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• W2005448107 abstract "The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor that regulates genes involved in cell proliferation and differentiation. Using a C-terminal region of the human AR in a yeast two-hybrid screen, we have identified RACK1 (receptor for activated Ckinase-1) as an AR-interacting protein. In this report we found that RACK1, which was previously shown to be a protein kinase C (PKC)-anchoring protein that determines the localization of activated PKCβII isoform, facilitates ligand-independent AR nuclear translocation upon PKC activation by indolactam V. We also observed RACK1 to suppress ligand-dependent and -independent AR transactivation through PKC activation. In chromatin immunoprecipitation assays, we demonstrate a decrease in AR recruitment to the AR-responsive prostate-specific antigen (PSA) promoter following stimulation of PKC. Furthermore, prolonged exposure to indolactam V, a PKC activator, caused a reduction in PSA mRNA expression in prostate cancer LNCaP cells. Finally, we found PKC activation to have a repressive effect on AR and PSA protein expression in androgen-treated LNCaP cells. Our data suggest that RACK1 may function as a scaffold for the association and modification of AR by PKC enabling translocation of AR to the nucleus but rendering AR unable to activate transcription of its target genes. The androgen receptor (AR), a member of the nuclear hormone receptor superfamily, functions as a ligand-dependent transcription factor that regulates genes involved in cell proliferation and differentiation. Using a C-terminal region of the human AR in a yeast two-hybrid screen, we have identified RACK1 (receptor for activated Ckinase-1) as an AR-interacting protein. In this report we found that RACK1, which was previously shown to be a protein kinase C (PKC)-anchoring protein that determines the localization of activated PKCβII isoform, facilitates ligand-independent AR nuclear translocation upon PKC activation by indolactam V. We also observed RACK1 to suppress ligand-dependent and -independent AR transactivation through PKC activation. In chromatin immunoprecipitation assays, we demonstrate a decrease in AR recruitment to the AR-responsive prostate-specific antigen (PSA) promoter following stimulation of PKC. Furthermore, prolonged exposure to indolactam V, a PKC activator, caused a reduction in PSA mRNA expression in prostate cancer LNCaP cells. Finally, we found PKC activation to have a repressive effect on AR and PSA protein expression in androgen-treated LNCaP cells. Our data suggest that RACK1 may function as a scaffold for the association and modification of AR by PKC enabling translocation of AR to the nucleus but rendering AR unable to activate transcription of its target genes. The androgen receptor (AR), 1The abbreviations used are: AR, androgen receptor; RACK1, receptor for activated C kinase-1; PKC, protein kinase C; PSA, prostate-specific antigen; DHT, dihydroxytestosterone; IndoV, indolactam V; GFX, GF109203X; GFP, green fluorescent protein; RFP, red fluorescent protein; RT-PCR, reverse transcriptase-polymerase chain reaction; SDM, steroid-depleted medium; ChIP, chromatin immunoprecipitation assays; TPA, 12-O-tetradecanoy phorbol-13 acetate; PBS, phosphate-buffered saline; SRC-1, steroid receptor coactivator-1. a member of the steroid hormone nuclear receptor superfamily, is a ligand-dependent transcription factor that regulates gene expression required for proliferation and differentiation of cells within the prostate and also has a role in the development and progression of prostate cancer. In its inactive state, unliganded AR is associated with heat-shock proteins from which it dissociates upon binding of the ligand, dihydrotestosterone (DHT), which is generated from testosterone by membrane-bound 5-α-reductase. Following homodimerization, AR translocates to the nucleus where it interacts with coactivators or corepressors and binds to androgen response elements (AREs) located in the promoter region of target genes triggering their transcriptional activation or repression (1Gnanapragasam V.J. Robson C.N. Leung H.Y. Neal D.E. BJU Int. 2000; 86: 1001-1013Crossref PubMed Google Scholar, 2Jenster G. Semin. Oncol. 