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• W2125858585 abstract "The androgen receptor (AR) is a ligand-dependent transcription factor that has an essential role in the normal growth, development, and maintenance of the prostate gland. The AR is part of a large family of steroid receptors that also includes the glucocorticoid, progesterone, and mineralocorticoid receptors. Steroid receptor family members share significant homology at their DNA and ligand-binding domains. However, these receptors exhibit a high degree of sequence variability at their NH2-terminal domain, which suggests the possibility of receptor-specific interactions with co-regulator proteins. Transcriptional co-regulators that interact with the AR may have a role in defining AR activity and may be involved in directing AR-specific responses. Here we have identified Ran-binding protein in the microtubule-organizing center (RanBPM) to be a novel AR-interacting protein by yeast two-hybrid assay and have confirmed this interaction by glutathione S-transferase- and His-tagged pull-down assays. In addition, transient overexpression of RanBPM in prostate cancer cell lines resulted in enhanced AR activity in a ligand-dependent fashion. Glucocorticoid receptor activity was also enhanced when RanBPM was overexpressed, whereas estrogen receptor activity remained unchanged. These data demonstrate that RanBPM interacts with steroid receptors to selectively modify their activity. The androgen receptor (AR) is a ligand-dependent transcription factor that has an essential role in the normal growth, development, and maintenance of the prostate gland. The AR is part of a large family of steroid receptors that also includes the glucocorticoid, progesterone, and mineralocorticoid receptors. Steroid receptor family members share significant homology at their DNA and ligand-binding domains. However, these receptors exhibit a high degree of sequence variability at their NH2-terminal domain, which suggests the possibility of receptor-specific interactions with co-regulator proteins. Transcriptional co-regulators that interact with the AR may have a role in defining AR activity and may be involved in directing AR-specific responses. Here we have identified Ran-binding protein in the microtubule-organizing center (RanBPM) to be a novel AR-interacting protein by yeast two-hybrid assay and have confirmed this interaction by glutathione S-transferase- and His-tagged pull-down assays. In addition, transient overexpression of RanBPM in prostate cancer cell lines resulted in enhanced AR activity in a ligand-dependent fashion. Glucocorticoid receptor activity was also enhanced when RanBPM was overexpressed, whereas estrogen receptor activity remained unchanged. These data demonstrate that RanBPM interacts with steroid receptors to selectively modify their activity. The androgen receptor (AR) 1The abbreviations used are: AR, androgen receptor; GR, glucocorticoid receptor; LBD, ligand-binding domain; DBD, DNA-binding domain; ER, estrogen receptor; AF-1, activation function-1; CREB, cAMP-response element-binding protein; CMV, cytomegalovirus; GST, glutathione S-transferase; FBS, fetal bovine serum; Luc, luciferase; TBS, Tris-buffered saline; ARR, androgen response regions; PSA, prostate-specific antigen; PB, probasin; TK, thymidine kinase; Dex, dexamethasone; ERK, extracellular signal-regulated kinase; RanBPM, Ran-binding protein in the microtubule-organizing center; SPRY, repeats in splA and RyR. is a ligand-dependent transcription factor that belongs to a family of steroid receptors along with the glucocorticoid (GR), progesterone, and mineralocorticoid receptors. These steroid receptors share similar domain structures and mechanism of action. Steroid receptors including the AR have three functional domains: a COOH-terminal ligand-binding domain (LBD), a central DNA-binding domain (DBD), and an NH2-terminal domain (1Olefsky J.M. J. Biol. Chem. 2001; 276: 36863-36864Google Scholar). In the absence of androgens, the AR is localized to the cytoplasm in an inactive complex that includes heat shock proteins (HSP). Upon binding to its cognate ligand, the AR undergoes a conformational change that results in a more compact and stable form of the AR. The activated AR dissociates from HSPs and translocates to the nucleus where it interacts with consensus DNA sequences as a homodimer to influence transcription of downstream genes (2Kuil C.W. Berrevoets C.A. Mulder E. J. Biol. Chem. 1995; 270: 27569-27576Google Scholar). The estrogen receptor (ER) belongs to a different steroid receptor subfamily because it resides predominantly in the nucleus, even in its unliganded form, and does not require translocation across the nuclear membrane following activation (3Greene G.L. Press M.F. J. Steroid Biochem. 