The Linkage of Kennedy’s Neuron Disease to ARA24, the First Identified Androgen Receptor Polyglutamine Region-associated Coactivator*

Although the linkage of polyglutamine (poly-Q) repeat expansion in the androgen receptor (AR) to Kennedy’s disease (X-linked spinal and bulbar muscular atrophy) was a major step forward, the detailed molecular mechanism of how the change in poly-Q length contributes to the disease remains unclear. Here we report the identification of a nuclear G-protein, Ras-related nuclear protein/ARA24, as the first AR coactivator that can bind differentially with different lengths of poly-Q within AR. In the yeast and mammalian reciprocal interacting assays, our data suggested the interaction of AR N-terminal domain with ARA24 diminishes as the poly-Q length increases. The coactivation of ARA24 also diminishes with the poly-Q expansion within AR. Deletion of the acidic hexapeptide (DEDDDL) at the C terminus of ARA24 further enhances its AR coactivation. Together, our data suggest that poor interaction and weaker coactivation of ARA24 to the longer poly-Q AR in the X-linked spinal and bulbar muscular atrophied AR could contribute to the weaker transactivation of AR. The consequence of poor interaction and weak coactivation may eventually lead to the partial androgen insensitivity during the development of Kennedy’s disease.

tial AR-associated proteins that may contribute to this disease. Using the yeast two-hybrid system we were able to find an AR coactivator, ARA24, that can differentially activate the AR with different poly-Q lengths within AR. Nucleotide sequencing revealed that this AR coactivator has an identical sequence with Ran (7).
Genetic results have identified that Ran/ARA24 is involved in nuclear transport of proteins and RNA, cell cycle progression, and nuclear structure in mitotic regulation, as well as RNA and DNA synthesis (8). Ran/ARA24 was purified as a complex with a chromatin-associated DNA-binding protein, RCC1 (regulator of chromosome condensation 1). RCC1 was first identified as the product of a gene mutated in the tsBN2 (temperature-sensitive baby hamster kidney) cell line (9). RCC1 has been shown to function as a guanine nucleotide exchange factor on Ran/ARA24 by increasing its rate ϳ5 ϫ 10 5 times. On the other hand, Ran GTPase-activating protein can also increase the GTPase activity ϳ1 ϫ 10 5 times (10). During biochemical identification of Ran-associated protein, previous reports mainly revealed its functions in nuclear transport (8).
Here we demonstrate that Ran/ARA24 can also interact with the AR N-terminal poly-Q region and enhance the AR transactivation.

MATERIALS AND METHODS
Yeast Two-hybrid Screening-The cDNA fragment encoding the AR poly-Q stretch and its flanking region (amino acids   (11) were inserted into a pAS2 yeast expression plasmid as bait. Expression of this AR bait in the yeast strain Y190 for the screening of associated proteins from human brain cDNA library was performed following the standard protocols described previously (12)(13)(14).
Purification of Recombinant AR and ARA24 Proteins-The Histagged full-length human AR (ARQ17 was used exclusively unless otherwise specified) was expressed by the baculovirus system described previously (15). The His-ARA24 was expressed in Escherichia coli strain BL21-(DE3)-LysS transformed with pET28c-ARA24 (Novagen). Both the His-tagged AR and His-tagged ARA24 proteins were partially purified by immobilized metal affinity chromatography using TALON metal affinity resin (CLONTECH) and quantified by Western blot analysis (16). The loading of nucleotide to ARA24 was done as described previously (17).
Yeast Two-hybrid Liquid Culture ␤-Galactosidase Assay-Yeast clones transformed with pAS2-ARN (amino acids 11-208) and pGAD10-ARA24 were streaked on Ϫ2SD selective medium (devoid of Trp and Leu), freshly inoculated in the Ϫ2SD medium, and grown at 30°C overnight. The yeast cells were diluted and regrown in Ϫ2SD medium to an A 600 value of ϳ0.6. The cells were lysed in Z buffer (60 mM * This work was supported by National Institutes of Health Grants CA55639, CA68518, and CA71570. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF052578.
