Male Germ Cell–Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

Research Article

Ai-Hong Ma,1 Liang Xia,1 Sonal J. Desai,1 David L. Boucher,1 Yi Guan,1 Hsiu-Ming Shih,3 Xu-Bao Shi,2 Ralph W. deVere White,2 Hong-Wu Chen,1 Cliff G. Tepper,1 and Hsing-Jien Kung1

1Department of Biochemistry and Molecular Medicine and University of California Davis Cancer Center and 2Department of Urology, School of Medicine, University of California at Davis, Sacramento, California and 3Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

Abstract majority of end-stage, hormone-refractory tumors, AR continues to Androgen receptor (AR) is a ligand-induced transcriptional be expressed and seems to be activated under androgen ablation factor, which plays an important role in the normal conditions. Understanding the mechanisms of AR activation and development of prostate as well as in the progression of the genes regulated by AR is of critical importance to appreciate prostate cancer. Numerous coactivators, which associate with the prostate cancer progression process and to identify possible AR and function to remodel chromatin and recruit RNA targets for intervention (7, 8). polymerase II to enhance the transcriptional potential of AR, AR mediates androgen action by being a transcriptional factor have been identified. Among these coactivators, few are that binds specific DNA sequences and consequently recruits RNA protein kinases. In this study, we describe the characteriza- polymerase II and a basal transcriptional complex for efficient tion of a novel protein kinase, male germ cell–associated transcription of cellular genes. The transcriptional activity of AR is kinase (MAK), which serves as a coactivator of AR. We present mediated by coregulators (coactivators and corepressors; refs. 9, 10), evidence, which indicates that (a) MAK physically associates which, in response to binding of androgen to AR and nuclear with AR (MAK and AR are found to be coprecipitated from translocation, are assembled in a dynamic way at androgen cell extracts, colocalized in nucleus, and corecruited to response elements (ARE) along the genome. The best recognized prostate-specific antigen promoter in LNCaP as well as in coactivators are the histone acetylases, such as p300/cyclic AMP– transfected cells); (b) MAK is able to enhance AR trans- responsive element binding protein–binding protein (11, 12) activation potential in an androgen- and kinase-dependent and the p160 steroid receptor coactivator (SRC) family (13–16). manner in several prostate cancer cells and synergize with These coactivators drive transcription by remodeling chromatin via ACTR/steroid receptor coactivator-3 coactivator; (c) small histone acetylation and by recruiting RNA polymerase II complex hairpin RNA (shRNA) knocks down MAK expression resulting to the promoter as we recently shown (17). The molecular basis in the reduction of AR transactivation ability; (d) MAK-shRNA for AR activation by androgen is a conformational change of AR or kinase-dead mutant, when introduced into LNCaP cells, induced by androgen binding, allowing the coactivators to reduces the growth of the cells; and (e) microarray analysis of associate. This process, however, is modulated and, in some cases, LNCaP cells carrying kinase-dead MAK mutant showed a overridden by phosphorylation, which has been postulated to be an significant impediment of AR signaling, indicating that underlying reason for the androgen-independent activation of AR endogenous MAK plays a general role in AR function in by nonsteroidal agonists (18–20). In vitro phosphorylation and prostate cancer cells and likely to be a general coactivator of activation of AR by serine/threonine kinases extracellular signal- AR in prostate tissues. The highly restricted expression of this regulated kinase (ERK; refs. 21, 22), AKT(23), protein kinase A (24), kinase makes it a potentially useful target for intervention of and protein kinase C (25, 26) have been reported. Thus, serine/ androgen independence. (Cancer Res 2006; 66(17): 8439-47) threonine kinases seem to be mediators of AR activation. Androgen treatment not only activates AR via conformational changes but Introduction also causes significant phosphorylation of AR (21, 27, 28), suggesting that kinase cascades are also being activated by Prostate cancer represents the most frequently diagnosed androgen. The in vivo phosphorylation sites of AR have recently malignancy and the second leading cause of cancer deaths among been identified (21, 27–29); none of which, however, are consensus men in the United States (1). Prostate cancer is a hormonally phosphorylation sites of ERK and AKT, raising the possibility that regulated malignancy, and androgen receptor (AR) plays an other novel kinases are involved. Studies to identify protein kinases important role in disease progression (2–4). One of the most associated with or induced by AR resulted in the discovery of troubling aspects of prostate cancer progression is the conversion several novel kinases: ANPK (30), SPAK (31), cyclin-dependent from an androgen-dependent to an androgen-independent state, kinase (CDK) 6 (32), CDK7/CAK (33), and PAK6 (34, 35). These which, at present, defies any effective treatment (5, 6). In the kinases modulate AR activity (ANPK, SPAK, CDK7, and CDK6 activate, whereas PAK6 represses) by direct binding and do not seem to directly phosphorylate AR or affect cellular growth Requests for reprints: Hsing-Jien Kung, Department of Biochemistry and Molecular Medicine and Cancer Center Basic Sciences, School of Medicine, induced by androgen. In this report, we describe a novel kinase University of California at Davis, Room 2400, 4645 Second Avenue, Sacramento, CA male germ cell–associated kinase (MAK), which modulates AR 95817. Phone: 916-734-1538; Fax: 916-734-2589; E-mail: [email protected]. I2006 American Association for Cancer Research. activity and whose kinase activity is important for the growth of doi:10.1158/0008-5472.CAN-06-1636 androgen-dependent LNCaP cells. www.aacrjournals.org 8439 Cancer Res 2006; 66: (17). September 1, 2006