1999; 26: 407-421PubMed Google Scholar). There is evidence to support AR cross-talk with other cellular signal transduction pathways resulting in AR ligand-independent activation and modulation of AR transactivation. There is also mounting evidence that ligand-independent activation of AR may play a role in hormone refractory prostate cancer. cAMP-dependent kinase (PKA) has been reported to mediate ligand-independent AR activation and cross-talk between AR and PKA signaling pathways resulting in androgen-independent induction of prostate-specific antigen (PSA) gene expression (3Nazareth L.V. Weigel N.L. J. Biol. Chem. 1996; 271: 19900-19907Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 4Sadar M.D. J. Biol. Chem. 1999; 274: 7777-7783Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). In contrast, protein kinase C (PKC) negatively regulates AR-dependent transcription (5Darne C. Veyssiere G. Jean C. Eur. J. Biochem. 1998; 256: 541-549Crossref PubMed Scopus (48) Google Scholar). AR activation in the absence of ligand has been reported in response to the peptide hormones epidermal growth factor (EGF), keratinocyte growth factor (KGF), and insulin-like growth factor I (IGF-I), which serve as ligands for receptor-tyrosine kinases and activate downstream intracellular kinase cascades (6Culig Z. Hobisch A. Cronauer M.V. Radmayr C. Trapman J. Hittmair A. Bartsch G. Klocker H. Cancer Res. 1994; 54: 5474-5478PubMed Google Scholar). These growth factors can activate transcription from an androgen-responsive reporter construct in the absence of ligand or synergistically in conjunction with androgens. Interleukin-6 (IL-6), a multifunctional cytokine, can also stimulate ligand-independent AR activation via mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription-3 (STAT-3) signaling pathways in LNCaP human prostate cancer cells (7Ueda T. Mawji N.R. Bruchovsky N. Sadar M.D. J. Biol. Chem. 2002; 277: 38087-38094Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Moreover, steroid receptor coactivator-1 (SRC-1) has been shown to enhance ligand-independent activation of the N-terminal domain (NTD) of the AR by IL-6 via a pathway that is dependent on MAPK in LNCaP (8Ueda T. Bruchovsky N. Sadar M.D. J. Biol. Chem. 2002; 277: 7076-7085Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). The mechanism by which these signaling pathways affect AR function is not known. They may modulate AR via phosphorylation of either AR itself or of AR coregulators. For example, activation of the HER2/Neu MAPK pathway results in AR phosphorylation, increasing its ability to recruit coregulators and enhancing AR transactivation (9Wen Y. Hu M.C. Makino K. Spohn B. Bartholomeusz G. Yan D.H. Hung M.C. Cancer Res. 2000; 60: 6841-6845PubMed Google Scholar). Pyk2 interacts with ARA55 protein and represses AR transactivation via phosphorylation of ARA55 (10Wang X. Yang Y. Guo X. Sampson E.R. Hsu C.L. Tsai M.Y. Yeh S. Wu G. Guo Y. Chang C. J. Biol. Chem. 2002; 277: 15426-15431Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In turn, these alternative pathways may affect AR translocation to the nucleus or transcriptional activity via interaction with scaffolding proteins that orchestrate the precise compartmentalization of signal transduction components. It is thought scaffolding proteins cluster signaling proteins thus allowing a tight control of cellular pathways as well as cross-talk between different cascades. Caveolin, a major component of caveolae membrane structures, has been implicated as a principal scaffold for many signal transduction pathways. In a study by Lu et al. (28Lu M.L. Schneider M.C. Zheng Y. Zhang X. Richie J.P. J. Biol. Chem. 2001; 276: 13442-13451Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), caveolin was shown to interact with AR and its overexpression potentiated ligand-dependent AR activation. Recently, we identified filamin, an actin-binding protein also thought to act as a scaffold, as an AR-interacting protein that facilitates ligand-dependent AR nuclear translocation (11Ozanne D.M. Brady M.E. Cook S. Gaughan L. Neal D.E. Robson C.N. Mol. Endocrinol. 