Transfection and Reporter Gene Assays-Human prostate cancer PC-3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. ϳ4 ϫ 10 5 cells (50 -70% confluency in 35-mm Petri dishes) growing in Dulbecco's modified Eagle's medium supplemented with 10% charcoal-dextran stripped fetal bovine serum were transfected by SuperFect transfection reagent (QIAGEN) with DNA as described in each figure legend. As a control of each expression plasmid, the same amount of parental expression plasmid was used and indicated as a minus in each figures 1, 2, 4, and 5. 24 h after transfection, the medium was changed to fresh medium containing different hormones as indicated in each figure legend for another 24 h. Luciferase activities were measured using the Dual-Luciferase TM reporter assay system and Turner Designs luminometer TD-20/20 (Promega). Relative luciferase activities were plotted using the activity of DHT-AR in the absence of coactivator as 1. The results were summarized from at least three sets of transfection and presented as mean Ϯ S.E. For antisense ARA24 experiments, human androgen non-responsive NCI H1299 cells were transfected by the calcium phosphate method, and CAT assays were performed as described previously (18 -22). Each final assessment represents the mean Ϯ S.E. of five independent experiments. For detecting the elimination of endogenous ARA24 protein levels by the introduction of antisense ARA24 (ARA2400as) into cells, H1299 cells were electroporated by Gene Pulser (Bio-Rad) with 10 g of either parental vector (pCMV) or pCMV-ARA24as. The cells were harvested 48 h posttransfection.

RESULTS
Cloning of ARA24 by Yeast Two-hybrid System-A hybrid protein bait, GAL4 DNA binding domain (GBD) fused with the human AR poly-Q region (amino acids 11-208), was expressed in the yeast strain Y190 to screen the human brain cDNA library. A clone that showed strong interaction with the AR poly-Q region was identified. This clone has an insert of 1566 base pairs with an open reading frame encoding a peptide of 216 amino acids. Based on the calculated molecular mass (24 kDa) and its ability to bind to AR, we named this clone ARA24. GenBank TM sequence comparison found ARA24 has the same amino acid sequence as Ran, a Ras-like small nuclear GTPase. The nucleotide and deduced amino acid sequences of the ARA24 clone from yeast two-hybrid screening are shown in Fig.  1A.
We used coimmunoprecipitation followed by Western blot analysis to confirm the interaction between ARA24 and AR. In the presence of AR, ARA24 can be coimmunoprecipitated by anti-AR antibody (NH27) conjugated to protein A-agarose beads (Fig. 1B, lanes 1-3), and neither AR nor ARA24 alone (Fig. 1B, lanes 4 and 5) showed a comparable level of coimmunoprecipitation. Binding of GTP or GDP to ARA24 did not cause substantial differences of ARA24 protein to interact with AR in the coimmunoprecipitation. These results strengthen the interaction of AR and ARA24 found in yeast screening. Our results also suggest that various guanine nucleotide binding states of ARA24 may not affect its interaction with AR in vitro. Furthermore, one-hybrid interaction in mammalian cells was applied to demonstrate the functional interaction of ARA24 with AR. Expression of GBD-fused ARA24 and AR in the presence of androgen forms a transactive complex that can activate the transcription of pG5-E1b-Luc. As shown in Fig. 1C, coexpression of GBD-fused ARA24 and AR in the presence of 1 nM DHT induces the luciferase activity. Taken together, results from each of the three experiments described in Fig. 1 provide strong evidence that AR can interact well with ARA24, the first identified AR poly-Q region-associated protein.
Expression of Either Wild Type ARA24 or Mutant ARA24 (C-del) Enhances AR Transactivation-It has been demonstrated that WT Ran/ARA24 or mutant Ran/ARA24, ARA24 C-del, with C-terminal hexapeptide deletion can be overexpressed up to 7-8-fold above endogenous background in transient transfection without affecting cell cycle progression (23). It was also suggested that the conserved acidic hexapeptide sequence, DEDDDL at the C-terminal end of Ran/ARA24, is not required for Ran/ARA24 to stay in the nucleus but might be required for interaction with an effector (23). We therefore examined the role of both WT ARA24 and ARA24 C-del in AR-mediated transactivation. Cotransfection of WT ARA24 enhanced AR-mediated transactivation ϳ4-fold in PC-3 cells at a transfection ratio of AR to WT ARA24 of 1:30 ( Fig. 2A). Unexpectedly, we found ARA24 C-del was a more potent AR coactivator. ARA24 C-del enhances AR transactivation up to 18-fold in PC-3 cells at a transfection ratio of AR to ARA24 C-del of 1:30 (Fig. 2B). Using antisense ARA24 expression plasmid (ARA24as), we also found that AR transactivation was significantly decreased by cotransfecting ARA24as (Fig. 2C). Western blot analysis data showed that the cotransfection of ARA24as could decrease the cellular ARA24 protein significantly 48 h after transfection, therefore confirming the reduced AR transactivation could be due to the reduced ARA24 protein caused by expression of ARA24as (Fig. 2D). Together, our results indicate that AR transactivation can be either enhanced or repressed by increasing or decreasing the cellular ARA24 protein amount.
