Androgen Receptor Specifically Interacts with a Novel p21-activated Kinase, PAK6*

Fajun YangDagger , Xiaoyu Li§, Manju SharmaDagger , Mark ZarnegarDagger , Bing Lim§, and Zijie SunDagger

From the Dagger  Liem Sioe Liong Molecular Biology Laboratory, Departments of Surgery and Genetics, Stanford University School of Medicine, Stanford, California 94305 and the § Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215

Received for publication, November 13, 2000, and in revised form, January 24, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The androgen receptor (AR) is a hormone-dependent transcription factor that plays important roles in male sexual differentiation and development. Transcription activation by steroid hormone receptors, such as the androgen receptor, is mediated through interaction with cofactors. We recently identified a novel AR-interacting protein, provisionally termed PAK6, that shares a high degree of sequence similarity with p21-activated kinases (PAKs). PAK6 is a 75-kDa protein that contains a putative amino-terminal Cdc42/Rac interactive binding motif and a carboxyl-terminal kinase domain. A domain-specific and ligand-dependent interaction between AR and PAK6 was further confirmed in vivo and in vitro. Northern blot analysis revealed that PAK6 is highly expressed in testis and prostate tissues. Most importantly, immunofluorescence studies showed that PAK6 cotranslocates into the nucleus with AR in response to androgen. Transient transfection experiments showed that PAK6 specifically repressed AR-mediated transcription. This report identifies a novel function for a PAK-homologous protein and suggests a potential unique mechanism by which other signal transduction pathways may cross-talk with AR pathways to regulate AR function in normal and malignant prostate cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effects of androgens are mediated by the androgen receptor (AR),1 which plays a critical role in inducing normal differentiation of tissues of the reproductive organs and in the development and progression of prostate cancer (1). AR belongs to the nuclear receptor superfamily, whose members regulate ligand-dependent gene transcription (2, 3). The AR and other receptors in this family possess identifiable activation domains that confer transactivation potential when fused to a heterologous DNA binding domain. However, an important feature of the AR and other nuclear receptors that distinguish them from other transcription factors is that they are activated by their ligand binding domains (LBD). The unbound AR forms a complex with heat-shock proteins (HSPs) (4, 5). Upon binding to ligand, the AR dissociates from the HSPs and translocates into the nucleus, where it binds to the androgen response element (ARE) and recruits cofactors to regulate transcription (6, 7).

Several coactivators that interact preferentially with the LBD of nuclear receptors have been identified, such as SRC1/NCoA1, TIF2/GRIP1/NCoA2, and ACTR/AIB1/TRAM1/RAC3 (8-11). These cofactors facilitate nuclear receptor-mediated, ligand-dependent transcription through chromatin remodeling and histone modification (12). A group of AR-associated (ARA) proteins, termed ARA54 (13), ARA55 (14), ARA70 (15), Tip60 (16), and HBO1 (17) was identified by using the DBD and LBD of AR as baits in a yeast two-hybrid screen and shown to modulate the ligand-dependent transactivation of AR. FHL2 is a novel AR-interacting protein that was identified recently by using a modified yeast one-hybrid screen and was shown to enhance AR-mediated transcription (18).

It has also been reported that certain growth factors or agents that stimulate protein kinase A or C can independently activate steroid hormone receptors in the absence of ligand or have a synergistic effect with ligand (19, 20). For example, stimulation of protein kinase A or C activates the AR (20, 21). Androgen-independent activation of the AR by the polypeptide growth factors insulin-like growth factor 1 (IGF-1), keratinocyte growth factor (KGF), and epidermal growth factor (EGF) has also been demonstrated (22). These observations demonstrate that the AR binds, directly or indirectly, to a large number of proteins and that signaling pathways may modulate AR activity differently in normal and tumor cells.

The p21-activated kinases (PAKs) are members of a growing class of Rac/Cdc42-associated Ste20-like Ser/Thr protein kinases, characterized by a highly conserved amino-terminal Cdc42/Rac interactive binding (CRIB) domain and a carboxyl-terminal kinase domain (23-27). PAK1, 2, and 3 are three closely related mammalian members of the family, whereas the recently reported members PAK4 and PAK5 share high homology with each other but have lower homology to PAKs 1-3 (28). The mammalian PAKs exert their effects by regulating kinase cascades, much like the Saccharomyces cerevisiae Ste20 protein kinase that controls the pheromone/mating factor pathway. Both Cdc42 and Rac initiate signaling cascades to the nucleus that involve activation of the stress response mitogen-activated protein kinases, c-jun kinase, and p38 (29, 30). Recent studies have shown that in some situations, this signaling is mediated via Cdc42/Rac stimulation of PAK activity (23, 31). Although the biological functions of PAKs remain unclear, PAKs are implicated in the regulation of a number of cellular processes, including rearrangement of the cytoskeleton, apoptosis, mitogen-activated protein kinase signaling pathway, growth factor-induced neurite outgrowth, and control of phagocyte NADPH oxidase (28, 32-35).