In search of protein kinases transcriptionally induced by Invitrogen) or along with pCMV-AR and pCMV-ACTR into DU145 and PC3 androgen, we previously reported the cloning and identification cells using the Fugene 6 reagent (Roche, Indianapolis, IN). The assay for the of human MAK (36), which shares homology to rat MAK (37, 38). luciferase activity was as described before (42). in vitro MAK was originally recognized as kinase with a highly restricted Coimmunoprecipitation and kinase assay. To study the associations between AR, MAK, and ACTR, various AR deletion mutants or expression primarily in adult testis and at specific stages of WTAR, along with Flag-tagged MAK expression constructs, were spermatogenesis (37, 38). We showed that human MAK is a 623- cotransfected into COS1 or 293Tcells by Fugene 6 reagent. At 48 hours amino acid protein, with an NH2-terminal kinase domain and a after cotransfection, cells were solubilized in 1 mL of modified radio- proline- and glutamine-rich domain and a putative nuclear immunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.8), localization signal (429KEKRKK434). The kinase domain of MAK 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Triton X-100, 10% glycerol] is most homologous to the mitogen-activated protein kinase supplemented with a protease inhibitor mixture (Complete, Roche). (MAPK) family (45% identity, 80% similarity) and to the CDK family Flag-tagged MAK, HA-tagged ACTR, or AR proteins were immunoprecipi- (40% identity, 75% similarity). We showed that MAK is induced by tated from equal amounts of cell lysates by incubation with anti-Flag androgen in a dose-dependent manner with a rapid kinetics. M2-agarose (Sigma-Aldrich), anti-HA (Covance, Berkeley, CA), or anti- Transactivation assay and protein inhibitor study showed that AR441 (NeoMarkers, Fremont, CA). Immunoblot analyses of precipitated proteins were done as described previously (36). The dilution of primary MAK promoter is a direct target of AR and identified two putative antibodies was as follows: AR PG-21 (1:1,000; Upstate, Charlottesville, VA), ARE sites within the promoter of MAK. Jia et al. (39) recently AR441 (1:500; NeoMarkers), AR C-19 (1 Ag/mL; Santa Cruz Biotechnology, extended this analysis and showed by chromatin immunoprecipi- Inc., Santa Cruz, CA), MAK antiserum (1:3,000), Flag M2 (1:1,000; Sigma- tation (ChIP) that AR is recruited to the MAK promoter after Aldrich), and HA (1:1,000; Covance). For the determination of endogenous 5a-dihydrotestosterone (DHT) stimulation. However, it is unclear protein interactions, LNCaP cells were subjected to the same coimmuno- how MAK plays a role in androgen signaling or prostate precipitation assay using AR441 to immunoprecipitate AR protein and carcinogenesis. Given the inducibility of MAK by androgen and blotting with MAK antiserum as described above. In vitro kinase assay was its putative nuclear location, we speculated that MAK might play a done as described previously (36). role as an effector or a feedback kinase to androgen/AR signaling. Immunofluorescence. Immunofluorescence staining was done as In this study, we present several lines of evidence showing that described previously (36, 40). Anti-AR polyclonal antibody N-20 (Santa Cruz Biotechnology) and anti-HA monoclonal antibody (Covance) were MAK is a coactivator of AR. We show that MAK is associated with used to examine endogenous AR and ectopically transfected HA-MAK, the AR transcriptional complex and able to enhance AR trans- respectively, followed by FITC-conjugated anti-mouse immunoglobulin G activation in an androgen- and kinase-dependent manner. (IgG) and Texas red–conjugated anti-rabbit IgG (Molecular Probes/ Significantly, knocking down MAK expression by small hairpin Invitrogen, Eugene, OR) staining. Nuclei were visualized by 4¶,6-diami- RNA (shRNA) or MAK activity with a kinase-dead mutant dino-2-phenylindole staining (10 Ag/mL). The cells were then examined for diminished the expression of prostate-specific antigen (PSA) as fluorescence staining under the Olympus BX61 fluorescence microscope well as other AR-responsive genes and resulted in the inhibition of (Olympus America, Inc., Melville, NY). androgen-induced growth. To our knowledge, MAK is the first ChIP assays. ChIP assays were done as described previously (17). protein kinase shown to be a direct transcriptional target of AR Chromatin fragments were immunoprecipitated with specific antibodies j and serves as a coactivator of AR in propagating the androgen overnight at 4 C. For a 5-mL diluted chromatin solution, the following amount of antibodies were used: 5 Ag of anti-AR PG-21, 30 Ag of anti-Pol II signal. (Santa Cruz Biotechnology), 30 Ag of anti-ACTR (Santa Cruz), and 50 ALof anti-MAK rabbit antiserum. Immunocomplexes were recovered and eluted. After reverse cross-linking at 65jC overnight, the DNA fragments were Materials and Methods purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and Plasmids and reagents. DHTwas purchased from Sigma-Aldrich eluted with 100 AL of TE1/10 (pH 8.0). PCR was done using 5 AL of purified (St. Louis, MO). Hemagglutinin (HA) or Flag epitope-tagged MAK wild- DNA for 28 cycles. The gel images of ChIP results are representative of type (WT) and the kinase-dead mutant KR were constructed as described independent experiments done at least thrice. before (36). The KR mutant carries a mutation at Lys33 in the ATP-binding RNA interference recombinant adenoviruses. The adenoviral RNA pocket, which renders the molecule catalytically inactive (36). AR full-length interference (RNAi) vector was described previously (43). Oligodeoxynu- and deletion mutant plasmids AR-N, AR-ND, AR-DL, and AR-L were cleotides encoding shRNA, which target MAK gene, were inserted described previously (40, 41). pCMV-ACTR was provided by Dr. Hong-Wu downstream of the human H1 gene promoter. Three regions of MAK were Chen (17). The full-length human MAK-related kinase (MRK) and targeted by shRNA: MAKi-1, 5¶-GTTGTTCCCTGAATCAGTCA-3¶; MAKi-2, ¶ ¶ ¶ MAK/MRK overlapping kinase (MOK) cDNAs tagged with HA at NH2 5 -CTCTTATTCCCAATGCCAGT-3 ; and MAKi-3, 5 -GCTATTCAGCTCAT- terminus were subcloned into the mammalian expression vector pcDNA3.1 GACCGA-3¶. The resulting pShuttle-RNAi constructs were used to generate (Invitrogen, Carlsbad, CA). The MAK rabbit polyclonal antibody targeting a recombinant adenoviruses following the protocols described previously 19-amino acid polypeptide (amino acid 577-595) was generated by Genemed (44). Viral particles were purified by centrifugation in a CsCl step gradient. Synthesis, Inc. (South San Francisco, CA). Viral titers were determined by the anti-hexon antibody-based Adeno-X Cell cultures. Prostate carcinoma cell lines LNCaP, CWR22Rv1, DU145, Rapid Titer kit (BD Biosciences, San Jose, CA). The control adenovirus and PC3 were obtained from the American Type Culture Collection green fluorescent protein (GFP)-shRNA was described previously (43). The (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal targeting sequence of GFP shRNA is 5¶-GAACTTCAGGGTCAGCTTG-3¶. bovine serum (FBS) from Life Technologies/Invitrogen (Rockville, MD). LNCaP cells were infected with MAK and GFP shRNA adenoviruses at COS1, 293T, and 293 cells were cultured in DMEM with 4.5 g/L glucose and multiplicities of infection (MOI) of 5, 10, and 20, and the cells were analyzed 4 mmol/L L-glutamine supplemented with 10% FBS. for the MAK gene expression. Dual-luciferase assay. Cells were seeded in 24-well plates at 1 Â 105 per Reverse transcription-PCR assay. Reverse transcription-PCR (RT-PCR) well (LNCaP and CWR22Rv1) or 5 Â 104 per well (PC3 and DU145) and was done as described previously (36). Primer sequences used to amplify incubated at 37jC with 5% CO2 for 24 hours. The PSA-Luc reporter plasmid MAK fragment are listed as follow: 5¶-TCCAAGATGAACCGATACACAACC- carrying 6 kb of the PSA promoter sequence (42) and the pRL-SV40 Renilla 3¶ (sense) and 5¶-CAGCAATTTTCACAAGCTCTGGAC-3¶ (antisense). PCRs luciferase plasmid (Promega, Madison, WI) were transfected into LNCaP were conducted at 95jC Â 1 minute, (95jC Â 30 seconds, 60jC Â 30 and CWR22Rv1 cells using the Lipofectin reagent (Life Technologies/ seconds, 72jC Â 1 minute) Â 35 cycles, 72jC Â 5 minutes. RT-PCR was