1986; 24: 1-7Google Scholar). There are two major transactivation regions in the AR. 1) The activation function-1 (AF-1) domain is found at the NH2 terminus. 2) AF-2 is located in the LBD. AF-2 is a weak transactivator that is dependent on the presence of androgens for its activation. AF-1, on the other hand, is capable of ligand-independent transactivation, and fragments of the AR that contain AF-1 show high levels of transcriptional activity when ectopically expressed in cell lines that are devoid of endogenous AR (4Snoek R. Bruchovsky N. Kasper S. Matusik R.J. Gleave M. Sato N. Mawji N.R. Rennie P.S. Prostate. 1998; 36: 256-263Google Scholar, 5Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Google Scholar). Upon DNA binding, AR recruits components of the basal transcriptional machinery and influences either the up-regulation or down-regulation of gene expression. The exact mechanism of AR-specific gene expression is not fully understood. Each steroid receptor regulates unique sets of genes. However, in vitro assays have shown that these receptors recognize similar DNA sequences known as steroid response elements. These elements are comprised of a palindrome that contains two half-sites based on the 5′-TGTTCT-3′ motif that are separated by a three nucleotide spacer (6Tsai S.Y. Carlstedt-Duke J. Weigel N.L. Dahlman K. Gustafsson J.A. Tsai M.J. O'Malley B.W. Cell. 1988; 55: 361-369Google Scholar). Detailed analysis has demonstrated that both GR and AR bind with highest affinity to a steroid response element that has an imperfect palindrome, 5′-GGTACAnnnTGTTCT-3′ (7Nelson C.C. Hendy S.C. Shukin R.J. Cheng H. Bruchovsky N. Koop B.F. Rennie P.S. Mol. Endocrinol. 1999; 13: 2090-2107Google Scholar). The quandary is that although activated steroid receptors bind to highly homologous response elements on DNA, they still demonstrate an ability to regulate the expression of unique gene sets. There are several mechanisms by which receptors can specifically regulate gene expression. One mechanism suggests that co-regulatory proteins interact with steroid receptors to direct their activity. The search for AR-specific co-regulatory molecules has led to the identification of several AR-interacting proteins. Early studies identified ARA70/ELE1 as a ligand-dependent co-activator of AR (8Yeh S. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5517-5521Google Scholar). Subsequently, CREB-binding protein/p300, which has histone acetyltransferase activity, was also shown to interact with AR and enhance receptor activity in prostate cells (9Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Google Scholar). More recent AR-interacting proteins that have been identified include β-catenin (10Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713Google Scholar), caveolin (11Lu M.L. Schneider M.C. Zheng Y. Zhang X. Richie J.P. J. Biol. Chem. 2001; 276: 13442-13451Google Scholar), BAG-1L (12Froesch B.A. Takayama S. Reed J.C. J. Biol. Chem. 1998; 273: 11660-11666Google Scholar), SMAD3 (13Hayes S.A. Zarnegar M. Sharma M. Yang F. Peehl D.M. ten Dijke P. Sun Z. Cancer Res. 2001; 61: 2112-2118Google Scholar), cyclin D1 (14Petre C.E. Wetherill Y.B. Danielsen M. Knudsen K.E. J. Biol. Chem. 2002; 277: 2207-2215Google Scholar), and several others. These proteins have been shown to either positively (caveolin and BAG-1L) or negatively (SMAD3 and cyclin D1) affect AR transactivation. Identification of proteins that specifically interact with the AR has been a challenge because the AR and other steroid receptors share a high degree of sequence homology at their DBD and LBD. The DBD of human AR shares as much as 80% homology with that of progesterone receptor and over 70% with GR (15Chang C.S. Kokontis J. Liao S.T. Science. 