2000; 14: 1618-1626Crossref PubMed Google Scholar). In filamin-deficient M2 cells AR remained cytoplasmic even after prolonged exposure to androgen. Previous studies have also identified filamin as an interacting partner for caveolin (12Stahlhut M. van Deurs B. Mol. Biol. Cell. 2000; 11: 325-337Crossref PubMed Scopus (266) Google Scholar). Furthermore, caveolin also appears to have a role in ligand-independent estrogen receptor α nuclear translocation (13Schlegel A. Wang C. Pestell R.G. Lisanti M.P. Biochem. J. 2001; 359: 203-210Crossref PubMed Scopus (66) Google Scholar). The aim of this study was to identify novel AR-interacting proteins involved in movement or signaling from the cytoplasm to the nucleus. A yeast-two hybrid screen identified RACK1 (receptor for activated protein kinase C-1) as an AR-interacting protein. RACK1, a 36-kDa homologue of the β subunit of G proteins, is a member of the WD-40 family of proteins characterized by highly conserved internal WD-40 repeats (Trp-Asp) (14Ron D. Chen C.H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (647) Google Scholar, 15van der Voorn L. Ploegh H.L. FEBS Lett. 1992; 307: 131-134Crossref PubMed Scopus (218) Google Scholar). It was initially identified as a protein that interacts with activated PKC and acts as a shuttling protein that coordinates the correct localization of βIIPKC isoform for its function (16Mochly-Rosen D. Khaner H. Lopez J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3997-4000Crossref PubMed Scopus (442) Google Scholar, 17Ron D. Jiang Z. Yao L. Vagts A. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Due to its association with a large number of signaling proteins such as ϵPKC (18Besson A. Wilson T.L. Yong V.W. J. Biol. Chem. 2002; 277: 22073-22084Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), Src (19Chang B.Y. Conroy K.B. Machleder E.M. Cartwright C.A. Mol. Cell. Biol. 1998; 18: 3245-3256Crossref PubMed Google Scholar), integrin β subunit (20Liliental J. Chang D.D. J. Biol. Chem. 1998; 273: 2379-2383Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), phosphodiesterase PDE4D5 (21Yarwood S.J. Steele M.R. Scotland G. Houslay M.D. Bolger G.B. J. Biol. Chem. 1999; 274: 14909-14917Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), STAT1 (22Usacheva A. Smith R. Minshall R. Baida G. Seng S. Croze E. Colamonici O. J. Biol. Chem. 2001; 276: 22948-22953Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), IGF-I receptor (23Hermanto U. Zong C.S. Li W. Wang L.H. Mol. Cell. Biol. 2002; 22: 2345-2365Crossref PubMed Scopus (174) Google Scholar, 24Kiely P.A. Sant A. O'Connor R. J. Biol. Chem. 2002; 277: 22581-22589Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), a central role of RACK1 as a scaffolding, anchor or adaptor protein in multiple intracellular signal transduction pathways has been suggested. We have found RACK1 to have a role in ligand-independent AR movement and transactivation via the PKC signaling pathway suggesting a possible function for this scaffolding protein in AR cross-talk with other signaling pathways. Materials—R1881 was purchased from PerkinElmer Life Sciences. Dihydrotestosterone, Indolactam V, and monoclonal anti-α tubulin antibody were purchased from Sigma. GF109203X was purchased from Calbiochem. Monoclonal anti-RACK1 antibody was purchased from Transduction Laboratories. Monoclonal anti-AR antibody was purchased from BD Pharmingen. Polyclonal anti-PSA antibody was purchased from Santa Cruz Biotechnology. Yeast Two-hybrid Analysis—Two-hybrid screening was performed as described previously (25Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). An hAR fragment comprising residues 559-918, encompassing the DNA-binding and ligand-binding domains was used as bait in the assay. Positive clones were isolated, sequenced and submitted into the GenBank™ data base. One positive clone (RACK1163-317), 0.5 kb in size, gave a 100% homology to the receptor for activated protein kinase C (RACK1, GenBank™ accession no NM_006098). Interaction of RACK1163-317 with truncated forms of the hAR was examined by liquid LacZ assays as previously described (25Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Deletion Constructs of RACK1 Clones—Four deletion constructs