Using a yeast two-hybrid screen, we identified a novel AR-interacting protein, provisionally termed PAK6, which shares a high degree of sequence similarity with PAK family members. Like other PAKs, PAK6 contains a putative amino-terminal CRIB domain and a carboxyl-terminal kinase domain. PAK6 is highly expressed in testis and prostate tissues. Protein-protein interaction between AR and PAK6 was further demonstrated both in vitro and in vivo. Significantly, PAK6 can cotranslocate to nuclei with AR in a ligand-dependent manner. Furthermore, cotransfection of PAK6 with AR was shown to specifically repress AR-mediated transcription. These results reveal a novel function for PAK proteins and suggest a unique mechanism by which other signal transduction pathways, such as the small GTPases, may impact AR-mediated transcription.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Two-hybrid Screen-- The LBD of human AR (amino acids 629-919) was fused in-frame to the GAL4 DBD in the pGBT9 vector for use as bait (CLONTECH, Palo Alto, CA). The construct was transformed into a modified yeast strain, PJ69-4A (36). A cDNA library from human prostate tissue fused to the GAL4 transactivation domain (TAD) was used in this screening (CLONTECH). Transformants were selected on Sabouraud dextrose (SD) medium lacking adenine, leucine, and tryptophan in the presence of 100 nM R1881.

The specificity of interaction with AR was determined by liquid beta -galactosidase (beta -gal) assay. pGBT9 constructs with two different AR fragments, including the partial TAD (amino acids 1-333) and LBD, were cotransformed with the GAL4 activation domain constructs. Specific interactions were measured by production of adenine and beta -gal.

Plasmid Construction-- The carboxyl-terminal region of PAK6 was isolated in the initial yeast two-hybrid screening. To isolate the entire coding region for PAK6, the rapid amplification of 5' cDNA ends (5'-RACE) method was used with human prostate Marathon Ready cDNA (CLONTECH). The specific reverse primer for 5'-RACE spans amino acid residues 371 and 378 (5'-AGCCAGGGCACCCTTGGCAACCGT-3') of PAK6. A full-length PAK6 cDNA was created by fusing a DNA fragment coding for the amino-terminal region, isolated by 5'-RACE, with a fragment encoding the carboxyl-terminal fragment, obtained from yeast two-hybrid screening, in the pcDNA3 vector containing an amino-terminal Flag epitope tag. Truncated PAK6 mutants were generated from the full-length clone in the same vector.

The reporter plasmid MMTVpA3-Luc, containing the luciferase gene under the control of the steroid hormone response elements in the MMTV-LTR, was provided by Dr. Richard Pestell (Albert Einstein Medical College, New York, NY) (37). The human AR cDNA, cloned into an SV40 promoter-driven expression vector, pSV-hAR, was kindly provided by Dr. Albert Brinkmann (Erasmus University, Rotterdam, The Netherlands). An SV40-driven beta -galactosidase reporter plasmid (pSV-beta -GAL) (Promega, Madison, WI) was used in this study as an internal control. A human GR expression construct (pSV-hGR) was the kind gift of Dr. David Feldman.

GST Pull-down Assay-- GST·AR fusion proteins were constructed in the pGEX-2TK vector (Amersham Pharmacia Biotech). Expression and purification of GST fusion proteins were performed as described previously (38). The full-length human PAK6 protein was generated and labeled in vitro by the TNT-coupled reticulocyte lysate system (Promega, Madison, WI). Equal amounts of GST·AR fusion proteins coupled to glutathione-Sepharose beads were incubated with 35S-labeled PAK6 proteins at 4 °C for 2 h in the modified binding buffer (0.1% Nonidet P-40, 0.05% nonfat milk, 0.5 mM EDTA, 20 mM Tris-HCl, 180 mM KCl, 10% glycerol, 50 mM ZnCl2, 5 mM MgCl2, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Beads were carefully washed three times with buffer, and bound proteins were analyzed by SDS-PAGE followed by autoradiography.