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. MAK Is a Coactivator of Androgen Receptor also done for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) LNCaP cells and monitored the subcellular localization of MAK as a control to check the amount and integrity of the total RNA samples. and AR. As shown in Fig. 1D, MAK is primarily localized in the ¶ GAPDH primer sequences are as follows: 5 -ACCACAGTCCATGCCATCAC- nucleus of LNCaP. Before DHTtreatment, diffusive stain of MAK in ¶ ¶ ¶ 3 (sense) and 5 -TCCACCACCCTGTTGCTGTA-3 (antisense). PCR ampli- the cytoplasm could be detected. After DHTtreatment, the images fication variables for GAPDH are the same as those for MAK, except that of both MAK and endogenous AR seem to be more ‘‘compacted’’ 25 cycles of amplification were done to ensure that the reaction remained in the log phase. and colocalized in the nucleus. We have extended the localization Selection of stable cell lines and cell proliferation assay. LNCaP cells studies of MAK to other prostate cancer cell lines, including were transfected with MAK WT, kinase-dead mutant (KR), and empty DU145, PC3, and CWR22Rv1. In every case, nuclear localization vector plasmids by Lipofectin reagent and selected with 0.8 mg/mL G418 was detected (data not shown). (Life Technologies/Invitrogen) for 2 weeks. The resistant clones were Having shown coprecipitation and colocalization of MAK and selected and expanded, and the MAK expression was confirmed by AR, we were interested in studying the functional outcome of their immunoblotting. CellTiter 96 nonradioactive cell proliferation assay interaction. On DHTtreatment, AR is recruited to the PSA (Promega) was used to analyze LNCaP/MAK cells. Specifically, cells were promoter, where it assembles an active transcriptional complex cultured for 3 days in 10% charcoal-stripped FBS medium and then plated leading to the loading of RNA polymerase II (17). ChIP experiments into 96-well plates at 5,000 per well in 100 AL medium. Cells were treated were carried out to examine whether MAK is recruited to the same with 1 nmol/L DHTand refed with fresh medium containing DHTevery D 2 days. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) site. As shown in Fig. 1 , MAK and AR are corecruited to the ARE cell proliferation assay was done according to the manufacturer’s site of the PSA promoter, suggestive of a functional role of MAK in instruction (Promega). AR transactivation. Likewise, p160SRC3/ACTR coactivator (14, 15) Microarray analysis. LNCaP/vector and LNCaP/MAK-KR cells were and RNA Pol II were also recruited to the same site after DHT grown to 60% to 70% confluence, switched to charcoal-stripped medium for treatment. This suggests a role for MAK as a cofactor participating 3 days, and then stimulated with 10 nmol/L DHTfor 6 hours or treated in the assembly of the AR transcription complex. with ethanol vehicle control for the same duration. Total RNA was MAK enhances the transactivation potential of AR. We next extracted using Trizol reagent (Invitrogen) and submitted to the University asked whether MAK functionally activates AR activity. A HA- of California Davis Cancer Center Gene Expression Resource (Sacramento, tagged MAK expression vector and a PSA promoter-luciferase CA). Microarray analysis was done using Affymetrix Human Genome U133A reporter were transiently cotransfected into CWR22Rv1 and (HG-U133A) GeneChip arrays (Affymetrix, South San Francisco, CA), which permit expression analysis of the entire Genbank RefSeq database. Probe LNCaP cells, and the reporter activity was assessed before and A top preparation, hybridization, and signal detection were done according to after DHTtreatment. Figure 2 ( ) shows that MAK can enhance standard protocol as described previously (45, 46). Array scanning and androgen-dependent, endogenous AR-mediated transactivation generation of raw signal data files were done with GeneChip Operating and that the kinase activity seems important, as the MAK-KR Software (Affymetrix). Subsequent data analysis was done using DNA-Chip mutant, which lacks the kinase activity, has much less potency. To Analyzer (dChip) software (47). Comparison analysis of the expression data generalize the finding that MAK is a coactivator of AR, we from the LNCaP/MAK-KR and LNCaP/vector cell lines was conducted to cotransfected MAK and WTAR into DU145 and PC3 cell lines. The evaluate the effects of MAK-KR on androgen-regulated gene expression. results (Fig. 2A, bottom) confirmed the ability of MAK to enhance Statistically significant differential expression changes were selected based the transactivation ability of AR. These experiments also showed on attaining PsofV0.05. that the enhancement effect can be seen with WTAR and, thus, is not a peculiarity of the mutant ARs associated with LNCaP and Results CWR22Rv1 cell lines. MAK associates with AR. We showed previously that MAK is The ChIP experiment described above suggested that MAK is transcriptionally activated by androgen. As such, we hypothesize recruited to the AR transcriptional complex, which occupies the that MAK is either an effector of AR signaling or a modulator of AR proximal ARE sequences in the PSA promoter. We also observed activity (36). To test the latter possibility, we studied the physical that coactivator ACTR is recruited, although it is not clear whether interaction between MAK and AR. LNCaP cells were treated with MAK and SRC3/ACTR are in the same complex. To test whether DHTto induce the expression of MAK. TheAR was then immuno- MAK and ACTR functionally interact, we conducted transactiva- precipitated and immunoblotted with MAK antibody. As shown in tion assay in PC3 cells using PSA-Luc as a reporter. WTAR was Fig. 1A, MAK protein is coprecipitated with AR in the presence of cotransfected with MAK and ACTR alone or in combination, and DHT. To extend this finding and to map the interacting domain(s), the cells were treated with DHT. Cells transfected with AR alone a series of AR deletion mutants were constructed (Fig. 1B). and treated with DHTwere used as a control. As shown in Fig. 2 B, They include the NH2-terminal domain (N), NH2-terminal domain in the presence of androgen, MAK and ACTR respectively plus DNA-binding/hinge domain (ND), ligand-binding domain (L), enhanced AR transactivation of PSA-Luc by 1.5- and 2.0-fold. and DNA-binding/hinge domain plus ligand-binding domain (DL). Together, they enhanced AR activity by 5-fold, indicating a The AR and Flag-tagged MAK plasmids were cotransfected into potential functional interaction between these two coactivators. COS1 cells and immunoprecipitated by anti-Flag antibody followed To further test whether MAK, AR, and ACTR form a complex, we by immunoblotting with anti-AR. The results showed that AR ND did coimmunoprecipitation experiment. 293Tcells were cotrans- and DL, but not N or L, coprecipitated with MAK, suggesting that fected with MAK, ACTR, and AR followed by immunoprecipitation the DNA-binding/hinge domain is responsible for the interaction with one antibody and blotted with the other two in all three with MAK (Fig. 1C). permutations. The results in Fig. 2D clearly showed that MAK, MAK is colocalized and corecruited with AR to PSA ACTR, and AR form a complex. promoter. If MAK and AR physically associate with each other, To define the domain(s) of AR, which functionally interacts with we anticipate colocalization of the molecules in androgen-treated MAK, we used the AR truncation mutants ND and DL in the cells. For the lack of an avid, immunohistochemical quality MAK transactivation assay. These constructs were cotransfected into PC3 antibody, we resorted to transfection of HA-tagged MAK into cells with MAK and PSA-Luc reporter gene in the presence or www.aacrjournals.org 8441 Cancer Res 2006; 66: (17). September 1, 2006