of RACK1163-326 in pACT2 were constructed via deletion of residues from C terminus of the fragment, sequentially deleting individual WD domains. Primers used in PCR to generate the deletion constructs were: WD4-7, 5′-AGCAGCAACCCTATCATCGT-3′; WD5-7, 5′-TCTCCAGATGGATCCCTCTG-3′; WD6-7, 5′-TGCTTCAGCCCTAACCGCTA-3′; WD7, 5′-AAGGCAGAACCACCCCAGT-3′; RACK1 Reverse, 5′-GTGTGCCTAG GTCACCTG-3′. Amplified inserts were cloned into pCR2.1 (Invitrogen) and subcloned into pACT2 via EcoRI site. Orientation was determined via restriction analysis and confirmed by sequencing. Deletion constructs were cotransformed with pAS2-1-AR559-918 into yeast strain PJ69-4A and resultant transformants were grown on media lacking adenine, leucine, and tryptophan. In Vitro Interaction of hAR and RACK1—RACK1163-317 cDNA was excised with EcoRI and XhoI digestion from pACT2 and directionally cloned into pRSETC (Invitrogen) via EcoRI and XhoI sites. hAR fragments used in the yeast two-hybrid analysis were cloned into the EcoRI site of pRSETC from pAS2-1. Constructs were sequenced to confirm the maintenance of the open reading frame. Template DNA was subjected to Geneclean (Anachem) to remove contaminant RNases and the coupled Transcription/Translation Kit (Promega) was used for in vitro interaction analysis. Templates were labeled with [35S]methionine, and the reaction was performed according to the manufacturer's guidelines. Equivalent amounts of labeled protein samples were mixed in immunoprecipitation buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.2 mm Na3VO4, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 25 μg/ml leupeptin, 25 μg/ml aprotinin, 25 μg/ml pepstatin). Interacting proteins were precipitated and visualized as previously described (25Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Cell Culture—Tissue culture ingredients were purchased from Sigma, unless stated otherwise. LNCaP and COS7 cells were maintained in RPMI 1640 media supplemented with 10% fetal calf serum (FCS) (Invitrogen), l-glutamine, 1% penicillin, and 1% streptomycin. Transient Transfections and Luciferase Assays—PC3M cells were seeded in 24-well culture plates (Corning) at densities of 1 × 104 cells/well. After 24 h, cells were transfected using Superfect (Qiagen) according to the manufacturer's recommendation with pPSAluc reporter (200 ng), pCMV-β-Gal control plasmid (200 ng), pEGFP-C1AR1-918 (50 ng) and pDSRed-1N1RACK11-326 (100 ng). After 2 h, cells were washed and incubated in RPMI 1640 medium supplemented with 10% fetal calf serum that had been depleted of steroids by treatment with dextran-coated charcoal (SDM), with the addition of activator/inhibitor as indicated. After 48 h, cells were harvested and assayed for luciferase activity according to the manufacturer's guidelines (Promega). Luciferase activity was corrected for the corresponding β-galactosidase activity to give relative activity. β-galactosidase assays were previously described (25Brady M.E. Ozanne D.M. Gaughan L. Waite I. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 1999; 274: 17599-17604Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Immunoprecipitations—Approximately 1 × 106 LNCaP cells were washed in PBS, harvested, and lysed in reaction lysis buffer on ice for 30 min. Lysates were centrifuged at 14,000 × g for 3 min. Supernatants were precleared with 20 μl of protein G-Sepharose (PGS) after three washes in reaction lysis buffer and rotated at 4 °C for 4 h. PGS and nonspecific bound protein were removed by centrifugation at 14,000 × g for 5 min. Immunoprecipitation was performed with 4 μg of polyclonal anti-AR antibody and incubated overnight at 4 °C with rotation. After incubation, a further 20 μl of PGS were added to immunoprecipitated samples and returned to 4 °C for 1 h with rotation. PGS with bound protein complexes was recovered by centrifugation at 14,000 × g for 5 min, and samples were washed once in wash buffer A (PBS, 0.2% Triton-X-100, 350 mm NaCl) and twice in wash buffer B (PBS, 0.2% Triton-X-100). Sample buffer (100 mm dithiothreitol, 125 mm Tris-HCl (pH 6.8), 2% SDS, 20% glycerol, 0.005% bromphenol blue) containing 10% β-mercaptoethanol was added to recovered fractions. Samples were denatured