Immunoprecipitation and Western Blotting-- The human AR expression vector pSV-hAR, alone or with the Flag-tagged PAK6 expression plasmid, was transfected into CV-1 cells. Transfected cells were cultured in the presence of 10 nM DHT for 24 h and then harvested in a buffer containing 20 mM HEPES (pH 8.0), 0.5% Nonidet P-40, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 1 mM CaCl2, 10 mM ZnCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and 5% glycerol). Whole cell lysates were incubated with mouse normal IgG or anti-Flag monoclonal antibody (Sigma) at 4 °C for 2 h. Pre-equilibrated protein-A-Sepharose beads were then added and, after an hour of incubation, collected by centrifugation; they were then gently washed three times with the same buffer as described above. Proteins were eluted by boiling in SDS-sample buffer and resolved on a 10% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane and probed with a 1:500 dilution of a polyclonal antibody against the amino terminus of AR (catalog number sc-816, Santa Cruz Biotechnology, Santa Cruz, CA). Proteins were detected using the ECL kit (Amersham Pharmacia Biotech).

Northern Blot Analysis-- A blot with RNA from multiple human tissues was obtained from CLONTECH Inc. Total RNA was prepared from prostate cancer cell lines (LNCaP, DU145, PC-3, ARCaP, and LAPC4) and nonprostate cancer cell lines (MCF7 and HeLa). For Northern blotting, 25 µg of total RNA were electrophoresed on a 1% agarose-formaldehyde gel, transferred to Hybond-N nylon membranes (Amersham Pharmacia Biotech) by capillary blotting in 20× SSC, and hybridized with a DNA fragment (encoding amino acids 1-349) derived from PAK6. beta -actin was used to normalize loadings.

Immunofluorescence-- CV-1 cells were plated onto gelatin-coated (2%) coverslips the day before transfection. The full-length or truncated Flag-tagged pcDNA3-PAK6 alone, or with pSV-hAR, was transiently transfected into cells with LipofectAMINE-PLUS reagent (Life Technologies, Inc.) according to the manufacturer's instructions. After 6 h, transfected cells were fed with fresh medium plus/minus 10-8 M DHT, incubated for 24 h, then fixed for 10 min with 3% paraformaldehyde in phosphate-buffered saline, and washed with 0.1% Nonidet P-40/phosphate-buffered saline buffer. All subsequent antibody dilutions and cell washings were done using the same Nonidet P-40/phosphate-buffered saline buffer. Nonspecific sites were blocked with 5% skim milk powder in phosphate-buffered saline for 30 min. The cells were then incubated with either anti-Flag monoclonal antibody (Sigma) or anti-AR polyclonal antibody (catalog number sc-805) for 1 h at room temperature. Cells were washed three times, followed by incubation with fluorescein isothiocyanate-conjugated anti-mouse (catalog number sc2010) or rhodamine-conjugated anti-rabbit secondary antibody (catalog number sc816). The coverslips were then inverted, mounted on slides, and sealed with nail polish. Pictures were taken using confocal microscopy.

Protein Kinase Assays-- The flag-tagged full-length and truncated PAK6 expression constructs were transfected into CV-1 cells. After 48 h of culture, cells were harvested, and the expressed PAK6 proteins were immunoprecipitated as described above. The immune complexes were then washed with the kinase buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM MnCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 10 mg/ml aprotinin). Kinase assays were performed in a 50-µl volume with 50 µCi of [gamma -32P]ATP, 5 µg of myelin basic protein (Sigma), and 5 µl of immunoprecipitated PAK6 proteins. The reactions were carried out at room temperature for 30 min and terminated by addition of SDS-PAGE loading buffer. Samples were analyzed by SDS-PAGE and autoradiography.

Cell Cultures and Transient Transfections-- A monkey kidney cell line (CV-1) containing no detectable levels of endogenous steroid hormone receptor activity was maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (HyClone, Denver, CO). Transient transfections were carried out using a LipofectAMINE transfection kit (Life Technologies, Inc.). Approximately 1.5-2 × 104 cells were plated in a 48-well plate 16 h before transfection. About 200 ng of total plasmid DNA per well were used in the transfection. Approximately 16 h after transfection, the cells were washed and fed medium containing 5% charcoal-stripped (steroid hormone-free) fetal calf serum (HyClone) in the presence or absence of steroid hormones. Cells were incubated for another 24 h, and luciferase activity was measured as relative light units, as we described previously (17, 39). The relative light units from individual transfections were normalized by beta -galactosidase activity in the same samples. Individual transfection experiments were done in triplicate, and the results are reported as mean relative light units/beta -galactosidase (± S.D.) from representative experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Isolation of Androgen Receptor-interacting Proteins-- To identify AR-interacting proteins, we screened a human prostate library with a bait containing full-length AR-LBD. Yeast transformants were selected for growth in medium containing 100 nM R1881. Of 8 × 106 transformants, 155 grew under selective conditions and showed an increased beta -gal production in a hormone-dependent manner. Rescue of the plasmids and sequencing of the inserts revealed several different cDNAs. Among them, 56 clones encoded a previously identified AR-associated protein, ARA70 (15), and 43 clones encoded gelsolin, a presumptive calcium-regulated actin filament-capping protein (40). Two cDNAs (clone numbers P18 and P56) encoded a novel sequence, and analysis of these clones with the NCBI GenBankTM sequence data base revealed homology with the carboxyl-terminal kinase domains of p21-activated kinases. We named the novel protein "PAK6." To confirm the interaction, we cotransformed the P18 clone with various constructs containing either GAL4DBD alone or the AR fusion proteins with LBD and a partial transactivation domain (Fig. 1). pACT2-PAK6 showed a specific interaction with GAL4DBD-AR-LBD by producing adenine in the presence of androgen, similar to the pACT2-ARA70 positive control (Fig. 1A). A liquid beta -gal assay was performed to quantify the interactions. The pACT2-PAK6 showed an ~170-fold induction with the pGBT9-AR-LBD in the presence of DHT (Fig. 1B). Our results demonstrate that the LBD of AR specifically interacts with PAK6 in a ligand-dependent manner in yeast.