Figure 1. MAK associates with AR. A, association of endogenous AR and MAK in LNCaP cells. LNCaP cells were cultured in charcoal/dextran-stripped FBSfor 3 days before treatment with 10 nmol/L DHT for 24 hours. Cell lysates were collected and immunoprecipitated with anti-AR (AR441) followed by immunoblotting with anti-MAK. Bottom, expression of AR from 1 of 20 of the total immunoprecipitated cell lysates used for coimmunoprecipitation experiments. B, schematic presentation of WT and deletion mutants of AR. NTD, NH2-terminal domain; DBD, DNA-binding/hinge domain; LBD, ligand-binding domain of the AR. C, MAK interacts with DNA-binding/hinge region of AR. COS1 cells were transiently transfected with Flag-tagged MAK and full-length or various deletion mutants of AR expression constructs. Top, after 48 hours, cells were lysed and subjected to immunoprecipitation (IP) followed by immunoblotting with AR antibodies; bottom, expression levels of MAK and AR from the transfected cell lysate. Left and right, anti-Flag antibody was used to detect MAK. Anti-AR PG-21 and C-19 antibodies, which specifically recognize the NH2-terminal and COOH-terminal domains of AR, were used. Asterisks, full-length and deleted forms of AR proteins. D, MAK is colocalized and corecruited with AR to the PSA promoter in LNCaP cells. LNCaP cells were transiently transfected with HA-MAK expression plasmid. Cells were treated with or without DHT for 24 hours and then subjected to immunostaining analyses with anti-HA monoclonal antibody and anti-AR rabbit antibody N-20. The secondary antibodies for these studies were FITC-conjugated anti-mouse IgG and Texas red–conjugated anti-rabbit IgG. After immunostaining and washing procedures, the samples were analyzed by immunofluorescence microscopy. For the ChIP assay, LNCaP cells were androgen deprived for 3 days followed by treatment with 10 nmol/L DHT. Cells were fixed with formaldehyde and sonicated, and protein-chromatin complexes were immunoprecipitated with 5 Ag of anti-AR (PG-21), 50 AL of anti-MAK, 30 Ag of anti-Pol II, and 30 Ag of anti-ACTR antibodies. DNA fragments were released by reverse cross-linking and then purified, and 10% of ChIP product or 1% of input was used in the PCR. absence of DHT. Transfection with AR alone was used as a control. ectopic MAK expression in LNCaP, CWR22Rv1, and PC3 prostate AsshowninFig.2C, the presence of MAK enhanced the cancer cells. Figure 3A shows an immunoblot using HA antibody to transactivation activity of the ND but not the DL domains, detect MAK at 48 hours after transfection. The suppression of suggesting that the enhancing effect of MAK requires its interaction MAK expression was nearly complete for MAK-shRNA1 and MAK- with coactivator complex residing at the NH2-terminal domain of shRNA2 and close to 90% for shRNA3 compared with a control AR. The data are consistent with the report that ATCR and p160SRC transfected with a scrambled sequence shRNA expression con- family of coactivators bind NH2-terminal domain of AR (48). The struct (scRNA). To define the specificity of the shRNA, we results also showed that ligand-bound AR is the target for MAK. cotransfected the shRNA constructs with MRK and MOK, two MAK-shRNA suppresses AR transactivation. As an additional related members of the MAK kinase family (49). We found that test for the role of MAK in AR transactivation, we developed MAK- shRNA1 to be most specific, only knocking down MAK expression. shRNAs. Of the six sets we constructed, three of them (shRNA1, This is followed by shRNA3, with shRNA2 being the least specific, shRNA2, and shRNA3) showed significant efficacies in silencing which also reduced the expression of MRK.