and separated by SDS-PAGE, followed by transfer to nitrocellulose membrane. The membrane was probed with monoclonal anti-RACK1 antibody. Immunoreaction was visualized using enhanced chemiluminescence (ECL) reagents (Amersham Biosciences). Western Blotting—LNCaP cells were seeded in 6-well cell culture plates at densities of 1 × 105 cells/well. After 24 h, cells were washed in PBS and incubated for 48 h in SDM in the presence or absence of activators/inhibitors. Adherent cells were lysed directly using SDS sample buffer containing 10% β-mercaptoethanol. Samples were denatured by boiling for 10 min and separated on 12% polyacrylamide gels, followed by transfer to nitrocellulose Hybond™ membrane (Amersham Biosciences). Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS, 50 mm Tris HCl, pH 7.5, 150 mm NaCl) for 1 h and then incubated with primary antibodies in diluant (1% nonfat milk in TBS-Tween) for 1 h. Primary antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies, and protein bands were visualized using ECL reagents. Microscopic Analysis—Full-length hAR (corresponding to residues 1-918) was cloned into the XbaI site of pEGFP-C1 (Clontech) to generate GFP-hAR1-918. Full-length RACK1 (corresponding to residues 1-326) was cloned into pDSRed-1N1 (Clontech) via HindIII and SalI to generate RFP-RACK11-326. Cells were seeded on ethanol-sterilized 22 × 22-mm microscope cover slips in 6-well cell culture plates at densities of 1 × 105 cells/well. After 24 h, cells were washed with PBS and transfected with 1 μg of GFP- and/or RFP-construct using Superfect reagent. After 3 h, cells were washed twice with PBS and incubated overnight in RPMI 1640 media supplemented with 10% fetal calf serum. Following a PBS wash, cells were placed in serum-free containing RPMI 1640 medium for a further 18 h and then exposed to activators or inhibitors. Cover slips were washed with PBS, fixed in methanol for 10 min at -20 °C, dried, inverted on microscope slides and mounted in mounting media (DAKO). Slides were analyzed using a Leica scanning laser confocal microscope. Reverse Transcription PCR—Total RNA was isolated from LNCaP cells using TRIzol reagent according to the manufacturer's recommendation (Invitrogen). Complementary DNA was synthesized from 2 μg of total RNA using MMLV reverse transcriptase (Promega) at 37 °C for 60 min. The sequences of the primers for the amplification of PSA cDNA were as follows: forward primer, 5′-CATCAGGAACAAAAGCGTGA-3′ and reverse primer, 5′-TGGGGTCAAGAACTCCTCCTG-3′. This primer pair amplified a 305-bp product. PCR cycle conditions were 94 °C for 30 s, 60 °C for 20 s and 72 °C for 30 s for 25 cycles with an initial denaturation at 94 °C for 3 min and a final extension at 72 °C for 5 min. β-Actin, a housekeeping control gene, was used to ensure equal initial RNA amounts. The PCR products were resolved by agarose gel electrophoresis and ethidium bromide staining and visualized by UV. Chromatin Immunoprecipitation Assays—LNCaP cells were grown on 150-mm dishes in fetal calf serum-containing RPMI 1640 media for 2 days until approx. 5 × 106 cells were present. 16 h prior to androgen and PKC activator treatment, cells were incubated in steroid-depleted media. Cells were placed in steroid-depleted media supplemented with or without 10 nm R1881 or 10 μm indolactam V for 15 min or 60 min. Following treatment, chromatin immunoprecipitation assays were carried out as described (26Gaughan L. Logan I.R. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2002; 277: 25904-25913Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Primer sequences for PSA were also described previously (26Gaughan L. Logan I.R. Cook S. Neal D.E. Robson C.N. J. Biol. Chem. 2002; 277: 25904-25913Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). RACK1 Is an AR-interacting Protein—Yeast two-hybrid screening using an AR construct AR559-918 comprising the DNA and ligand-binding domains (DBD and LBD respectively) as bait, identified RACK1 as an AR-interacting protein (Fig. 1). The clone isolated in the screen spanned a region of 154 amino acids from amino acids 163 to 317, designated RACK1163-317. The interaction of AR and RACK1 is enhanced in the presence of ligand, demonstrated