Cloning and characterization of ART-27. (A) Amino acid sequence of ART-27. The amino acid sequence of ART-27 is shown with an asterisk, representing the stop codon. Potential phosphorylation sites for protein kinase C (pKC), casein kinase II (CKII), and protein kinase A (pKA) are marked by a dot below the target residue. Lines above the sequence represent predicted α-helical regions as determined by Chou-Fasman and GarnierOsguthorpe-Robson algorithms. (B) Expression of ART-27 protein. Equal amounts (50 μg) of nuclear extracts prepared from HeLa cells (lane 1), treated with TPA for 2 h (lane 2), serum-starved and stimulated with serum for 2 h (lane 3), PC-3 cells (lane 4), and whole cell extracts from COS1 cells transfected with pcDNA3 (lane 5) or pcDNA3:ART-27 (lanes 6) were analyzed by immunoblotting with affinity-purified anti–ART-27 antibody. No ART-27 immunoreactivity is observed with preimmune serum (Markus, Taneja, Logan, Li, Ha, Hittelman, Rogatsky, and Garabedian, unpublished results). (C) ART-27 mRNA expression in human tissues and prostate cancer cell lines. A human multiple tissue Northern blot (CLONTECH: human 12-lane MTN Blot I and II) containing 2 μg of poly(A+) mRNA from the indicated tissues was hybridized with ³²P-labeled probes corresponding to ART-27 (top) and β-actin (bottom). Total RNA was extracted from PC-3 and LNCaP cells cultured in the absence or presence of the indicated doses of androgen (R1881) for 72 h. Equal amounts of RNA were separated on denaturing formaldehyde-agarose gels (see MATERIALS AND METHODS), transferred to a Duralon nylon membrane, and hybridized to ³²P-labeled cDNA probes corresponding to ART-27 (right). Equal loading for each lane was determined by ethidium bromide staining of the 28S rRNA (Markus, Taneja, Logan, Li, Ha, Hittelman, Rogatsky, and Garabedian, unpublished results). (D) ART-27 subcellular localization. HeLa cells were transfected with an FLAG–ART-27 expression construct, fixed, permeabilized, and incubated with an anti-FLAG primary antibody and a corresponding fluorescein-conjugated secondary antibody, and the DNA in nucleus was stained with Hoechst dye H334211. Cells were visualized using a Zeiss Axioplan 2 fluorescence microscope. No signal above background was observed when the primary antibody was omitted (Markus, Taneja, Logan, Li, Ha, Hittelman, Rogatsky, and Garabedian, unpublished results) or in nontransfected cells. Note that the FLAG–ART-27 fluorescence is localized predominantly to the nucleus. (E) Expression pattern of endogenous ART-27 in human PC-3 and LNCaP prostate cancer cell xenografts. Immunohistochemical analysis of paraffin-embedded PC-3 (left) and LNCaP (right) human prostate cancer cell xenografts hybridized with affinity-purified ART-27 antibody is shown (200×). Immunoreactivity, seen as brown staining within the nuclei of these cells, is blocked by coincubation of the antibody with the ART-27 peptide immunogen (Markus, Taneja, Logan, Li, Ha, Hittelman, Rogatsky, and Garabedian, unpublished results).

ART-27 interacts with AR in vitro and in cell extracts. (A) Full-length human AR and HA–ART-27 were synthesized in vitro using a coupled transcription/translation system in the presence of [³⁵S]methionine and in the absence or presence of 100 nM R1881, as indicated, and incubated with an antibody against HA. Bound proteins were collected on Protein A Sepharose beads, washed, eluted, resolved by SDS-PAGE, and visualized by autoradiography. Twenty percent of the input AR and ART-27 translation reaction are shown. (B) ART-27 interacts with endogenous AR in LNCaP cell extracts. Nuclear extracts from untreated and 100 nM R1881-treated LNCaP-Tet-on-ART-27-FLAG cells were prepared as described in MATERIALS AND METHODS and incubated with an antibody against either preimmune or ART-27 immune sera. Immune complexes were collected on protein A Sepharose beads, washed, eluted, resolved by SDS-PAGE, and transferred to a membrane. The filter was probed with antibody against AR and ART-27.