1988; 240: 324-326Google Scholar). The LBD of the steroid receptors are highly homologous as well with up to 55% similarity at the amino acid level. Therefore, many co-regulatory proteins that interact with the AR at the LBD and DBD are promiscuous in their ability to interact with and influence activity of other steroid receptors. A report by Alenet al. (16Alen P. Claessens F. Schoenmakers E. Swinnen J.V. Verhoeven G. Rombauts W. Peeters B. Mol. Endocrinol. 1999; 13: 117-128Google Scholar) has demonstrated that ARA70 is not specific to the AR and that this protein interacts with the ER and GR as well. Likewise, the steroid receptor co-activator-1/NCoA1, which is the founding member of the p160 family of transcriptional co-activators, interacts indiscriminately with the LBD of steroid receptors to enhance activity (17Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Google Scholar). Other members of the p160 family such as TIF-2/GRIP-1 enhance AR, GR and ER activity alike (18Ding X.F. Anderson C.M. Ma H. Hong H. Uht R.M. Kushner P.J. Stallcup M.R. Mol. Endocrinol. 1998; 12: 302-313Google Scholar). Nevertheless, steroid receptor family members show the greatest degree of sequence variability at the NH2-terminal domain (<15%). Little is known regarding the role of the NH2-terminal domain in AR transactivation. Therefore, we used an NH2-terminal fragment of the AR, which is devoid of transcriptional activity (AR1–232), as bait in a yeast two-hybrid assay, and RanBPM was identified as an AR-interacting protein. Although RanBPM was initially described as a 55-kDa protein (BPM55), a subsequent report has shown it to be a 90-kDa protein (BPM90) (19Nakamura M. Masuda H. Horii J. Kuma K. Yokoyama N. Ohba T. Nishitani H. Miyata T. Tanaka M. Nishimoto T. J. Cell Biol. 1998; 143: 1041-1052Google Scholar, 20Nishitani H. Hirose E. Uchimura Y. Nakamura M. Umeda M. Nishii K. Mori N. Nishimoto T. Gene (Amst.). 2001; 272: 25-33Google Scholar). Here we demonstrate that the larger form of RanBPM, BPM90, is able to bind to multiple domains of the AR and that this interaction occurs in vivo. Furthermore, overexpression of RanBPM in prostate cancer cell lines shows that RanBPM can enhance AR transactivation. This property of RanBPM does not appear to be exclusive to the AR because BPM90 also enhances GR activity, although neither ER-α nor ER-β activity is affected. These experiments clearly demonstrate that RanBPM is capable of interacting with and modifying the activity of selective steroid receptors. A 696-bp fragment coding for the first 232 amino acids of the human AR (AR1–232) was cloned into the BamHI site of the pGBT9 vector for expression as a fusion protein with the GAL4 DBD (Clontech). A cDNA library made from normal human prostate tissue that was fused to the transactivation domain of GAL4 in the pACT2 expression vector was used for screening (Clontech). Expression plasmids were transformed into the Y190 yeast strain, and transformants were selected on SD minimal medium lacking tryptophan, leucine, and histidine. Clones that grew on minimal medium agar plates were subjected to β-galactosidase assays by colony filter-lift according to the manufacturer's instructions. Clones that tested positive for β-galactosidase were sequenced using Big Dye Terminator cycle sequencing reactions (Applied Biosystems) and were then compared with known sequences available in GenBankTM. The full-length RanBPM expression vector, pcDEBΔ-BPM90, and empty vector, pcDEBΔ, were provided by Dr. H. Nishitani (Kyushu University) (20Nishitani H. Hirose E. Uchimura Y. Nakamura M. Umeda M. Nishii K. Mori N. Nishimoto T. Gene (Amst.). 2001; 272: 25-33Google Scholar). The longest RanBPM library clone that was isolated in the yeast two-hybrid assay contained a 2152-bp fragment that coded for amino acids 148–729 of BPM90 and the 3′-untranslated region. This fragment of BPM90, herein referred to as BPML, was cloned into the pRC/cytomegalovirus (CMV) mammalian expression vector (Invitrogen) in which transcription is driven by the CMV promoter. BPML was cloned in-frame with an upstream ATG for translation initiation and a Kozak sequence to enhance translation efficiency. The NH2-terminal region of the human AR spanning the NH2-terminal domain and DBD (hAR1–646) was generated by PCR and cloned into the multiple cloning site of pRC/CMV for expression in mammalian cells. The full-length rat AR cDNA was expressed from the pRC/CMV mammalian expression plasmid, pCMV/AR6 (21Rennie P.S. Bruchovsky N. Leco K.J. Sheppard P.C. McQueen S.A. Cheng H. Snoek R. Hamel A. Bock M.E. MacDonald B.S. Nickel B.E. Chang C. Liao S. Cattini P.A. Matusik R.J. Mol. Endocrinol. 