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Fig. 1.   Specific interaction of PAK6 with the AR-LBD in yeast. The PAK6 clone (P18) from the yeast two-hybrid screen, an ARA70 clone, or an empty library vector (pACT2) was cotransformed into yeast strain PJ69 with AR-LBD, AR-partial TAD, or the bait vector (pGBT9). Transformed cells were plated on SD-Ade-Leu-Trp plates (with or without 1 × 10-7 M R1881 for samples with AR-LBD) and SD-Leu-Trp plates (for monitoring the transformation efficiency). The plates were incubated at 30 °C for 5 days. A, appearance of yeast colonies on SD-Ade-Leu-Trp plates indicated the specific interaction between two fusion proteins. B, liquid beta -gal assay was performed to check the interaction. Yeast colonies from the SD-Leu-Trp plates were cultured in SD-Leu-Trp medium with or without 1 × 10-7 M R1881 for 16 h. The beta -gal activities were measured using the Galacto-light plus kit (Tropix Inc., Bedford, MA) and normalized by cell density (A600). The data represent the mean ± S.D. of three independent colonies.

PAK6 Is a Novel Member of the PAK Protein Family-- Sequence analysis revealed that the PAK6 clones isolated from the yeast two-hybrid screen were truncated proteins. To isolate the amino-terminal fragment of PAK6, we performed 5'-RACE. Three positive clones covering different lengths of the 5' region were isolated, and the longest was used to generate a full-length clone by fusion with the DNA fragment containing the carboxyl-terminal region obtained from the original yeast screening (Fig. 2A). Sequence analysis of the full-length clone revealed a methionine initiation codon at nucleotide 198, followed by an open reading frame encoding a 681-amino acid protein with a predicted molecular mass of 75 kDa (GenBankTM accession number AF276893). Using in vitro transcription and translation, a 75-kDa protein was generated by several different PAK6 plasmids (Fig. 2B), confirming the identity of the predicted initiation codon.



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Fig. 2.   Nucleotide and predicted amino acid sequence of human PAK6. A, the amino-terminal portion of human PAK6 was obtained by 5'-RACE from a human prostate cDNA library (CLONTECH). Positive polymerase chain reaction products were identified by Southern blotting. Three positive clones containing different length cDNA fragments were obtained from this screen. aa, amino acid; bp, base pair. B, the three positive clones containing the amino-terminal portion of PAK6 were ligated with the carboxyl-terminal portion from the yeast clone and subcloned into pcDNA3. The clones were transcribed and translated in vitro and analyzed by SDS-PAGE. Kd, kilodalton. C, the full-length cDNA and predicted amino acid sequence of human PAK6 are shown (GenBankTM accession number AF276893). The putative CRIB motif and the kinase domain are underlined. D, comparison of the putative CRIB motif and inhibitory region of PAK6 with those of other PAK proteins. The numbers on the right and left correspond to the amino acid of the respective proteins. E, alignment of the kinase domain of PAK6 with those of other PAK proteins. As indicated above, the numbers correspond to the amino acid of the PAK proteins.

The PAK family of proteins share a highly conserved amino-terminal CRIB domain (underlined in Fig. 2C) and a carboxyl-terminal protein kinase domain (boxed in Fig. 2C). A high degree of sequence similarity was observed when PAK6 was aligned with these two regions of other human PAKs. PAK6 shares eight consensus residues in the CRIB domain with other PAK proteins (Fig. 2D). The protein kinase domain of PAK spans 300 amino acids and is highly conserved among the family members. Comparison of PAK6 with human PAKs 1-5 showed that the kinase domain of PAK6 has more than a 50% sequence similarity with PAKs 1-5, with PAK6 having the highest similarity in this region to PAK4 (78%) and PAK5 (78%).