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We then proceeded to test the effect of knocking down MAK-defective LNCaP exhibited reduced growth and endogenous MAK in the AR transactivation potential. Taking impeded androgen response. The adenovirus shRNA experi- advantage of the constitutive expression of MAK in PC3 cells (36), ments described above provide preliminary indication that MAK we cotransfected the shRNA constructs, a WTAR cDNA expression expression is involved in androgen-dependent growth of LNCaP. construct, and PSA-Luc into PC3 cells in the presence of DHT Due to the transient nature of the experiment, it was difficult to (1 nmol/L). The luciferase activities after 48 hours are shown in conduct detailed molecular analysis. To this end, we generated Fig. 3B. Knockdown of the endogenous MAK by any of the shRNA LNCaP clones ectopically expressing the kinase-dead MAK-KR as constructs significantly compromised the ability of AR to trans- an alternative means to stably knock down the MAK activity. activate, suggesting that MAK is an intrinsic cofactor of AR LNCaP cells ectopically expressing WTMAK were also obtained to activation. serve as a positive control. Although we had no problem in MAK-shRNA adenovirus infection inhibits the growth of attaining LNCaP-MAK clones with a high level of MAK expression, LNCaP cells. If MAK is a coactivator of AR, we surmised knocking we experienced difficulty in isolating MAK-KR clones perhaps due down MAK should compromise the androgen response of LNCaP, to the deleterious effect of MAK knockdown in LNCaP cells. We, thereby reducing the androgen-dependent growth. Because of the nevertheless, were able to isolate one LNCaP/MAK-KR clone with a high specificity of shRNA1 and shRNA3, we structured the moderate level of MAK-KR expression (Fig. 5A, inset). In vitro constructs into an adenovirus vector to generate ad-MAKi-1 kinase assay confirmed the kinase-negative nature of MAK-KR (Mi1) and ad-MAKi-3 (Mi3) vectors. An adenovirus carrying shRNA (Fig. 5A, inset). When the growth curves are compared with targeting GFP (Gi) was used as a control. To show the efficacies of LNCaP/vector cells, LNCaP/MAK-WTexhibited a similar, if not the viruses, LNCaP cells were infected with either Mi or Gi viruses slightly increased, growth rate that is dependent on androgen. The and the MAK expression was determined by RT-PCR. The Mi1 and LNCaP/MAK-KR clone, on the other hand, grew at a much reduced Mi3 viruses effectively reduced the MAK expression in infected rate (Fig. 5A). The availability of LNCaP/MAK-KR subline also cells, whereas Gi virus had no effect, confirming the specificity of afforded us an opportunity to study the influence of endogenous the approach (Fig. 4A). Importantly, both Mi1 and Mi3 viruses MAK on global gene expression using Affymetrix HG-U133A significantly slowed down the growth of LNCaP, whereas the Gi GeneChip oligonucleotide arrays. The microarray analysis yielded virus-infected LNCaP exhibited a similar growth rate as uninfected an expression signature that was indeed consistent with the cells (Fig. 4B). These data together suggest that knockdown of MAK observed phenotype for MAK knockdown, thereby imparting expression has a significant effect on the androgen-dependent credibility to our data. A representative subset of the results is growth of LNCaP cells, implicating MAK in AR signaling. presented in Fig. 5B and Table 1. Consistent with our findings that