1993; 7: 23-36Google Scholar). The rat glucocorticoid receptor was expressed from the pGR mammalian expression vector as described elsewhere (22Miesfeld R. Rusconi S. Godowski P.J. Maler B.A. Okret S. Wikstrom A.C. Gustafsson J.A. Yamamoto K.R. Cell. 1986; 46: 389-399Google Scholar). The human ER-α expression vector (pSVMT:wER) has also been described previously (23Smith C.L. Conneely O.M. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6120-6124Google Scholar). The pcDNA4/HisMax-hERβ1 vector (a gift from Dr. L. Murphy, University of Manitoba) was used for the expression of human ER-β in mammalian cells. Multiple tissue Northern blots (Clontech) were probed with [32P]dCTP-labeled RanBPM cDNA. Poly(A) RNA was prepared from the LNCaP, PC3, MCF7, and HeLa cell lines using an Oligotex mRNA kit (Qiagen). For the Northern blot, 5 μg of poly(A) RNA were separated by electrophoresis on a 1% agarose gel with 30% formaldehyde and transferred to a Biodyne B nylon membrane (Pall Corporation) by capillary action in 10 mm NaOH. The membrane was hybridized with a [32P]dCTP-labeled 1100-bp cDNA fragment coding for RanBPM. Glyceraldehyde-3-phosphate dehydrogenase was used to normalize the loading of poly(A) RNA. Various NH2-terminal and DBD fragments of the human AR (AR1–232, AR1–559, and AR559–646) and a COOH-terminal fragment of rat AR (ARDBD/LBD) were cloned into the pGEX vector (AmershamBiosciences) for expression as GST fusion proteins. GST fusion proteins were expressed in the BL21 Escherichia coli strain and purified as described previously (5Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Google Scholar). Radiolabeled RanBPM protein was prepared from the pRC/CMV-BPML vector using the Quick Coupled T7 TnT in vitrotranscription/translation kit (Promega Corporation) in the presence of [35S]Met. Equimolar amounts of GST-AR fusion protein coupled to glutathione-agarose beads were incubated with radiolabeled RanBPM at 4 °C for 2 h in binding buffer (20 mm HEPES, pH 7.6, 150 mm KCl, 5 mmMgCl2, 1 mm EDTA, 0.05% Nonidet P-40). Beads were washed four times with binding buffer, and bound proteins were eluted into protein sample buffer (2% SDS, 5% β-mercaptoethanol) for analysis by SDS-PAGE followed by autoradiography. A fragment spanning the NH2-terminal domain and DNA-binding domain of the AR (AR1–646) was cloned into the pTrcHisC vector (Invitrogen) for expression with an NH2-terminal His tag, which consists of six histidine residues in tandem. His-tagged proteins were expressed in bacteria and purified using the nickel-nitrilotriacetic acid-agarose column according to the manufacturer's protocol (Qiagen). [35S]Met-RanBPM fragments were incubated with His-AR1–646 at 4 °C for 4 h in binding buffer (see above). His-AR1–646 was immunoprecipitated using an anti-His antibody (Qiagen) as described below. PC3, HeLa, and MCF7 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% fetal bovine serum (FBS) (Invitrogen) at 37 °C in 5% CO2. The LNCaP prostate carcinoma cell line was cultured in RPMI 1640 medium containing 5% FBS. For transient transfection, 3 × 105 cells were seeded in six-well plates and were transfected the following day using Lipofectin reagent (Invitrogen) as described previously (24Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Google Scholar). Transfection occurred for 16 h at 37 °C. Following transfection, cells were re-fed with fresh medium containing 5% dextran-coated charcoal-stripped FBS ± 1 nm R1881, 10 nm dexamethasone (Dex), 10 nm E2 or vehicle alone and incubated at 37 °C for an additional 24 h. After induction with hormone, cells were harvested and lysed in passive lysis buffer (Promega Corporation) for luciferase assay and for Western blot analysis. LNCaP cells were grown to 80% confluency in RPMI 1640 medium + 5% FBS. Cells were then cultured in RPMI 1640 medium containing 5% dextran-coated charcoal-stripped FBS for 16 h at 37 °C. The following day, cells were induced in the presence or absence of 10 nmR1881 for 4 h at 37 °C before scraping and lysis