PAK6 Interacts with AR in Vitro and in Vivo-- To confirm that the interaction observed in the yeast two-hybrid system is biologically relevant, GST pull-down experiments were carried out with a series of GST·AR fusion proteins (Fig. 3A) (38). The binding of [35S]methionine-labeled full-length PAK6 protein to GST·AR fusion proteins was analyzed by SDS-PAGE and detected by autoradiography. As seen in Fig. 3B, a specific retention of PAK6 protein was observed for the samples with GST·AR LBD. A stronger interaction was detected using two small fragments of LBD consisting of partial LBD and activation domain 2 (AF2) truncated mutants, pLBD and activation domain 2, suggesting that parts of each domain are dispensable for binding to PAK6. The binding between the LBD of AR and PAK6 was not significantly enhanced by androgen in our GST pull-down experiments. Similar results have been reported for other nuclear receptor cofactors, perhaps due to improper protein folding of the GST fusions (9, 11, 16, 17). Our results from the GST pull-down experiments further support those of the yeast-two hybrid experiments and suggest that the LBD of AR is required for interaction with PAK6.


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Fig. 3.   PAK6 interacts with the ligand binding domain of androgen receptor in vitro. A, as shown, AR-TAD, AR-DBD, and AR-LBD were fused to GST in pGEX-2TK and expressed in Escherichia coli strain BL21. The expressed fusion proteins were then purified with GST beads. HR, hinge region. B, in vitro translated, 35S-labeled, full-length PAK6 was incubated with various GST proteins on beads in the presence or absence of 1 × 10-7 M R1881. The beads were washed three times, and proteins were resolved by SDS-PAGE and visualized by autoradiography.

To determine whether an interaction between PAK6 and AR occurs in vivo, we tagged PAK6 at its amino terminus with a Flag epitope and expressed tagged PAK6 protein together with AR protein in CV-1 cells, which contain low levels of endogenous steroid hormone receptor activity. Both AR and Flag-PAK6 proteins were detected in the transfected cells (Fig. 4A). Whole cell lysates containing equal amounts of AR proteins were immunoprecipitated with normal mouse IgG or an anti-FLAG monoclonal antibody. As shown in Fig. 4B, Flag-PAK6 proteins were detected in the anti-Flag immunoprecipitates from cells cotransfected with AR and Flag-PAK6 in the presence or absence of DHT (lanes 1 and 3). With the specific AR antibody, AR protein was only detected in immunoprecipitates from cells cotransfected with AR and Flag-PAK6 in the presence of DHT (Fig. 4C). These data demonstrated that the interaction between AR and PAK6 occurs in mammalian cells in a ligand-dependent manner.


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Fig. 4.   AR is coimmunoprecipitated with PAK6. CV-1 cells were transiently cotransfected with AR alone or with Flag-tagged PAK6 and cultured in the presence or absence of 1 × 10-8 M DHT. A, whole cell lysates were precipitated with AR or Flag antibodies to detect expression of the two proteins. Equal amounts of cell lysate were immunoprecipitated with normal mouse IgG or anti-Flag monoclonal antibody at 4 °C. The precipitated fractions were then resolved by SDS-PAGE and analyzed by Western blot (Wb) using anti-Flag antibody (B) or anti-AR antibody (C). IP, immunoprecipitation.

We further examined interactions between the full-length AR and PAK6 in vivo (Fig. 5A) using a modified mammalian one-hybrid system, in which AR protein binds to the authentic AREs on the DNA via its own DNA binding domain. An AR expression vector and a luciferase reporter driven by the MMTV promoter (MMTVpA3-Luc) were cotransfected with the expression vector in which the partial PAK6 clone isolated originally from the yeast two-hybrid screen was fused to the VP16 activation domain (Fig. 5A). These constructs were tested for their ability to interact with the AR protein and to activate transcription of the MMTVpA3-Luc construct. Transfection of the PAK6-VP16AD showed a strong induction of luciferase activity, higher than that of VP16 alone or of a random yeast clone (ZF18) isolated from this screening as a negative control (Fig. 5B). Taken together, the results indicate that the carboxyl-terminal domain of PAK6 interacts with AR in a natural AR-ARE transcriptional complex on the promoter.


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Fig. 5.   PAK6 interacts with AR in CV-1 cells. The PAK6 clone from the yeast two-hybrid screen was fused to the VP16 transactivation domain in pVP16 vector and transiently transfected into CV-1 cells along with the full-length AR, MMTV-luciferase reporter, and beta -gal. Transfected cells were cultured in Dulbecco's modified Eagle's medium supplemented with 5% charcoal-stripped fetal calf serum for 24 h in the presence or absence of 1 × 10-8 M DHT. Luciferase activities were normalized using beta -gal activities. The data shown are the mean ± S.D. of three independent samples from one experiment. However, similar results were obtained from three independent experiments.