by an approximate 5-fold increase in β-galactosidase activity in its presence. Co-transformation of RACK1163-317 with AR deletion constructs in yeast revealed an interaction between RACK1163-317 and the AR fragment AR624-918 comprising the LBD alone upon treatment with ligand. The approximate 3-fold increase in β-galactosidase levels in the presence of 1 μm DHT suggested that additional residues in the DBD further contribute to the interaction or influence the conformational change upon ligand binding. These preliminary data imply that AR interacts with RACK1 in the presence of ligand. RACK1 Interacts with the AR in Vitro and in Vivo—To confirm the data obtained from the yeast two-hybrid system, we sought to determine whether RACK1 could interact with AR in vitro. The interaction was analyzed using in vitro transcription and translation of the proteins labeled with [35S]methionine. Fig. 2A represents in vitro transcribed/translated protein products before immunoprecipitation. Fig. 2B demonstrates that RACK1163-326 interacts with full-length AR (amino acids 1-918), the DBD and LBD (amino acids 559-918) and LDB alone (amino acids 624-918). No interaction was observed between RACK1163-317 and the transactivation domain and DBD (amino acids 1-624). In agreement with the results from the two-hybrid analysis the site of the RACK1 interaction on AR appeared to be around the LBD (amino acids 624-918). Immunoprecipitation and subsequent Western analysis were used to confirm the interaction between RACK1 and AR in vivo. A rabbit polyclonal antibody was used to immunoprecipitate AR protein from prostate LNCaP cells prior to detection of RACK1 by Western blotting. LNCaP cells were also treated with androgen to study the influence of ligand on the interaction. As shown in Fig. 2C, a 36-kDa band, corresponding to RACK1 was observed in the absence (lane 4) and presence of ligand (lane 5) but was absent in control lanes omitting extract (lanes 2 and 3) or antibody (lane 1). Minimal Domain Mapping of AR-RACK1 Interaction—The presence of WD-40 repeats, which have been implicated in protein-protein interactions, within RACK1 suggested that one of these motifs may be responsible for the interaction. The clone isolated in the two-hybrid system, RACK1163-317, contains five out of the seven WD repeat sequences. The minimal domain of RACK1 required to interact with AR was investigated by deleting residues from the C terminus of RACK1163-317. The deletion constructs were tested for their ability to interact with AR559-918 in yeast. Resultant transformants were grown on media lacking adenine, leucine, and tryptophan selecting for both plasmids and for an interaction between the proteins encoded by these plasmids. Interaction between AR559-918 and all RACK1 deletion constructs was observed apart from the RACK1 deletion construct solely encoding the seventh WD40 repeat (RACK1WD7) (Fig. 3). Combined these results implicate the sixth WD40 repeat in the interaction with the AR. RACK1 Is Involved in Ligand-independent AR Nuclear Translocation—To assess the functional significance of AR-RACK1 interaction, the role of RACK1 in AR movement to the nucleus was investigated by fluorescence (confocal) microscopy tagging the interacting proteins with GFP or red fluorescent protein (RFP). A recent report (17Ron D. Jiang Z. Yao L. Vagts A. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) proposed RACK1 to act as a PKC shuttling protein that moves βIIPKC upon activation from one intracellular site to another. It has been previously demonstrated using a GFP-AR chimera that unliganded AR is cytoplasmic but rapidly translocates to the nucleus upon addition of ligand (27Georget V. Lobaccaro J.M. Terouanne B. Mangeat P. Nicolas J.C. Sultan C. Mol. Cell. Endocrinol. 