in radioimmune precipitation buffer (150 mm NaCl, 50 mmTris-Cl, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Whole cell extracts (1 mg of protein) were incubated with a polyclonal rabbit anti-AR antibody or with normal rabbit IgG as a negative control (Santa Cruz Biotechnology, Inc.). Immunocomplexes were pulled down using protein A/G-agarose beads (Santa Cruz Biotechnology, Inc.) and washed four times with radioimmune precipitation buffer. After the final wash, proteins were solubilized in SDS sample buffer and analyzed by Western blot. Western blots were carried out as described previously (21Rennie P.S. Bruchovsky N. Leco K.J. Sheppard P.C. McQueen S.A. Cheng H. Snoek R. Hamel A. Bock M.E. MacDonald B.S. Nickel B.E. Chang C. Liao S. Cattini P.A. Matusik R.J. Mol. Endocrinol. 1993; 7: 23-36Google Scholar). Protein samples were resolved on a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore). Membranes were blocked in TBS (20 mm Tris-Cl, pH 7.6, 137 mm NaCl) with 5% skim milk. Blots were incubated with appropriate primary antibody, diluted to 2 μg/ml in TBS + 5% milk for 4 h at room temperature, washed three times in TBS + 0.5% Tween 20, and then incubated for 45 min in horseradish peroxidase-conjugated secondary antibody (1:10,000) (Santa Cruz Biotechnology, Inc.). Blots were developed using the ECL chemiluminescence kit (AmershamBiosciences). AR and GR constructs were co-transfected with the pARR3-tk-Luc reporter construct in which the promoter has three androgen response regions (ARRs) in tandem (5Snoek R. Rennie P.S. Kasper S. Matusik R.J. Bruchovsky N. J. Steroid Biochem. Mol. Biol. 1996; 59: 243-250Google Scholar). In addition, the prostate-specific antigen (PSA) and probasin (PB) luciferase reporter constructs were used to determine transcriptional activity of the AR (4Snoek R. Bruchovsky N. Kasper S. Matusik R.J. Gleave M. Sato N. Mawji N.R. Rennie P.S. Prostate. 1998; 36: 256-263Google Scholar, 24Sato N. Sadar M.D. Bruchovsky N. Saatcioglu F. Rennie P.S. Sato S. Lange P.H. Gleave M.E. J. Biol. Chem. 1997; 272: 17485-17494Google Scholar). ER expression vectors were co-transfected with the pERE-Luc reporter plasmid as described previously. The pERE-Luc plasmid contains a single vitellogenin estrogen response element upstream of the thymidine kinase (TK) promoter. 2J. L. Ralph, M. C. Orgebin-Crist, J.-J. Lareyre, and C. C. Nelson, manuscript in preparation. Transfected cells were incubated in the presence or absence of hormone at 37 °C for 24 h prior to analysis. Transfection efficiency was normalized using the Renilla luciferase expression vector, pRL-TK (Promega Corp.). Firefly and Renilla luciferase activities were assayed with the Dual Luciferase assay kit (Promega Corp.). 20 μl of cell lysate were analyzed for luciferase activity using MicroLumiatPlusluminometer (EG&G Berthold). A fragment coding for the first 232 amino acids of the AR (AR1–232) was used as bait in a yeast two-hybrid assay to screen a human prostate library for interacting proteins. Over 3 × 105 transformants were screened on selective media, and positive clones were identified by β-galactosidase assay. Sequence analysis of three independent positive clones revealed a gene that was highly homologous to a known human protein, RanBPM (GenBankTM accession number AB055311) (19Nakamura M. Masuda H. Horii J. Kuma K. Yokoyama N. Ohba T. Nishitani H. Miyata T. Tanaka M. Nishimoto T. J. Cell Biol. 1998; 143: 1041-1052Google Scholar). Both 55- and 90-kDa forms of RanBPM have been reported in the literature and are referred to as BPM55 and BPM90, respectively (19Nakamura M. Masuda H. Horii J. Kuma K. Yokoyama N. Ohba T. Nishitani H. Miyata T. Tanaka M. Nishimoto T. J. Cell Biol. 1998; 143: 1041-1052Google Scholar, 20Nishitani H. Hirose E. Uchimura Y. Nakamura M. Umeda M. Nishii K. Mori N. Nishimoto T. Gene (Amst.). 