PAK6 Is Selectively Expressed in Human Testis and Prostate-- Northern blot analysis was carried out to detect the expression of PAK6 in human tissues using a probe covering 1-349 amino acid residues of the PAK6 protein. An ~4.5-kilobase transcript of PAK6 was detected most abundantly in testis and at lower levels in prostate (Fig. 6A). There was little or no detectable signal in ovary, intestine, spleen, thymus, peripheral blood leukocytes, and colon. In this study, we also examined PAK6 expression in RNA samples isolated from human cancer cell lines, including AR-positive cells (ARCaP (41), LNCaP (42), and LAPC4 (43)), AR-negative cells (PC-3 (44) and DU145 (45)), and nonprostate cancer cells (MCF7 and HeLa) (Fig. 6B). Using the same DNA probe as above, a 4.5-kilobase transcript of PAK6 was detected in most of the cell lines tested, but not in LNCaP and HeLa cells (Fig. 6B).


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Fig. 6.   Expression pattern of PAK6 in human tissues. A Northern blot containing mRNA samples from various human tissues (A) or the total RNA samples from prostate and nonprostate cancer cell lines (B) were probed with a cDNA containing the amino-terminal fragment of PAK6 (amino acids 1-349). The hybridized transcripts of PAK6 are marked with arrows. kb, kilobases.

PAK6 Contains a Potent Kinase Domain-- Other PAK proteins have been shown to phosphorylate substrates such as histone H4 and myelin basic protein (28). To assess whether the kinase domain of PAK6 is functional, we performed in vitro kinase assays. The amino-terminal Flag-tagged full-length and truncated PAK6 constructs were transfected into CV-1 cells, and the PAK6 proteins were immunoprecipitated from whole cell lysates using the flag antibody. As shown in Fig. 7, the truncated construct containing the kinase domain exhibited a strong kinase activity with myelin basic protein, whereas the full-length PAK6 showed a lower level of activity. These results suggest that PAK6 contains an active kinase domain but that its activity is regulated by other regions of the protein, perhaps by the binding of other proteins to the amino-terminal CRIB motif.


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Fig. 7.   Kinase activity of full-length and truncated PAK6. CV-1 cells were transfected with the Flag-tagged full-length (F), amino-terminal (amino acids 1-250) (N), or carboxyl-terminal (amino acids 382-681) (C) fragment of PAK6 as indicated. Whole cell lysates were immunoprecipitated using the Flag antibody and used for in vitro kinase assays by using myelin basic protein (MBP) as the substrate (see "Experimental Procedures" for details).

PAK6 Translocates into the Nucleus with AR in Response to Androgen Induction-- To mediate transcription, AR has to translocate into the nucleus in response to induction by androgen. To assess the biological function of PAK6 as an AR-interacting protein, it is important to determine what happens to the AR-PAK6 complex upon androgen induction. First, we examined the subcellular localization of PAK6 protein by immunofluorescence. As shown in Fig. 8A, the full-length PAK6 is mainly localized in the cytoplasm. Treatment with DHT, as previously reported, resulted in a clear induction of AR nuclear translocation but had no effect on the localization of PAK6 when the PAK6 vector alone was expressed in cells (Fig. 8A). When AR and PAK6 were cotransfected into cells, they were localized in the cytoplasm in the absence of DHT (Fig. 8B). However, with DHT treatment, the full-length PAK6 protein was totally translocated into the nucleus and colocalized with AR. These results provide additional data demonstrating that PAK6 specifically interacts with AR in a ligand-dependent manner and, through this interaction, translocates into the nucleus as part of an AR complex.


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Fig. 8.   Nuclear cotranslocation of PAK6 and AR. A, CV-1 cells were transfected with the Flag-tagged full-length PAK6 or AR expression construct and cultured in the presence or absence of DHT. AR protein was detected with a polyclonal anti-AR antibody (Ab) and revealed by rhodamine-conjugated secondary antibody. Flag-tagged PAK6 protein was detected with monoclonal anti-FLAG antibody and revealed with fluorescein isothiocyanate-conjugated secondary antibody. Pictures of cells were taken with confocal microscopy. B, CV-1 cells were cotransfected with the Flag-tagged full-length PAK6 and AR expression constructs and then stained as described above.

