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[Cancer Research 66, 8439-8447, September 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

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

Ai-Hong Ma1, Liang Xia1, Sonal J. Desai1, David L. Boucher1, Yi Guan1, Hsiu-Ming Shih3, Xu-Bao Shi2, Ralph W. deVere White2, Hong-Wu Chen1, Cliff G. Tepper1 and Hsing-Jien Kung1

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

Requests for reprints: Hsing-Jien Kung, Department of Biochemistry and Molecular Medicine and Cancer Center Basic Sciences, School of Medicine, University of California at Davis, Room 2400, 4645 Second Avenue, Sacramento, CA 95817. Phone: 916-734-1538; Fax: 916-734-2589; E-mail: hkung{at}ucdavis.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen receptor (AR) is a ligand-induced transcriptional factor, which plays an important role in the normal development of prostate as well as in the progression of prostate cancer. Numerous coactivators, which associate with AR and function to remodel chromatin and recruit RNA polymerase II to enhance the transcriptional potential of AR, have been identified. Among these coactivators, few are protein kinases. In this study, we describe the characterization of a novel protein kinase, male germ cell–associated kinase (MAK), which serves as a coactivator of AR. We present evidence, which indicates that (a) MAK physically associates with AR (MAK and AR are found to be coprecipitated from cell extracts, colocalized in nucleus, and corecruited to prostate-specific antigen promoter in LNCaP as well as in transfected cells); (b) MAK is able to enhance AR transactivation potential in an androgen- and kinase-dependent manner in several prostate cancer cells and synergize with ACTR/steroid receptor coactivator-3 coactivator; (c) small hairpin RNA (shRNA) knocks down MAK expression resulting in the reduction of AR transactivation ability; (d) MAK-shRNA or kinase-dead mutant, when introduced into LNCaP cells, reduces the growth of the cells; and (e) microarray analysis of LNCaP cells carrying kinase-dead MAK mutant showed a significant impediment of AR signaling, indicating that endogenous MAK plays a general role in AR function in prostate cancer cells and likely to be a general coactivator of AR in prostate tissues. The highly restricted expression of this kinase makes it a potentially useful target for intervention of androgen independence. (Cancer Res 2006; 66(17): 8439-47)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer represents the most frequently diagnosed malignancy and the second leading cause of cancer deaths among men in the United States (1). Prostate cancer is a hormonally regulated malignancy, and androgen receptor (AR) plays an important role in disease progression (2–4). One of the most troubling aspects of prostate cancer progression is the conversion from an androgen-dependent to an androgen-independent state, which, at present, defies any effective treatment (5, 6). In the majority of end-stage, hormone-refractory tumors, AR continues to be expressed and seems to be activated under androgen ablation conditions. Understanding the mechanisms of AR activation and the genes regulated by AR is of critical importance to appreciate the prostate cancer progression process and to identify possible targets for intervention (7, 8).

AR mediates androgen action by being a transcriptional factor that binds specific DNA sequences and consequently recruits RNA polymerase II and a basal transcriptional complex for efficient transcription of cellular genes. The transcriptional activity of AR is mediated by coregulators (coactivators and corepressors; refs. 9, 10), which, in response to binding of androgen to AR and nuclear translocation, are assembled in a dynamic way at androgen response elements (ARE) along the genome. The best recognized coactivators are the histone acetylases, such as p300/cyclic AMP–responsive element binding protein–binding protein (11, 12) and the p160 steroid receptor coactivator (SRC) family (13–16). These coactivators drive transcription by remodeling chromatin via histone acetylation and by recruiting RNA polymerase II complex to the promoter as we recently shown (17). The molecular basis for AR activation by androgen is a conformational change of AR induced by androgen binding, allowing the coactivators to associate. This process, however, is modulated and, in some cases, overridden by phosphorylation, which has been postulated to be an underlying reason for the androgen-independent activation of AR by nonsteroidal agonists (18–20). In vitro phosphorylation and activation of AR by serine/threonine kinases extracellular signal-regulated kinase (ERK; refs. 21, 22), AKT (23), protein kinase A (24), and protein kinase C (25, 26) have been reported. Thus, serine/threonine kinases seem to be mediators of AR activation. Androgen treatment not only activates AR via conformational changes but also causes significant phosphorylation of AR (21, 27, 28), suggesting that kinase cascades are also being activated by androgen. The in vivo phosphorylation sites of AR have recently been identified (21, 27–29); none of which, however, are consensus phosphorylation sites of ERK and AKT, raising the possibility that other novel kinases are involved. Studies to identify protein kinases associated with or induced by AR resulted in the discovery of several novel kinases: ANPK (30), SPAK (31), cyclin-dependent kinase (CDK) 6 (32), CDK7/CAK (33), and PAK6 (34, 35). These 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 induced by androgen. In this report, we describe a novel kinase male germ cell–associated kinase (MAK), which modulates AR activity and whose kinase activity is important for the growth of androgen-dependent LNCaP cells.

