-3βΔ9 is a deletion mutant that has the first 9 amino acids removed and is constitutively active because it is missing Ser9, the inhibitory phosphorylation site for Akt. 3, GSK-3βY216F is an enzymatically inactive point mutant that has an essential tyrosine phosphorylation site mutated to a phenylalanine. 4, GSK-3βK85M is an enzymatically suppressed point mutant that has an essential lysine mutated to a methionine. C, GSK-3β variants are expressed in AR+ cells. Only endogenous GSK-3β is expressed in the PCMV4 vector control samples (lanes 1, 5, and 9). In general, the HA tag on these GSK-3β variants caused them to migrate above the endogenous GSK-3β. GSK-3βwt (lanes 2, 6, and 10) has migrated above the endogenous GSK-3β. Constitutively active GSK-3βΔ9 (lanes 3, 7, and 11) has migrated above and below the endogenous GSK-3β. The inactive point mutant GSK-3βY216F (lanes 4, 8, and 12) has migrated above the endogenous GSK-3β. Heavy phosphorylation of Ser9 occurs primarily on the endogenous GSK-3β in the presence of all variant proteins. Relative density represents the mean pixel density of the (P)Ser9-GSK-3β normalized to that of the total GSK-3β. D, enzymatically active GSK-3β variants phosphorylate AR. Overexpression of constitutively active GSK-3βΔ9 (lanes 3, 8, and 13) in AR+ cell lines increased the phosphorylation of AR to its greatest extent, followed by the phosphorylation produced by GSK-3βwt (lanes 2, 7, and 12). Vector controls (PCMV4, lanes 1, 6, and 11) and inactive GSK-3βY216F (lanes 4, 9, and 14) point mutant-expressing cells showed less phosphorylation of AR, whereas treatment with GSK-3β inhibitor LiCl (2.0 mm, lanes 5, 10, and 15) actively suppressed the phosphorylation of AR. Relative density represents the mean pixel density of the 32P-labeled AR normalized to that of the total AR.View Large Image Figure ViewerDownload (PPT)To determine the effect of an androgen-independent cell background on AR activity, we selected PC-3 cells that stably express AR from a PC-3 cell background. When we selected PC-3 prostate cells that stably expressed a control vector (C3 cells), AR plasmid (A103 cells) or VP16-AR (V28 cells), Ser9 did not increase to the same levels in these cells as observed in the androgen-expressing LNCaP cells (Fig. 1A, lanes 4-7).Phosphorylation of GSK-3β Variants in AR-expressing Cells—We next overexpressed HA-tagged GSK-3β variants (29Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Google Scholar) (Fig. 1B) containing various point mutations or deletions in the AR-expressing LNCaP, A103, and V28 cells to determine how these molecular changes affected GSK-3β expression (Fig. 1C). Deletion of the first 9 amino acids of GSK-3β resulted in the formation of the GSK-3βΔ9 protein, which is constitutively active and cannot be inactivated by Akt. Ser9 phosphorylation was present on endogenous GSK-3β but not to the same degree on the transfected HA-tagged variants (Fig. 1C). The position of the HA-tagged variants was verified using an anti-HA antibody. 3T. R. Salas, S. M. Lippman, and D. G. Menter, unpublished observations. Prevention of GSK-3β-induced Phosphorylation of AR by LiCl—The phosphorylation of AR increased above that in control cells in the presence of GSK-3βwt (Fig. 1D, lanes 2, 7, and 12) and to a greater extent in the presence of constitutively active GSK-3βΔ9 (Fig. 1D, lanes 3, 8, and 13). In contrast, the phosphorylation of AR was limited in the presence of PCMV4 (Fig. 1D, lanes 1, 6, and 11) and inactive GSK-3βY216F (Fig. 1D, lanes 4, 9, and 14). Phosphorylation was further decreased in cells treated with 2.0 mm lithium chloride (LiCl), a GSK-3β inhibitor (Fig. 1D, lanes 5, 10, and 15). These data suggest that GSK-3β is involved in phosphorylating AR.Distribution of GSK-3β Variants and AR in Cell Nuclei—We examined the intracellular distribution of the different GSK-3β (green) proteins, the AR (blue) and actin (red) in immunofluorescence studies to determine whether these molecules could interact within cells (Fig. 2). Untransfected PC-3 and C3 cells contained low levels of endogenous GSK-3β that were located in both the cytoplasm and nucleus of the PC-3 cells. Overexpressed GSK-3βwt was also present in both the cytoplasm and nucleus of transfected cells. However, the