Tat-binding protein-1 (TBP-1), an ATPase of 19S regulatory particles of the 26S proteasome, enhances androgen receptor function in cooperation with TBP-1-interacting protein/Hop2

Abstract

The 26S proteasome, which degrades ubiquitinated proteins, appears to contribute to the cyclical loading of androgen receptor (AR) to androgen response elements of target gene promoters; however, the mechanism whereby the 26S proteasome modulates AR recruitment remains unknown. Using yeast two-hybrid screening, we previously identified Tat-binding protein-1 (TBP-1), an adenosine triphosphatase of 19S regulatory particles of the 26S proteasome, as a transcriptional coactivator of thyroid hormone receptor. Independently, TBP-1-interacting protein (TBPIP) was also identified as a coactivator of several nuclear receptors, including AR. Here, we investigated whether TBP-1 could interact with and modulate transcriptional activation by AR cooperatively with TBPIP. TBP-1 mRNA was ubiquitously expressed in human tissues, including the testis and prostate, as well as in LNCaP cells. TBP-1 directly bound TBPIP through the amino-terminal domain possessing the leucine zipper structure. AR is physically associated with TBP-1 and TBPIP in vitro and in LNCaP cells. TBP-1 similarly and additively augmented AR-mediated transcription upon coexpression with TBPIP, and the ATPase domain, as well as leucine zipper structure in TBP-1, was essential for transcriptional enhancement. Overexpression of TBP-1 did not alter AR protein and mRNA levels. In the chromatin immunoprecipitation assay, TBP-1 was transiently recruited to the proximal androgen response element of the prostate-specific antigen gene promoter in a ligand-dependent manner in LNCaP cells. These findings suggest that a component of 19S regulatory particles directly binds AR and might participate in AR-mediated transcriptional activation in cooperation with TBPIP.

Figure 1

Northern blot analysis of TBP-1 gene expression in multiple human tissues and prostate cancer cells. One microgram of poly(A⁺) RNA isolated from 16 human tissues (A) or 25 μg total RNA isolated from human normal prostate and the LNCaP cell line (B) was hybridized with the ³²P-labeled TBP-1 cRNA probe. Positions of RNA size markers (kb) or 28S and 18S ribosomal RNA are indicated. Arrows indicate specific hybridization signals.

Figure 2

TBP-1 and TBPIP physically associate in mammalian cells. Flag-tagged TBP-1 and Xpress-tagged TBPIP cDNA vectors were cotransfected into CV-1 cells using Lipofectamine 2000. Forty-eight hours after transfection, WCL was prepared as described, and 500 μg WCL was subjected to immunoprecipitation (IP) using 1 μg anti-Flag Ab (αFlag) (A) or anti-Xpress Ab (αXpress) (B). Western blot analysis (IB) was performed using αXpress or αFlag as described in Materials and Methods. The same amount of nontransfected WCL (control) was subjected to immunoprecipitation in parallel, and preimmune IgG was used as a negative control. The experiments were repeated with identical results. Input represents 1% of input WCL. The positions of molecular mass markers are indicated (kDa). IgH, Ig heavy chain.

Figure 3

TBP-1 interacts with TBPIP in GST pull-down assays. A, SDS-PAGE of GST-fused TBP-1 and TBPIPs. GST fusion proteins were expressed in bacteria and purified as described. Ten and 20 μl fusion proteins and 10 μl GST protein were subjected to electrophoresis and visualized by Coomassie blue staining. B, GST pull-down assays were performed using in vitro translated ³⁵S-labeled TBP-1 proteins and equivalent amounts of GST, GST-fused TBP-1, or TBPIPs as described. Input represents 10% of input proteins. C, In vitro translated ³⁵S-labeled TBPIP was incubated with equivalent amounts of GST, GST-fused TBP-1N, or TBP-1 C proteins. Input represents 10% of input proteins. The experiments were repeated with identical results. Arrows indicate the input and pulled-down samples.