PAK6 Selectively Represses AR-mediated Transactivation-- Because PAK6 has been shown to interact with the AR-ARE complex (Fig. 5), we next sought to determine whether the interaction of PAK6 and AR had demonstrable effects on AR-mediated transcription. AR and PAK6 expression plasmids, along with a luciferase reporter plasmid regulated by the steroid hormone responsive elements in the MMTV-LTR (MMTVpA3-Luc) (46, 47), were transfected into CV-1 cells, and the effects on luciferase activity were measured. When the AR expression construct alone was transfected with the luciferase reporter, an ~20-fold induction of luciferase activity was observed in the presence of 10 nM DHT (Fig. 9). This ligand-dependent AR activation was repressed, in a dose-dependent manner, by more than 60% when the PAK6 expression vector was cotransfected. In contrast, cotransfection of PAK6 and GR showed no repression of MMTV promoter activity in the presence of 10 nM dexamethasone. The repression by PAK6 was not associated with a change in the intracellular steady state level of AR protein (data not shown), suggesting that repression resulted from a direct effect of PAK6 on AR activity and not from the down-regulation of AR expression.


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Fig. 9.   Effect of PAK6 on AR-mediated transcription. CV-1 cells were transiently transfected in 48-well plates with 100 ng of pMMTV-Luc, 50 ng of pSV40-beta -gal (beta -gal), 10 ng of pSV-hAR or pSV-hGR, where indicated, and 10 or 20 ng of pCMV-PAK6 (F-PAK6) constructs. White bars represent the absence of ligand, and black bars represent the addition of ligand. 10 nM DHT or DEX was used as the ligand for AR or GR, respectively. Luciferase activity is reported as relative light units and represented as the mean + S.D.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we identified a new p21-activated kinase, PAK6, and demonstrated a specific interaction between PAK6 and AR. These findings link PAK-mediated signaling to the steroid hormone receptor pathway, suggesting a novel function for this new PAK member. Many AR-interacting proteins have been identified thus far, and in general they are transcription factors that play direct roles in regulating transcription. The identification of PAK6, a protein kinase, as an AR-interacting protein is therefore quite interesting and novel. Consequently, we have spent a considerable amount of effort to prove that the interaction between PAK6 and AR is a truly physiological protein-protein interaction. We showed that the LBD of AR is necessary and sufficient for the interaction with PAK6 in an androgen-dependent manner in the yeast two-hybrid system, suggesting that conformational changes in the LBD of AR upon binding to ligand are necessary for PAK6 to bind to AR protein. The interaction was further confirmed in vivo in mammalian cells. In the coimmunprecipitation experiments, the AR protein specifically interacted with Flag-tagged PAK6. The interaction between AR and PAK6 proteins was also observed in the mammalian one-hybrid experiment, in which PAK6 interacted with AR in the active AR-ARE transcription complex. In both cases, androgen was absolutely required for the interaction. Finally, we demonstrated a dynamic interaction between AR and PAK6 in the immunofluorescence studies. As shown in Fig. 7, PAK6 cotranslocates into nuclei with AR in response to androgen induction. Taken together, the data clearly indicate that the interaction between AR and PAK6 is biologically significant.

To search for the biological consequences of the interaction between PAK6 and AR, we tested whether PAK6 was able to directly alter AR-mediated transcription. In transient transfection experiments, we showed that PAK6 inhibited ligand-dependent, AR-mediated transactivation. Importantly, PAK6 was shown to have no effect on another steroid hormone receptor, the GR, under identical experimental conditions (Fig. 9). These data suggest that the specific repression of AR by PAK6 may be one of the biological consequences of their protein-protein interaction. In the future, it will be interesting to determine whether PAK6 interacts with other nuclear receptors.

Although both physical and functional interactions between PAK6 and AR have been demonstrated, the mechanism by which PAK6 represses AR function is unknown. Given the fact that PAK6 contains a functional carboxyl-terminal kinase domain, we examined whether PAK6 can alter AR activity by the phosphorylation of AR protein. Using recombinant AR proteins, however, we failed to detect phosphorylation of AR by PAK6 (data not shown). Previous studies have suggested that AR phosphorylation states might not significantly enhance AR transcription activity (48, 49). Recent reports have shown that acetylation of AR can enhance AR transactivation (50). Taken together, it appears that the repression of AR-mediated transcription by PAK6 may not be due to an alteration of AR phosphorylation states.

To determine the mechanism by which PAK6 affected transcription, we fused PAK6 cDNAs into a heterologous DNA binding domain (GAL4-DBD) to test intrinsic transcriptional activity. Both full-length and truncated PAK6 containing either amino- or carboxyl-terminal fragments showed no transcriptional activity (data not shown). These data are consistent with the protein structure of PAK6, suggesting that PAK6 may not contain a transcriptional inhibitory domain or may not interfere with the recruitment of other transcription repressors. Because several AR coactivators have been shown to enhance AR-mediated transcription through a specific interaction with the LBD of AR, one possible mechanism for PAK6 repression of AR could be due to direct competition with the binding of the coactivators to AR. Supporting this hypothesis is our preliminary data showing that coexpression of the AR coactivator, ARA70, can partially alleviate the repression of AR by PAK6 (data not shown). Because the mechanism of ARA70 enhancement of AR transactivation is unclear, further analyses with other AR coactivators need to be done to understand the effect of PAK6 on AR.