In search of protein kinases transcriptionally induced by androgen, we previously reported the cloning and identification of human MAK (36), which shares homology to rat MAK (37, 38). MAK was originally recognized as kinase with a highly restricted expression primarily in adult testis and at specific stages of spermatogenesis (37, 38). We showed that human MAK is a 623-amino acid protein, with an NH2-terminal kinase domain and a proline- and glutamine-rich domain and a putative nuclear localization signal (429KEKRKK434). The kinase domain of MAK is most homologous to the mitogen-activated protein kinase (MAPK) family (45% identity, 80% similarity) and to the CDK family (40% identity, 75% similarity). We showed that MAK is induced by androgen in a dose-dependent manner with a rapid kinetics. Transactivation assay and protein inhibitor study showed that MAK promoter is a direct target of AR and identified two putative ARE sites within the promoter of MAK. Jia et al. (39) recently extended this analysis and showed by chromatin immunoprecipitation (ChIP) that AR is recruited to the MAK promoter after 5{alpha}-dihydrotestosterone (DHT) stimulation. However, it is unclear how MAK plays a role in androgen signaling or prostate carcinogenesis. Given the inducibility of MAK by androgen and its putative nuclear location, we speculated that MAK might play a role as an effector or a feedback kinase to androgen/AR signaling.

In this study, we present several lines of evidence showing that MAK is a coactivator of AR. We show that MAK is associated with the AR transcriptional complex and able to enhance AR transactivation in an androgen- and kinase-dependent manner. Significantly, knocking down MAK expression by small hairpin RNA (shRNA) or MAK activity with a kinase-dead mutant diminished the expression of prostate-specific antigen (PSA) as well as other AR-responsive genes and resulted in the inhibition of androgen-induced growth. To our knowledge, MAK is the first protein kinase shown to be a direct transcriptional target of AR and serves as a coactivator of AR in propagating the androgen signal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids and reagents. DHT was purchased from Sigma-Aldrich (St. Louis, MO). Hemagglutinin (HA) or Flag epitope-tagged MAK wild-type (WT) and the kinase-dead mutant KR were constructed as described before (36). The KR mutant carries a mutation at Lys33 in the ATP-binding pocket, which renders the molecule catalytically inactive (36). AR full-length and deletion mutant plasmids AR-N, AR-ND, AR-DL, and AR-L were described previously (40, 41). pCMV-ACTR was provided by Dr. Hong-Wu Chen (17). The full-length human MAK-related kinase (MRK) and MAK/MRK overlapping kinase (MOK) cDNAs tagged with HA at NH2 terminus were subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA). The MAK rabbit polyclonal antibody targeting a 19-amino acid polypeptide (amino acid 577-595) was generated by Genemed Synthesis, Inc. (South San Francisco, CA).

Cell cultures. Prostate carcinoma cell lines LNCaP, CWR22Rv1, DU145, and PC3 were obtained from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) from Life Technologies/Invitrogen (Rockville, MD). COS1, 293T, and 293 cells were cultured in DMEM with 4.5 g/L glucose and 4 mmol/L L-glutamine supplemented with 10% FBS.

Dual-luciferase assay. Cells were seeded in 24-well plates at 1 x 105 per well (LNCaP and CWR22Rv1) or 5 x 104 per well (PC3 and DU145) and incubated at 37°C with 5% CO2 for 24 hours. The PSA-Luc reporter plasmid carrying 6 kb of the PSA promoter sequence (42) and the pRL-SV40 Renilla luciferase plasmid (Promega, Madison, WI) were transfected into LNCaP and CWR22Rv1 cells using the Lipofectin reagent (Life Technologies/Invitrogen) or along with pCMV-AR and pCMV-ACTR into DU145 and PC3 cells using the Fugene 6 reagent (Roche, Indianapolis, IN). The assay for the luciferase activity was as described before (42).