Expression of ARA24 and ARA160 Increases DHT Activity Over 10-fold-Because ARA160, another identified AR N-terminal coactivator, 2 can also enhance AR transactivation, it is important to know the profile of DHT-induced AR transactivation in the presence or absence of these two AR N-terminal ARAs. In the absence of ARA160 and ARA24, DHT starts to induce AR-mediated transactivation between 10 Ϫ9 and 10 Ϫ10 M (Fig. 3, lanes 1-4). In the presence of ARA24 or ARA160, DHT significantly induces AR-mediated transactivation from 10 Ϫ10 to 10 Ϫ11 M (Fig. 3, lanes 5-12). These data suggest that these AR N-terminal coactivators can increase DHT sensitivity over 10-fold. This 10-fold increase may allow the low concentration of androgen (10 Ϫ10 -10 Ϫ11 M) that exists in the serum of prostate cancer patients undergoing the androgen ablation therapy to induce androgen target genes. This may imply that these AR N-terminal coactivators could play very important roles in the hormone-refractory prostate cancer.
Expansion of AR Poly-Q Length Diminishes AR-ARA24 Interaction-As ARA24 associates to the AR poly-Q region, we then used yeast two-hybrid and mammalian two-hybrid assays to test if the different lengths of poly-Q in AR can influence this interaction. As shown in Fig. 4A, the interaction between ARA24 and the AR poly-Q region in yeast cells decreased with the increase of AR poly-Q length. This result was further confirmed in mammalian Chinese hamster ovary cells by reciprocal two-hybrid assay with exchanged fusion partners (GBDfused ARA24 versus VP16-ARN in mammalian systems as compared with GAD-ARA24 versus GBD-ARN in yeast systems) (Fig. 4B). Together, these results consistently show that the interaction between ARA24 and the AR poly-Q region is inversely correlated with AR poly-Q length in both yeast and mammalian Chinese hamster ovary cells. These data represent the first evidence to show that WT AR (Q-25) and SBMA AR (Q-49) can bind differentially to ARA24, the first identified AR poly-Q region coactivator.
Coactivation of ARA24 to AR Decreases with the Expansion of AR Poly-Q Length-We then compared the coactivator activity of ARA24 to AR with different poly-Q lengths (Q-25 versus Q-49). In the absence of ARA24 cotransfection, AR Q-49 shows a little less activity than AR Q-25 as previously reported (Fig.  5, lane 6 versus lane 2). Cotransfection of ARA24 enhances AR Q-25 transactivation 4-fold (Fig. 5, lane 4 versus lane 2). However, cotransfection of ARA24 enhances AR Q-49 transactivation only 2-fold (Fig. 5, lane 8 versus lane 6). Together, results shown in Figs. 4 and 5 provide strong evidence to suggest that the lower AR transactivation of SBMA AR (Q-49) as compared with normal AR (Q-25) could be because of a weaker interaction with ARA24. The consequence of this weaker interaction could also result in the weaker AR transactivation. DISCUSSION Previous studies concerning the transactivation activity of AR with different poly-Q lengths have consistently shown their inverse relationship (21,25). The present study demonstrates that ARA24 interacts with the AR poly-Q region and functions as an AR coactivator. The interaction between AR and ARA24 decreases with the expansion of AR poly-Q, as well as the coactivation of cotransfected ARA24 to the SBMA AR. These results provide a potential mechanism for the partial androgen-insensitive syndrome in Kennedy's disease.
It is well documented that a continual cycling of binding and hydrolyzing GTP from ARA24 is required in multiple cellular processes (reviewed in Ref. 8). Yeast mutations prp20 and rna1, which are homologues of mammalian Ran-guanine nucleotide exchange factor and Ran-GTPase-activating protein, respectively, have been shown to exhibit the phenotype of reduced accuracy in positioning the correct transcription initiation site (26). These phenotypes are readily explained by either a change in chromatin structure or by certain defects in the transcription regulation. The ability to enhance AR transactivation by ARA24 may therefore provide a bridge for ARA24 to link to the transcriptional regulation.