Figure 2. MAK enhances AR-mediated gene activation. A, androgen-dependent activation of AR activity by MAK in prostate cancer cells. PSA-Luc was transfected with vector, MAK-WT, or MAK-KR into LNCaP, CWR22Rv1, PC3, and DU145 cells. Transfected cells were incubated in charcoal-depleted FBSmedium overnig ht and then washed and treated with 1 nmol/L DHT for another 24 hours. Luciferase activity with or without 1 nmol/L DHT treatment was measured. Columns, mean; bars, SE. White columns, luciferase activities without DHT treatment; black columns, luciferase activities in the presence of DHT. Asterisks, statistically significance based on attaining Ps of < 0.05. B, MAK and ACTR/SRC3 cooperate to enhance AR transactivation. AR and PSA-Luc were cotransfected with MAK and ACTR/SRC3 alone or in combination into PC3 cells. After 24 hours, the cells were treated with 0.1 or 1.0 nmol/L DHT. Luciferase reporter readings were recorded. Data are expressed as fold induction over the cells transfected with AR protein alone. C, MAK functionally interacts with AR. PC3 cells were cotransfected with WT MAK, PSA-Luc, and AR full-length or mutant plasmids as indicated. Cells were treated with 1 nmol/L DHT for 24 hours, and the dual-luciferase activities were measured. White columns, luciferase activities in the absence of MAK; black columns, luciferase activities in the presence of MAK. D, MAK, ACTR, and AR form a complex. 293T cells were transiently transfected with Flag-tagged MAK, HA-tagged ACTR, and AR as indicated. After 48 hours, cells were lysed and subjected to immunoprecipitation with one antibody followed by immunoblotting with the other two antibodies in all three permutations. Anti-Flag, anti-HA, and anti-AR441 antibodies were used to detect MAK, ACTR, and AR, respectively. www.aacrjournals.org 8443 Cancer Res 2006; 66: (17). September 1, 2006

MCM7)andDNAmetabolism(ribonucleotide reductase and thymidylate synthetase) were down-regulated. The data also implicate a role for MAK in survival, as LNCaP/MAK-KR has elevated expression of several proapoptotic regulatory molecules (PAWR and BAD) and execution phase constituents (APAF1)as well as concurrent decreased expression of prosurvival genes, such as survivin.

Discussion In this article, we report the characterization of an androgen- induced and male-specific protein kinase, MAK. To our knowledge, this is one of the only two androgen-induced kinases (31, 36) that are associated with AR and the only one that is the direct target of AR. We showed that MAK is a downstream modulator of AR signaling: inhibition of its expression or kinase activity leads to significant attenuation of androgen-induced growth and the

Figure 3. shRNA knockdown of MAK expression and activity. A, knockdown expression of androgen-responsive genes in LNCaP cells. This of MAK protein expression by MAK-shRNA. MAK-shRNAs were cotransfected conclusion was drawn not only by a functional assay on PSA with HA-MAK, HA-MRK, or HA-MOK into 293 cells. scRNA was used as a promoter but also based on results of global gene expression control. Immunoblot analysis was used to detect the protein levels. Anti-HA was used to detect MAK, MRK, and MOK, and anti-actin was used to detect actin profiling. We also showed that MAK physically associates with AR expression as an internal control. B, suppression of AR activity by MAK-shRNA. and enhances the transcriptional potential of AR. Although we do PC3 cells were cotransfected with WT AR, PSA-Luc, and MAK-shRNA. Cells not know whether AR and MAK directly contact each other, our were treated with 1 nmol/L DHT for 24 hours, and the luciferase activity was measured. scRNA was similarly cotransfected with AR and PSA-Luc and used evidence suggests a strong interaction between them. Their as a control. interactions were shown by its coprecipitation, colocalization, and corecruitment to the PSA promoter. As such, MAK behaves like a coactivator of AR. Given the lack of understanding of how MAK-shRNA decreased AR transactivation in reporter assays, a androgen transmits signals to induce cell growth or differentia- diminished AR response was clearly evident as indicated by the tion, the discovery of a protein kinase, which is a direct target decreased expression of classically androgen-induced genes (e.g., gene of AR and which modulates the AR activity, is thus PSA/KLK3, NKX3.1, FKBP5, and VEGF) and restored expression of significant. Furthermore, the male tissue-selective expression of androgen-repressed genes (clusterin/TRPM-2, CHES1, and T-cell MAK makes it a potential target for therapeutic attenuation of AR receptor c transcript). Growth inhibition was indicated by an activities. Cip1 induction of p53 target gene p21 and decreased expression of The coactivator role of MAK was defined first by transactivation genes driving progression through various phases of the cell cycle, assay on PSA promoter in a variety of prostate cancer cell lines, such as CDC2 and CDC6. Notably, several genes encoding critical indicating the generality of the phenomenon. It is further enforced components of the DNA replication machinery (MCM2/MCM4/ by the expression profile analysis using a kinase-dead MAK