Figure 4

AR interacts with TBP-1 and TBPIP in vitro and in LNCaP cells. A, GST pull-down assays were performed between in vitro translated ³⁵S-labeled full-length AR and GST, GST-fused TBP-1, or TBPIPs in the absence and presence of 10 nm DHT as described. Input represents 10% of input proteins. The AR binding to TBP-1 and TBPIP relative to input protein levels was expressed as the mean ± se from four separate experiments. Asterisks indicate significant difference from the AR binding to GST alone (*, P < 0.05; **, P < 0.01; ***, P < 0.001). No significant difference (NS) was observed in AR binding to TBP-1 or TBPIP in the absence or presence of DHT. B, WCL was prepared from LNCaP cells treated with 10 nm DHT for 1 h. A total of 500 μg WCL was immunoprecipitated (IP) using 1 μg preimmune IgG, anti-TBP-1 Ab (αTBP-1), or anti-AR Ab (αAR) ON at 4 C in the presence of 10 nm DHT. After washing, samples were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Western blot analysis (IB) was performed using αAR or αTBP-1. Input represents 4% of input WCL. An asterisk indicates nonspecific binding. The experiments were repeated with identical results. IgH, Ig heavy chain.

Figure 5

TBP-1 and TBPIP cooperatively enhance AR-mediated transactivation. A, Expression vectors for AR (333 ng/well), TBP-1 (1.67 μg/well), or TBPIP (1.67 μg/well) were cotransfected with MMTV promoter-driven luciferase reporter (1.67 μg/well) into CV-1 cells using the calcium phosphate precipitation method. Total amounts of transfected plasmids were adjusted by adding empty pKCR2 vector. Luciferase assay was performed after 24 h treatment with 10 nm DHT. Fold induction calculated from three independent transfection experiments is shown as the mean ± se (*, P < 0.05; **, P < 0.01; ***, P < 0.001). NS, Not significant. B, Expression vectors for mutant TBP-1 (K233H, D286A, or ΔLZ) were cotransfected with MMTV luciferase reporter in the presence of AR expression vector into CV-1 cells. Fold induction relative to that in the absence of cotransfected TBP-1 vector was calculated from three independent transfection experiments and shown as the mean ± se (*, P < 0.05; **, P < 0.01). NS, Not significant.

Figure 6

A, TBP-1 did not increase the steady-state level of AR proteins in the absence and presence of DHT. CV-1 cells were transfected with pKCR2AR in the presence of pKCR2TBP-1 or pKCR2K233H by lipofection. TBPIP was overexpressed as a control protein by cotransfection of pcDNA4HisMaxC-TBPIP in the absence of cotransfected TBP-1. Twenty-four hours after adding 10 nm DHT, WCL was prepared and subjected to Western blot analyses of AR, TBP-1, and TBPIP levels. Cyclophilin (Cyclo) was simultaneously immunoblotted as a loading reference. The experiments were repeated with identical results. B, TBP-1 did not affect TBPIP levels. CV-1 cells were transfected with pcDNA4HisMaxC-TBPIP in the absence and presence of pKCR2TBP-1. Forty-eight hours after transfection, WCLs were prepared and subjected to Western blot analysis for TBPIP, TBP-1, and cyclophilin proteins. The experiments were repeated with identical results. C, TBP-1 did not increase steady-state levels of AR mRNA. CV-1 cells were transfected, and total RNA was isolated using the same time course as in A. First-strand cDNA synthesis and RT-PCR were performed as described in Materials and Methods. PCR products were subjected to electrophoresis on a 2% agarose gel, and band intensities were measured using an image analyzer. D, TBP-1 did not alter AR protein stability. CV-1 cells were transfected with pKCR2AR in the absence and presence of pKCR2TBP-1 by lipofection. pcDNA4HisMaxC-TBPIP was transfected as an overexpressed control protein. Twenty-four hours after transfection, CHX (final concentration at 50 μm) was added, and WCLs were prepared after 0, 4, 6, 8, and 12 h. Western blot analyses were performed to quantitate AR, TBP-1, TBPIP, and αtubulin proteins. AR protein levels were normalized by tubulin levels, and the relative protein levels in the presence of cotransfected TBP-1 or TBPIP at time zero were set at 100%. Data represent the mean ± se from three separate experiments.

Figure 7

TBP-1 was recruited to ARE I of the human PSA gene promoter in LNCaP cells in a ligand-dependent manner. The position of ARE I in the PSA gene relative to the transcription start site (+1) is schematically shown. ChIP assays were performed as described in Materials and Methods using preimmune IgG, anti-AR, and anti-TBP-1 antibodies. PCR was performed for 35 cycles using primer pairs flanking ARE I. PCR products were subjected to electrophoresis in a 2% agarose gel and visualized with ethidium bromide staining. Representative PCR products are shown, and the graph indicates relative occupancy of AR and TBP-1 to ARE I at each time point expressed as a percentage of input DNA levels (mean ± se) from three independent experiments.