Specificity of AR:ART-27 interactions. Interaction of ART-27 with the AR N terminus (N-T; rat AR_18–500), AR LBD (rat AR_579–901), and other transcriptional regulatory factors was analyzed using the modified yeast two-hybrid assay. Regulatory factors examined include the GR AF-1 (rat GR_107–237), GR 30IIB (rat GR_107–237 E219K, F220L, W234R), SRC-1 (SRC-1_374–800), GR LBD (rat GR_525–795), TAF_II130 (TAF_II130_270–700), Sp1 A (Sp1_83–262), Sp1 B (Sp1_263–524), CREB-N (CREB_3–296), ERα(ERα_1–595), and VP16. The strength of interaction was determined by a quantitative liquid β-galactosidase assay after a 24-h incubation in galactose medium at 30°C and normalized to protein expression using the common HA epitope resident on each protein. The background from the vector-only control was subtracted from each sample. The interaction with ERα was observed in the presence, but not the absence (Markus, Taneja, Logan, Li, Ha, Hittelman, Rogatsky, and Garabedian, unpublished results), of 100 nM 17β-estradiol. No interaction was observed with AR LBD and the GR LBD in either the presence or absence of 100 nM R1881 and 10 μM deoxycorticosterone, the preferred GR ligand in yeast (Garabedian and Yamamoto, 1992).

Domains of AR and ART-27 mediating interaction. (A) Quantitative analysis of ART-27 interaction with AR N-terminal derivatives. The relative affinity of ART-27 for the AR N-terminal subdomains 336–500, 153–336, and 18–156 was assessed using the yeast two-hybrid assay. The dark gray boxes show the location of the AR N-terminal activation functions AF-1a and AF-1b and the light gray box denotes the glutamine (Q) repeat region. Data represent the means of triplicate data points normalized to cell number. (B) Only full-length ART-27 interacts with the AR N terminus. ART-27 deletion derivatives fused to LexA were tested for their ability to interact with the AR N terminus (AR_18–500) using a qualitative β-galactosidase plate assay after a 24-h incubation on galactose X-gal plates at 30°C. +, strong interactions (blue colonies); −, no interactions above background vector-only control (white colony). Left, an immunoblot of the ART-27 derivatives expressed in yeast and probed with an antibody against the LexA moiety common to all ART-27 truncations.

ART-27 enhances steroid and thyroid receptor-dependent transcriptional activation. HeLa cells (1.2 × 10⁵ cells/35-mm dish) were transiently transfected using Lipofectamine with paired receptor (0.2 μg/dish) and reporter constructs (0.1 μg/dish) for hAR + MMTV-Luc (A), GR + MMTV-Luc (B), ERα and ERβ + XETL (C), and human TRβ-1 + pGL3-DR4 (D) along with the indicated amount of ART-27 or empty expression vector to equalize the total amount of DNA per dish and 0.05 μg of pCMV-LacZ per dish as an internal control for transfection efficiency. Cells were treated with 100 nM R1881 (A), 100 nM dexamethasone (Dex; B), 10 nM 17β-estradiol (E2; C), 100 nM triac (T3, D), or the ethanol vehicle (white bars) for 12 h, and receptor transcriptional activation was assayed as described in MATERIALS AND METHODS, normalized to β-galactosidase activity, and expressed as relative luminescence units (RLU). The average of three independent experiments and SE is shown. (E) ART-27 does not affect transactivation by VP16. HeLa cells were transfected as above with an expression construct for GAL4-VP16 and a reporter plasmid containing five Gal4-binding sites upstream of the E1b promoter, in the absence (white bars) or presence (gray bars) of 1 μg of ART-27, and assayed for luciferase activity. (F) ART-27 failed to activate transcription when tethered to DNA. HeLa cells were transfected as above with expression constructs for LexA, LexA–ART-27, or LexA-AR_18–500 and the pΔ4X-LALO reporter plasmid and assayed for luciferase activity.

The effect of ART-27 on AR transcriptional activation is dependent on the AR-interacting region. (A) HeLa cells were transfected with 0.5 μg of the pcDNA3LexA:AR N-terminal derivatives indicated and either 1.0 μg of the empty expression vector (white bars) or 1.0 μg pcDNA3-HA–ART-27 (gray bars) along with 1.0 μg of the LexA responsive-luciferase reporter (pΔ4X-LALO) and 0.25 μg of pCMV-LacZ, and AR activity was determined as described in Figure 5. Data represent the means of duplicate data points normalized to β-galactosidase units. Error bars represent the range of the duplicate data points. (B) The expression of the LexA:AR derivatives was analyzed by immunoblotting from a parallel set of transfections using a polyclonal antibody to LexA.

Native ART-27 is part of a high molecular weight complex. Nuclear extracts were prepared from HeLa cells and loaded onto linear 15–35%glycerol gradients with or without 2.4 M urea. Fractions were collected and analyzed by Western blot using affinity-purified ART-27 polyclonal or TBP monoclonal antibodies. Migration of molecular mass markers is indicated: 45- and 68-kDa albumin, 158-kDa aldolase, 240-kDa catalase. The molecular mass estimate of TFIID is 700 kDa (Tanese et al., 1991).