Figure 4. Biological effects of MAK knockdown. A, MAK gene expression inhibited by adenovirus carrying shRNA in LNCaP cells. Cells were hormone deprived for 3 days before infection with adeno-MAK-RNAi adenovirus Mi1, Mi3, or Gi at an estimated MOI of 20. Uninfected (U) LNCaP was used as a control. Different days after infection, cells were harvested and RNA samples were isolated for RT-PCR analysis of MAK gene expression. GAPDH was used as an internal control. Cells were treated with 1 nmol/L DHT 1 day after infection and refed with fresh medium containing DHT every 2 days. Lane 1, molecular weight marker. B, growth inhibition of LNCaP cells by adenovirus carrying shRNA. LNCaP cells were treated as described in (A), and the cell proliferation was assessed by MTT assay as described in Materials and Methods.

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Figure 5. Growth and androgen responses of LNCaP harboring kinase-dead mutant of MAK. A, growth kinetics of LNCaP cells stably transfected with LNCaP/MAK-WT, LNCaP/MAK-KR, and LNCaP/vector. Experiments were carried out in the presence of 1 nmol/L DHT. The absorbance reading is based on MTT assay. Insets, expression of MAK-WT and KR proteins as well as tubulin in the respective LNCaP sublines as analyzed by immunoblots. In vitro kinase activities of immunoprecipitated MAK proteins were measured as described previously (36). Same amount of MAK-WT and KR proteins was used for kinase reactions. B, microarray Affymetrix HG-U133A oligo microarray analysis of LNCaP/MAK-KR RNA samples. Samples were isolated from cells 6 hours after DHT treatment. Expression profiles were compared with those without DHT treatment. Comparison analysis was done with dChip software. LNCaP/vector samples served as controls. Right, representative androgen-inducible genes whose expressions are attenuated in LNCaP/MAK-KR compared with the LNCaP/vector.

mutant, which blunts the expression of a large number, but not all, terminal domain of AR. Interestingly, thus far, none of these of androgen-induced genes. We interpret this to mean that MAK is kinases have been shown to directly phosphorylate AR, presumably part of a general transcriptional complex of AR, which recognizes a the kinase activity, if required, is directed toward cofactors or other large subset of the ARE-containing promoters. One consequence of chromatin proteins. the significant attenuation of androgen responsiveness in LNCaP MAK shares some similarities with these kinases but also has would be the attenuation of the growth responses. This is indeed distinct features. First, it seems to be the only one shown to be a what was observed. This conclusion was further confirmed by the direct transcriptional target of AR. We previously showed that adenovirus-shRNA experiment. We selected shRNA species, which androgen augments the transcription of MAK in the presence of target only MAK but not its related kinases MOK and MRK (49). cycloheximide and indicated the presence of three AREs in MAK The growth of LNCaP in the presence of at least two different promoter, which respond to androgen stimulation (36). In a shRNAs against MAK is significantly slowed down. This suggests recent publication, AR was found to be recruited to the major that MAK is an important component in the AR complex in LNCaP ARE site of the MAK promoter we defined (39). By contrast, SPAK cells. MAK endogenous protein expression level is generally low as was shown to be transcriptionally activated by androgen but assessed by the presently available antibody, and reliable likely to be a secondary target (31). Second, the highly restricted quantitation comes primarily from RT-PCR (36). It is interesting tissue expression only in testis and prostate among the tissues that overexpression of MAK in LNCaP cells only marginally tested is rivaled only by SPAK (31) and ANPK (30). Third, increased the growth rate and did not overcome the androgen transactivation potential of MAK largely depends on its kinase dependence of the cells. This is, however, not entirely surprising, as activity, whereas the kinase activity of CDK6 is apparently not most of the coactivators, while significantly enhancing the action required for its augmentation of AR activity (32). It is noteworthy, of AR, are not sufficient to cause androgen-independent growth of however, that the kinase-dead mutant of MAK is not completely the cells. Thus, as a modulator of AR, MAK is required for full null for AR activation, suggesting some kinase-independent androgen response but in itself is not sufficient for causing function. Finally, direct in vitro or in vivo phosphorylation assays androgen independence. and transactivation assays with AR phosphorylation site mutants Numerous coactivators and corepressors for AR have been (29) failed to show AR as a direct phosphorylation target of MAK identified. Very few are protein kinases. Before this study, five nor do we see the effect of MAK on the stability of AR and AR kinases, SPAK (31), ANPK (30), PAK6 (34, 35), CDK6 (32), and CDK7 dimerization.4 This, together with the observation that MAK and (TFIIH; ref. 33) were identified as cofactors, which bind AR. Their AR are primarily localized in the nucleus and that they are both modes of action differ significantly. SPAK, ANPK, CDK6, and CDK7 recruited to the PSA promoter, suggests that MAK first assembles are coactivators of AR, whereas PAK6 behaves like a corepressor. The kinase activity of SPAK and ANPK is important for its activation, whereas that of CDK6 is not. ANPK interacts with the 4 DNA-binding/hinge region of AR, whereas CDK7 binds the NH2- Unpublished data. www.aacrjournals.org 8445 Cancer Res 2006; 66: (17). September 1, 2006