Coimmunoprecipitation and in vitro kinase assay. To study the associations between AR, MAK, and ACTR, various AR deletion mutants or WT AR, along with Flag-tagged MAK expression constructs, were cotransfected into COS1 or 293T cells by Fugene 6 reagent. At 48 hours after cotransfection, cells were solubilized in 1 mL of modified radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.8), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Triton X-100, 10% glycerol] supplemented with a protease inhibitor mixture (Complete, Roche). Flag-tagged MAK, HA-tagged ACTR, or AR proteins were immunoprecipitated from equal amounts of cell lysates by incubation with anti-Flag M2-agarose (Sigma-Aldrich), anti-HA (Covance, Berkeley, CA), or anti-AR441 (NeoMarkers, Fremont, CA). Immunoblot analyses of precipitated proteins were done as described previously (36). The dilution of primary antibodies was as follows: AR PG-21 (1:1,000; Upstate, Charlottesville, VA), AR441 (1:500; NeoMarkers), AR C-19 (1 µg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), MAK antiserum (1:3,000), Flag M2 (1:1,000; Sigma-Aldrich), and HA (1:1,000; Covance). For the determination of endogenous protein interactions, LNCaP cells were subjected to the same coimmunoprecipitation assay using AR441 to immunoprecipitate AR protein and blotting with MAK antiserum as described above. In vitro kinase assay was done as described previously (36).

Immunofluorescence. Immunofluorescence staining was done as described previously (36, 40). Anti-AR polyclonal antibody N-20 (Santa Cruz Biotechnology) and anti-HA monoclonal antibody (Covance) were used to examine endogenous AR and ectopically transfected HA-MAK, respectively, followed by FITC-conjugated anti-mouse immunoglobulin G (IgG) and Texas red–conjugated anti-rabbit IgG (Molecular Probes/Invitrogen, Eugene, OR) staining. Nuclei were visualized by 4',6-diamidino-2-phenylindole staining (10 µg/mL). The cells were then examined for fluorescence staining under the Olympus BX61 fluorescence microscope (Olympus America, Inc., Melville, NY).

ChIP assays. ChIP assays were done as described previously (17). Chromatin fragments were immunoprecipitated with specific antibodies overnight at 4°C. For a 5-mL diluted chromatin solution, the following amount of antibodies were used: 5 µg of anti-AR PG-21, 30 µg of anti-Pol II (Santa Cruz Biotechnology), 30 µg of anti-ACTR (Santa Cruz), and 50 µL of anti-MAK rabbit antiserum. Immunocomplexes were recovered and eluted. After reverse cross-linking at 65°C overnight, the DNA fragments were purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and eluted with 100 µL of TE1/10 (pH 8.0). PCR was done using 5 µL of purified DNA for 28 cycles. The gel images of ChIP results are representative of independent experiments done at least thrice.

RNA interference recombinant adenoviruses. The adenoviral RNA interference (RNAi) vector was described previously (43). Oligodeoxynucleotides encoding shRNA, which target MAK gene, were inserted downstream of the human H1 gene promoter. Three regions of MAK were targeted by shRNA: MAKi-1, 5'-GTTGTTCCCTGAATCAGTCA-3'; MAKi-2, 5'-CTCTTATTCCCAATGCCAGT-3'; and MAKi-3, 5'-GCTATTCAGCTCATGACCGA-3'. The resulting pShuttle-RNAi constructs were used to generate recombinant adenoviruses following the protocols described previously (44). Viral particles were purified by centrifugation in a CsCl step gradient. Viral titers were determined by the anti-hexon antibody-based Adeno-X Rapid Titer kit (BD Biosciences, San Jose, CA). The control adenovirus green fluorescent protein (GFP)-shRNA was described previously (43). The targeting sequence of GFP shRNA is 5'-GAACTTCAGGGTCAGCTTG-3'. LNCaP cells were infected with MAK and GFP shRNA adenoviruses at multiplicities of infection (MOI) of 5, 10, and 20, and the cells were analyzed for the MAK gene expression.

Reverse transcription-PCR assay. Reverse transcription-PCR (RT-PCR) was done as described previously (36). Primer sequences used to amplify MAK fragment are listed as follow: 5'-TCCAAGATGAACCGATACACAACC-3' (sense) and 5'-CAGCAATTTTCACAAGCTCTGGAC-3' (antisense). PCRs were conducted at 95°C x 1 minute, (95°C x 30 seconds, 60°C x 30 seconds, 72°C x 1 minute) x 35 cycles, 72°C x 5 minutes. RT-PCR was also done for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control to check the amount and integrity of the total RNA samples. GAPDH primer sequences are as follows: 5'-ACCACAGTCCATGCCATCAC-3' (sense) and 5'-TCCACCACCCTGTTGCTGTA-3' (antisense). PCR amplification variables for GAPDH are the same as those for MAK, except that 25 cycles of amplification were done to ensure that the reaction remained in the log phase.