Although we have determined that PAK6 represses AR activity, the protein sequence and structure of PAK6 suggest that the protein may possess other biological functions comparable with other PAK members. With a human multiple tissue blot, we demonstrated that PAK6 is selectively expressed in testis and prostate. This unique tissue distribution is similar to that of PAK4 but differs from other known PAK members (27). Except for PAK1 and PAK3, which have been shown to be expressed predominantly in human brain, other known PAKs appear to be ubiquitously expressed in human tissues (27, 28). The selective expression of PAK6 in testis and prostate tissues is consistent with the interaction between AR and PAK6 that we have found, which further supports the hypothesis that PAK6 plays a potential biological role in AR function. These results also raise the possibility that each PAK transduces its signal via distinct substrates.

PAK proteins are the direct effectors of the Rho family of GTPases, Rac1 and Cdc42. The GTPases bind to a conserved p21 binding domain, also known as CRIB, thereby stimulating their serine/threonine kinase activities by a mechanism involving autophosphorylation (28, 51). Sequence analysis showed that PAK6 contains CRIB and kinase domains that are homologous to the other known PAKs, especially to PAK4 and PAK5. However, outside of these domains, PAK6 diverges significantly from PAKs 1-3. Based upon low sequence homology with human PAKs 1-3 and the absence of SH3 motifs, it appears that the mechanism of regulation of PAK6 activity may differ from other PAK proteins. Using GST pull-down experiments, PAK6 was shown to interact only with activated Cdc42 through its CRIB domain, but PAK6 kinase activity was not activated upon binding to Cdc42.2 This raises the question as to whether other Rho family members specifically mediate PAK6 activity or whether PAK6 does in fact interact with members of the Rho GTPase family in vivo.

Recent studies have shown that the CRIB domain of PAK1 interacts intramolecularly with its catalytic domain to inhibit the kinase activity by forming a closed and inactive configuration (52). We have shown that the full-length PAK6 has much less kinase activity than the carboxyl-terminal kinase domain, suggesting that a similar auto-inhibition mechanism for kinase activity may also exist in PAK6. Thus far, all known PAKs are found to be localized mainly in the cytoplasm. PAK4 was shown to translocate from the perinuclear cytoplasm to Golgi membranes with activated Cdc42 (27). Our finding that PAK6 cotranslocates into the nucleus with AR suggests that PAK6 may have a novel function unlike other PAKs. It will be interesting to determine whether AR affects PAK6 functions through their interaction. Further characterization of PAK6 interaction with AR should clarify these issues.

In conclusion, this study demonstrates for the first time that a novel PAK protein, PAK6, specifically interacts with AR. Most importantly, the data indicate a mechanism whereby AR pathways can cross-talk with signaling pathways of the Rho-related small GTPase family. Further studies of the regulation of AR-mediated transcription by PAK6 may provide new insight into the biology of normal prostate and prostate cancer.

    ACKNOWLEDGEMENTS

We are especially grateful for the various reagents received from Drs. A. Brinkmann, Richard Pestell, and David Feldman. We thank Dr. Steven Balk for scientific input and Homer Abaya for administrative assistance and help in preparing this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA70297 (to Z. S.) and DK 47636 (to B. L.) and American Cancer Society Grant RPG98213 (to Z. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The 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 GenBankTM/EMBL Data Bank with accession number(s) AF276893.

To whom correspondence should be addressed: Depts. of Surgery and Genetics, R135, Edwards Building, Stanford University School of Medicine, Stanford, CA 94305-5328. Tel.: 650-498-7523; Fax: 650-725-8502; E-mail: zsun@stanford.edu.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010311200

2 Steven P. Balk (Beth Israel Deaconess Medical Center, Boston, MA), personal communication.

    ABBREVIATIONS

The abbreviations used are: AR, androgen receptor; LBD, ligand binding domain; ARE, androgen response element; ARA, androgen receptor-associated; DBD, DNA binding domain; PAK, p21-activated kinase; CRIB, Cdc42/Rac interactive binding; TAD, transcription activation domain; beta -gal, beta -galactosidase; 5'-RACE, rapid amplification of 5' cDNA ends; MMTV, mouse mammary tumor virus; GR, glucocorticoid receptor; GST, glutathione S-transferase; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; DHT, dihydrotestosterone; SD, Sabouraud dextrose.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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