GSK-3βY216F protein was predominantly found in the cytoplasm, and the GSK-3βΔ9 protein was predominantly found in the nucleus of transfected cells. In addition, AR was not present in the PC3-C3 control cells that expressed the empty vector but was present in the nuclei of all R1881-treated AR-expressing cells. In the absence of R1881 by contrast, much less AR was found in the nuclei of AR-expressing cells (data not shown). These data show that the intracellular distribution of both AR and GSK-3β did not influence each other. AR was predominantly in the nuclei of R1881-treated prostate cells and was not affected by the distribution of GSK-3β. In contrast, the intracellular distribution of GSK-3β appears to rely in part on its phosphorylation status, in particular, the absence of the Tyr216 phosphorylation site prevented the distribution of GSK-3βY216F into cell nuclei. These data suggest that although the intracellular distribution of AR and GSK-3β do not depend on each other, these two molecules are capable of co-localizing in cancer cell nuclei, which would enable GSK-3β to phosphorylate AR.Fig. 2Activated GSK-3β and AR distribution in prostate cells. We examined the localization of AR (blue) and GSK-3β (green) in R1881-treated prostate cancer cells after counterstaining samples for actin (red). In C3 cells stably expressing an empty transfection vector, no AR was observed (asterisk). The intracellular distribution of GSK-3β (green) in C3 cells varied with the variant that was transfected into cells. The GSK-3βwt was distributed in both the nucleus and cytoplasm of C3 cells, whereas the GSK-3βΔ9 variant was predominantly in the nucleus (narrow arrows) and the GSK-3βY216F variant was found in the cytoplasm (wide arrows). In A103, V28, and LNCaP cells that express AR, the AR (blue) was almost exclusively in the nucleus of R1881-treated cells. The distribution of GSK-3β (green) protein in cells that express AR (A103, V28, and LNCaP) was the same as cells that do not express AR (C3).View Large Image Figure ViewerDownload (PPT)Low GSK-3β Activity in COS-1 Cells—We used COS-1 cells to characterize the interactions between GSK-3β and AR deletion variants because they are easy to transfect and do not express endogenous AR. First, however, we determined that the expression pattern and phosphorylation status of GSK-3β were similar to those in the LNCaP cells, in that Ser9 was phosphorylated (Fig. 3A) and the kinase activity was low (data not shown). Similar to prostate cancer cells, when we expressed GSK-3β variants in COS-1 cells, the endogenous GSK-3β was phosphorylated on Ser9, but was decreased in the GSK-3β variants (Fig. 3B).Fig. 3The AR is phosphorylated by active forms of GSK-3β in COS-1 cell nuclei. A, the phosphorylation status of GSK-3β was examined in COS-1 cells. The total levels of GSK-3β (bottom row), (P)Tyr216-GSK-3β (top row), and (P)Ser9-GSK-3β (middle row) were similar to those in LNCaP, A103, and V28 cells. B, COS-1 cells co-transfected with AR and GSK-3β variants showed similar results to those in prostate cancer cells. Only endogenous GSK-3β was expressed in vector control samples (lane 1). GSK-3βwt (lane 2) migrated above the endogenous GSK-3β, constitutively active GSK-3βΔ9 (lane 3) migrated above and below the endogenous GSK-3β, and the inactive point mutant GSK-3βY216F (lane 4) migrated above the endogenous GSK-3β. The endogenous GSK-3β was phosphorylated on Ser9, but the variant GSK-3β proteins were not. Relative density represents the mean pixel density of the (P)Ser9-GSK-3β normalized to that of the total GSK-3β. C, AR (blue) was observed in the nuclei (narrow arrows) of R1881-treated COS-1 cells that were co-transfected with AR and GSK-3β variants. The pattern of GSK-3β (green) expression in COS-1 cells was the same as that in the cancer cells. AR was present in the cell nuclei with GSK-3βwt and GSK-3βΔ9, but only the AR was observed in the nuclei (narrow arrows) of cells transfected with GSK-3βY216F, which appeared in the cytoplasm (wide arrows). D, the co-transfection of enzymatically active GSK-3β variants with AR into R1881-treated COS-1 cells increased the phosphorylation of AR. In contrast, vector controls (PCMV4,
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