2001; 272: 25-33Google Scholar). Sequences isolated from the three library clones isolated by the yeast two-hybrid assay had sequences upstream of the published BPM55 translation start codon (Fig. 1). The longest sequence of the three library clones (ARBP1) was 2346 bp in length and coded for a protein from amino acid 148 of BPM90 to the termination codon. This fragment is referred to as BPML. To confirm the interaction between RanBPM and AR that was identified by yeast two-hybrid screening, various AR constructs were expressed as GST fusion proteins and coupled to glutathione-agarose beads for use in GST pull-down assays. The 2346-bp fragment from ARBP1 (BPML) was cloned into pRC/CMV to allow for in vitro transcription/translation from the T7 promoter. [35S]Methionine-labeled BPML protein was allowed to interact with the GST-AR fusion proteins and analyzed by SDS-PAGE followed by autoradiography. As seen in Fig. 2A, RanBPM interacts with the 232-amino acid fragment that was originally used as bait in the yeast two-hybrid screen (AR1–232) (Fig. 2A,lanes 3 and 4). Equimolar amounts of AR-GST fusion proteins were used to determine relative binding between RanBPM and various domains of AR, and the results show that RanBPM interacts most strongly with the DBD (AR559–646) (Fig. 2A, lane 2). In addition, RanBPM interacts with the AR fragment that spans the DBD and LBD (ARDBD/LBD) (Fig. 2, lane 5). However, presence of the LBD does not enhance interaction, which suggests that the LBD does not have additional domains for RanBPM binding. To assess the relevance of this interaction in vivo, we performed a co-immunoprecipitation assay using LNCaP cell lysates. The LNCaP prostate carcinoma cell line expresses high levels of functional AR and displays androgen-dependent gene expression activity. Cells were treated for 4 h with 10 nm R1881 or with vehicle alone prior to preparation of protein lysates. Whole cell lysates were immunoprecipitated with normal rabbit IgG as a negative control (Fig. 2B, lanes 3 and5) or with an antibody that recognizes the COOH terminus of the AR (lanes 4 and 6) (Santa Cruz Biotechnology, Inc.). Protein complexes were pulled down with protein A/G coupled to agarose beads. Proteins of the AR complex were resolved by SDS-PAGE prior to Western blotting with an anti-BPM90 antibody (provided by Dr. H. Nishitani). A 90-kDa protein that is seen in the input lanes (Fig. 2B, lanes 1 and 2) is BPM90. The results in Fig. 2B show that BPM90 interacts with AR specifically in the presence of hormone (lanes 3–6). We confirmed that AR was pulled-down in this assay by blotting the same membrane with an antibody that recognizes the NH2 terminus of the AR (Affinity BioReagents) (Fig. 2B). RanBPM encodes a putative SPRY domain at the NH2 terminus (Fig. 1) (19Nakamura M. Masuda H. Horii J. Kuma K. Yokoyama N. Ohba T. Nishitani H. Miyata T. Tanaka M. Nishimoto T. J. Cell Biol. 1998; 143: 1041-1052Google Scholar). This domain has been implicated in protein-protein interactions. To determine the importance of the SPRY domain, His-tag pull-down assays were carried out to map the interacting domains between RanBPM and the AR. Radiolabeled COOH-terminal truncations of RanBPM were generated by restriction digest of the pRC/CMV-BPML vector with EcoRI orNdeI followed by in vitrotranscription/translation in the presence of [35S]methionine. The truncated RanBPM proteins (BPM148–408 and BPM148–251) were incubated with purified recombinant His-tagged AR1–646, which includes the NH2-terminal domain and DBD. An antibody that recognizes the His-tag (Qiagen) was used to immunoprecipitate AR and its interacting proteins. After sufficient washing, samples were eluted into protein sample buffer, resolved by SDS-PAGE, and analyzed by autoradiography. Fig. 2C, Input lanes, show that radiolabeled proteins have molecular masses of 64, 28, and 11 kDa (BPML, BPM147–408, and BPM147–251, respectively). Both BPML and the truncated peptide generated by EcoRI digest, BPM147–408, interacted with the AR (Fig. 2C,top and middle panels). However, BPM147–251 in which the SPRY domain is disrupted was no longer able to interact with AR (Fig. 2C, bottom panel). This finding agrees with yeast two-hybrid results in which β-galactosidase activity and, therefore, interaction with AR1–232 are lost when an NH2-terminal truncation of RanBPM, which does not have a complete SPRY domain, is used as bait (data not shown). To determine expression levels of RanBPM, a [32P]cDNA probe was generated from the ARBP1 yeast two-hybrid library clone and hybridized to a multiple tissue Northern blot (Clontech). This Northern blot allows for analysis of RanBPM expression in several different tissue types. Fig. 3A shows that RanBPM is expressed as a 2.9-kb transcript in