1997; 129: 17-26Crossref PubMed Scopus (159) Google Scholar). This ligand-induced nuclear translocation was also observed in this study in COS7 cells (Fig. 4). In the absence of R1881, GFP-AR is predominantly cytoplasmic with background nuclear fluorescence (column 1, row 1). Following a 20-min exposure to steroid, GFP-AR is predominantly nuclear (column 1, row 2). We next determined the localization of RFP-RACK1 in COS7 cells. RFP-RACK1 was found to be cytoplasmic but also localized to a perinuclear structure in unstimulated cells (column 2, row 1). In the basal state, both constructs colocalized in the cytoplasm, displayed by a yellow fluorescent pattern (column 3, row1). Activation by synthetic androgen R1881 induced GFP-AR to move from the cytoplasm to the nucleus while RFP-RACK1 remained in the cytoplasm (column 3, row 2). Ron et al. (17Ron D. Jiang Z. Yao L. Vagts A. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) showed RACK1 movement upon PKC activation from the Golgi apparatus to the cell periphery alongside βIIPKC. Also, cross-talk has been reported between the AR and PKC signaling pathways. The PKC signaling pathway has been reported to negatively regulate AR-dependent transcription. Thus, we next sought to examine whether AR translocates to the nucleus in the presence of RACK1 and upon PKC activation. Upon treatment with PKC activator indolactam V, unliganded GFP-AR remained cytoplasmic (column 1, row 3). However, in the presence of RFP-RACK1 androgen-independent nuclear translocation of GFP-AR was observed upon exposure to indolactam V (column 3, row 3). Furthermore, this movement was reduced in the presence of PKC inhibitor GF109203X (column 3, row 4). These results suggest that RACK1 has a role in ligand-independent movement of AR induced by activation of the PKC signaling pathway. RACK1 and Activation of PKC Signaling Pathway Suppress AR Transactivation—We next determined the potential influence of RACK1 on AR transactivation in reporter assays using the pPSALuc reporter. Briefly, AR was transiently transfected into the AR-negative and androgen-independent prostate cancer PC3M cell line in the absence and presence of RACK1 together with pPSALuc. As shown in Fig. 5, R1881 induces AR transactivation up to 6-fold (compare bars 1 and 2). However, the addition of RACK1 repressed androgen-induced AR transactivation to a level comparable to that observed in the absence of R1881 (compare bars 2 and 8). Because we observed androgen-independent AR movement upon PKC activation and in the presence of RACK1, we also investigated their potential effect on androgen-dependent and -independent transcriptional activation of AR. Upon treatment with PKC activator indolactam alone, AR activity was comparable to that observed in the absence of any stimulus (compare bars 1 and 3) whereas upon treatment with PKC inhibitor GF109203X alone, AR activity was enhanced 7.5-fold (compare bars 1 and 4), suggesting a potential role for PKC signaling pathway in ligand-independent AR transactivation. Similarly, GF109203X enhanced R1881-dependent AR transactivation a further 3-fold (compare bars 2 and 6) but indolactam V slightly reduced reporter activity (compare bars 2 and 5). In the presence of GF109203X, the enhancement of reporter activity observed in the presence of AR alone was reduced 5-fold upon cotransfection with RACK1 (compare bars 4 and 10). A reduction was also observed upon treatment with indolactam V (compare bars 3 and 9). The addition of RACK1 also reduced androgen-dependent AR transactivation upon treatment with indolactam V and GF109203X (compare bars 5 and 11, 6 and 12). These results suggest a possible inhibitory role for RACK1. However, GF109203X appears to reverse the repression of both androgen-dependent and -independent AR activity observed upon treatment with indolactam V. PKC Activation Causes a Decrease in AR Recruitment to PSA Promoter—Because PKC activation induces AR movement to the nucleus aided by RACK1 but has a repressive effect upon AR-mediated transcription, we next sought to determine whether the AR is still" @default.
• W2005448107 created "2016-06-24" @default.
• W2005448107 creator A5044115567 @default.
• W2005448107 creator A5062478414 @default.
• W2005448107 creator A5075404586 @default.
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• W2005448107 date "2003-11-01" @default.
• W2005448107 modified "2023-10-18" @default.
• W2005448107 title "The Scaffolding Protein RACK1 Interacts with Androgen Receptor and Promotes Cross-talk through a Protein Kinase C Signaling Pathway" @default.
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