Table 1. Genes differentially expressed by MAK-KR overexpression in LNCaP cells

Biological process Gene symbol MAK-KR vs vector (fold change) Gene title

Androgen regulated (repressed) TRG@ 1.89 T-cell receptor g locus SMARCD3 1.82 SWI/SNF complex 60-kDa subunit C CHES1 1.81 Checkpoint suppressor 1 CLU 3.39 Clusterin (TRPM-2) Antiangiogenesis THBS1 5.99 Thrombospondin 1 Proapoptosis PAWR 2.04 PRKC, apoptosis, WT1, regulator BAD 1.88 BCL2 antagonist of cell death PDCD10 1.72 Programmed cell death 10 APLP1 2.24 Amyloid h (A4) precursor-like protein 1 APAF1 1.59 Apoptotic protease activating factor Cell cycle arrest/p53 target CDKN1A 2.15 p21 Cip1 WIG1 1.86 p53 target zinc finger protein CUL4A 1.91 Cullin 4A Tumor suppressor VHL 2.58 von Hippel-Lindau syndrome TUSC3 1.60 Prostate cancer tumor suppressor candidate 3 Hormone activity RLN2 3.76 Relaxin 2 (H2) Inflammatory response IL1RN 2.80 Interleukin-1 receptor antagonist Transcriptional regulation, corepressor SIN3B 1.63 SIN3 homologue B (yeast) Androgen regulated (induced) KLK3 À2.35 PSA/kallikrein 3 SPDEF À2.10 Prostate-derived Ets factor FKBP5 À2.00 FK506-binding protein 5 KLK2 À1.85 Kallikrein 2, prostatic NKX3-1 À1.60 NK3 transcription factor related, locus 1 Angiogenesis, androgen regulated (induced) VEGF À1.53 Vascular endothelial growth factor Antiapoptosis BIRC5 À1.96 Baculoviral IAP repeat-containing 5 (survivin) PRDX4 À1.55 Peroxiredoxin 4 Cell cycle progression CDC6 À2.38 CDC6 cell division cycle 6 homologue

CDC2 À1.95 Cell division cycle 2, G1 to S and G2 to M PRC1 À1.95 Protein regulator of cytokinesis 1 CKS2 À1.74 CDC28 protein kinase regulatory subunit 2 SKP2 À1.54 S-phase kinase-associated protein 2 (p45) DNA replication MCM2 À1.82 MCM2 minichromosome maintenance deficient 2 MCM4 À1.81 MCM4 minichromosome maintenance deficient 4 MCM7 À1.59 MCM7 minichromosome maintenance deficient 7 Pfs2 À1.57 DNA replication complex GINS protein PSF2 DNA metabolism RRM2 À2.33 Ribonucleotide reductase M2 polypeptide TYMS À1.55 Thymidylate synthetase DHFR À1.54 Dihydrofolate reductase TK1 À1.53 Thymidine kinase 1, soluble Proliferation PCNA À1.62 Proliferating cell nuclear antigen Chromatin remodeling RCBTB1 À1.68 RCC1 and BTB domain containing protein 1 Oncogene MYC À1.52 c-myc

into an AR complex and is then carried to the chromosomal site has not yielded positive results.4 An intriguing recent report by Fu where MAK phosphorylates other coactivators and chromatin et al. (50) revealed that ICK (also called MRK), a related family proteins. The conclusive demonstration of this model awaits the member of MAK, is a substrate of CDK7. Although we have not identification of physiologic relevant substrate of MAK in the tested whether CDK7 is an activator of MAK, the nuclear future. In this regard, it is interesting that MAK and ACTR seem localization of MAK and the association of both CDK7 and to be in proximity and synergize with each other in the MAK with AR make it an appealing hypothesis that MAK is transactivation of AR. In addition, MAK and ACTR seem to be activated by CDK7 in situ. assembled in the same AR complex based on coimmunoprecipi- Being a protein kinase transcriptionally regulated by AR, MAK tations. is likely to be a feedback modulator of AR or a downstream The kinase domain of MAK contains TDY, a phosphorylation effector for androgen signaling or both. Our data presented in this motif recognized by MAPK kinase. We showed that it is active, article showed that MAK is a part of the AR transcriptional capable of autophosphorylation and phosphorylation of exoge- complex and a positive modulator of the transactivation activity nous substrate. However, the unstimulated MAK activity is of AR. We think likely that MAK also serves as a downstream relatively low. Our effort to show its activation and phosphor- effector of AR by phosphorylating critical substrates involved in ylation by MAPK/ERK kinase, MAPK kinase 3, and MAPK kinase 6 proliferation and differentiation of prostate cells. This awaits the

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. MAK Is a Coactivator of Androgen Receptor identification of cellular substrates of MAK. The possibility that Grant support: NIH grants RO1CA114575-01 and RO1DK52659 and Department of Defense grant W81XWH0410835 (H-J. Kung). The University of California Davis Cancer MAK may require an additional agonist to fully activate its kinase Center Gene Expression Resource is supported by National Cancer Institute Cancer activity suggests that MAK may be an integrator of multiple signal Center grant P30 CA93373-01 (R.W. deVere White). pathways. 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. Acknowledgments We thank Dr. June Zou and Alina Rabinovich for technical help, Dr. M.J. Weber for providing phosphor-mutant AR plasmids, and Dr. T. Sturgill for communicating data Received 5/4/2006; revised 6/19/2006; accepted 7/7/2006. before publication.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Male Germ Cell−Associated Kinase, a Male-Specific Kinase Regulated by Androgen, Is a Coactivator of Androgen Receptor in Prostate Cancer Cells

Ai-Hong Ma, Liang Xia, Sonal J. Desai, et al.

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