As shown in Fig. 1B, AR forms complexes with ARA24 regardless of the bound guanine nucleotide, which seems different from what has been observed for a GTPase effector protein that exhibits selective binding to either a GTPase-GTP or a GTPase-GDP. Upon inactivation of RCC1, ARA24 was shown dispersed in the nucleus and cytosol, suggesting that GTPbound ARA24 is the major form in the nucleus (27). It seems that the AR-ARA24 interaction is not regulated by the guanine binding status of ARA24 in vitro. It is not clear whether the guanine binding status of ARA24 plays any role when mediating AR transactivation in cells.
ARA24 is a very abundant cellular protein. It is estimated that HeLa cells contain about 10 7 ARA24 molecules per cell (7). This high abundance does cause great difficulty when attempting to show its biochemical function by the addition of exogenous ARA24. This is probably the reason we need to use a relatively high dose of exogenous ARA24 to show its coactivator activity ( Fig. 2A). Nevertheless, the ratios of receptor to coactivator we used here (1:10 or 1:30) are close to the ratios of other steroid receptors to their coactivators. For example, full- length SRC-1 requires the addition ratio of 1:20 or 1:40 to see the coactivator effects (28). On the other hand, cotransfection of ARA24as to decrease the cellular ARA24 protein level can also repress the AR transactivation. As shown in Fig. 2B, deleting the six amino acid residues of the acidic C terminus in ARA24 results in a dominant positive AR coactivator. A lower (500 ng) level of ARA24 C-del is enough to exert a coactivation effect to AR compared with 1500 ng of WT ARA24. Moreover, increasing ARA24 C-del to a higher dose (1500 ng) further enhances DHT-induced AR transactivation from 4-fold to 18-fold. As the C-terminal hexapeptide sequence of ARA24 is conserved across species, it is likely that a repressor-like factor may bind to the conserved hexapeptide sequence, and deletion of this hexapeptide may therefore allow ARA24 C-del to function more efficiently as a coactivator to enhance AR transactivation.
The AR coactivator effects of ARA24 and ARA160 are not only enhancement of AR transactivation at the normal physiological concentration of DHT but also at the low DHT level (10 Ϫ10 -10 Ϫ11 M) that is found in prostate cancer patients undergoing androgen ablation therapy. The increase of androgen sensitivity by the non-androgen factors could be one of the reasons why the patients' prostate-specific antigen level might increase again even though the patients' serum testosterone is low (10 Ϫ10 M). Our findings that either ARA24 or ARA160 can increase androgen sensitivity so that 10 Ϫ10 M DHT can still efficiently stimulate androgen target genes, including prostatespecific antigen, may provide one molecular mechanism to explain the failure of androgen ablation therapy.
The polymorphic poly-Q stretch of AR has been specially recognized for being clinically associated with Kennedy's disease. Subsequent in vitro studies also show that the longer poly-Q stretch may result in weaker AR transactivation (21,25). Recently, pathological statistics have further correlated the shorter poly-Q AR with higher prostate cancer risk and a higher chance of developing distant metastasis (24,29). Our results showed that ARA24 can interact differentially with different lengths of poly-Q within AR, and its ability to enhance AR transactivation also decreases with expansion of poly-Q length in AR. A very interesting question to pursue in the future might be whether expression of ARA24 or its ability to interact with the AR poly-Q region could become an indicator to predict prostate cancer risk.
As ARA24 is also involved in nuclear transportation that can promote the nuclear localization of proteins, it is possible that the weaker interaction of ARA24 to longer poly-Q AR (SBMA AR) may result in aggregation of SBMA AR in cytoplasm, instead of translocation into the nucleus to function as a transcriptional activator. A detailed analysis of subcellular distribution of ARA24 and SBMA AR in Kennedy's disease cells should be able to answer such an interesting question.
In summary, our results suggested that ARA24 is the first identified AR N-terminal associated coactivator that can interact differentially with AR bearing different poly-Q lengths. This finding may help us to further understand the potential roles of AR function in Kennedy's disease and progression of prostate cancer.