multiple different tissue types. High levels of RanBPM message are seen in the prostate and ovaries, and highest levels of expression are observed in the testes (lanes 3, 5, and 4, respectively). RanBPM expression levels were determined for various cancer cell lines by both Northern and Western blots (Fig. 3, B andC). Cell lines that were used included the ovarian carcinoma HeLa cell line from which BPM90 was first identified, two prostate cancer cell lines, PC3 and LNCaP, and the MCF7 breast cancer cell line. Both Northern and Western blots show that RanBPM is highly expressed in all of the cell lines. In Fig. 3C, the 90-kDa protein seen in lanes 1–4 is BPM90. Although BPM90 is expressed in all of the cell lines tested, the highest levels were observed in HeLa cells (lane 4). PC3 and MCF7 cells expressed lower levels of BPM90 as compared with the LNCaP cell line (comparelanes 1 and 3 with lane 2). The membrane was probed with an antibody for β-actin (Sigma) to demonstrate that loading efficiency was consistent in all lanes (Fig. 3C). Our results have demonstrated that RanBPM interacts with the androgen receptor at the NH2 terminus and at the DNA-binding domain (Fig. 2A). To determine whether this interaction has a biological impact on AR activity, transcriptional assays were carried out using the PC3 cell line. PC3 cells represent a relatively undifferentiated stage of prostate cancer. These cells express very little to no androgen receptor and do not demonstrate androgen-regulated growth. Cells were transiently transfected with an AR expression vector (pCMV/AR6), the pARR3-tk-Luc reporter plasmid, and increasing amounts of RanBPM (pcDEBΔ-BPM90) and then were induced in the presence or absence of 1 nm R1881 for 24 h prior to harvesting for luciferase assays (Fig. 4A). In the absence of hormone, negligible AR-mediated transcriptional activity was observed from the pARR3-tk-Luc reporter plasmid. An addition of hormone resulted in an ∼9-fold induction of AR activity. In the presence of hormone, the overexpression of BPM90 resulted in an AR activity that was three times greater than in the absence of RanBPM. AR activity in the absence of hormone was unchanged by the addition of RanBPM, even with a 20-fold excess of BPM90 (Fig. 4B). Western blot analysis of cell lysates was carried out to ensure equivalent levels of AR expression in all samples (data not shown). Additional transactivation assays were carried out using an NH2-terminal truncated form of RanBPM (BPML) in which expression is under the control of a CMV promoter (Fig. 4A). The presence of BPMLincreases basal AR activity by 4-fold when androgens are present. BPML did not affect the activity of AR in the absence of ligand when the AR was transcriptionally inactive. Because RanBPM was identified by its interaction with the NH2 terminus of the AR, similar transactivation experiments were carried out with a form of the AR that is deleted for the LBD, pRC/CMV-AR1–646. This region of the AR has the AF-1 site and is capable of ligand-independent transcriptional activity. PC3 cells were transiently transfected with the truncated AR construct and treated with or without 1 nm R1881. AR1–646 is capable of high levels of transcriptional activity both in the presence and absence of ligand (Fig. 4C). Increasing amounts of BPM90 resulted in increased AR1–646 activity up to 2.5-times greater than basal levels. This increase in AR activity was independent of added hormone. The ARR3 promoter of the reporter plasmid is a synthetic highly active promoter that has three ARRs in tandem. The PB and PSA promoters, however, may be more physiologically relevant. In vivo, the expression of the rat probasin g" @default.
• W2125858585 created "2016-06-24" @default.
• W2125858585 creator A5008699306 @default.
• W2125858585 creator A5029529592 @default.
• W2125858585 creator A5039236851 @default.
• W2125858585 creator A5058530861 @default.
• W2125858585 creator A5085690109 @default.
• W2125858585 creator A5089480920 @default.
• W2125858585 date "2002-12-01" @default.
• W2125858585 modified "2023-10-16" @default.
• W2125858585 title "RanBPM, a Nuclear Protein That Interacts with and Regulates Transcriptional Activity of Androgen Receptor and Glucocorticoid Receptor" @default.
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