Selection of stable cell lines and cell proliferation assay. LNCaP cells were transfected with MAK WT, kinase-dead mutant (KR), and empty vector plasmids by Lipofectin reagent and selected with 0.8 mg/mL G418 (Life Technologies/Invitrogen) for 2 weeks. The resistant clones were selected and expanded, and the MAK expression was confirmed by immunoblotting. CellTiter 96 nonradioactive cell proliferation assay (Promega) was used to analyze LNCaP/MAK cells. Specifically, cells were cultured for 3 days in 10% charcoal-stripped FBS medium and then plated into 96-well plates at 5,000 per well in 100 µL medium. Cells were treated with 1 nmol/L DHT and refed with fresh medium containing DHT every 2 days. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay was done according to the manufacturer's instruction (Promega).

Microarray analysis. LNCaP/vector and LNCaP/MAK-KR cells were grown to 60% to 70% confluence, switched to charcoal-stripped medium for 3 days, and then stimulated with 10 nmol/L DHT for 6 hours or treated with ethanol vehicle control for the same duration. Total RNA was extracted using Trizol reagent (Invitrogen) and submitted to the University of California Davis Cancer Center Gene Expression Resource (Sacramento, CA). Microarray analysis was done using Affymetrix Human Genome U133A (HG-U133A) GeneChip arrays (Affymetrix, South San Francisco, CA), which permit expression analysis of the entire Genbank RefSeq database. Probe preparation, hybridization, and signal detection were done according to standard protocol as described previously (45, 46). Array scanning and generation of raw signal data files were done with GeneChip Operating Software (Affymetrix). Subsequent data analysis was done using DNA-Chip Analyzer (dChip) software (47). Comparison analysis of the expression data from the LNCaP/MAK-KR and LNCaP/vector cell lines was conducted to evaluate the effects of MAK-KR on androgen-regulated gene expression. Statistically significant differential expression changes were selected based on attaining Ps of ≤0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MAK associates with AR. We showed previously that MAK is transcriptionally activated by androgen. As such, we hypothesize that MAK is either an effector of AR signaling or a modulator of AR activity (36). To test the latter possibility, we studied the physical interaction between MAK and AR. LNCaP cells were treated with DHT to induce the expression of MAK. The AR was then immunoprecipitated and immunoblotted with MAK antibody. As shown in Fig. 1A , MAK protein is coprecipitated with AR in the presence of DHT. To extend this finding and to map the interacting domain(s), a series of AR deletion mutants were constructed (Fig. 1B). They include the NH2-terminal domain (N), NH2-terminal domain plus DNA-binding/hinge domain (ND), ligand-binding domain (L), and DNA-binding/hinge domain plus ligand-binding domain (DL). The AR and Flag-tagged MAK plasmids were cotransfected into COS1 cells and immunoprecipitated by anti-Flag antibody followed by immunoblotting with anti-AR. The results showed that AR ND and DL, but not N or L, coprecipitated with MAK, suggesting that the DNA-binding/hinge domain is responsible for the interaction with MAK (Fig. 1C).


Figure 1
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  		Figure 1. MAK associates with AR. A, association of endogenous AR and MAK in LNCaP cells. LNCaP cells were cultured in charcoal/dextran-stripped FBS for 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 µg of anti-AR (PG-21), 50 µL of anti-MAK, 30 µg of anti-Pol II, and 30 µg 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.

 
MAK is colocalized and corecruited with AR to PSA promoter. If MAK and AR physically associate with each other, we anticipate colocalization of the molecules in androgen-treated cells. For the lack of an avid, immunohistochemical quality MAK antibody, we resorted to transfection of HA-tagged MAK into LNCaP cells and monitored the subcellular localization of MAK and AR. As shown in Fig. 1D, MAK is primarily localized in the nucleus of LNCaP. Before DHT treatment, diffusive stain of MAK in the cytoplasm could be detected. After DHT treatment, the images of both MAK and endogenous AR seem to be more "compacted" and colocalized in the nucleus. We have extended the localization studies of MAK to other prostate cancer cell lines, including DU145, PC3, and CWR22Rv1. In every case, nuclear localization was detected (data not shown).

Having shown coprecipitation and colocalization of MAK and AR, we were interested in studying the functional outcome of their interaction. On DHT treatment, AR is recruited to the PSA promoter, where it assembles an active transcriptional complex leading to the loading of RNA polymerase II (17). ChIP experiments were carried out to examine whether MAK is recruited to the same site. As shown in Fig. 1D, MAK and AR are corecruited to the ARE site of the PSA promoter, suggestive of a functional role of MAK in AR transactivation. Likewise, p160SRC3/ACTR coactivator (14, 15) and RNA Pol II were also recruited to the same site after DHT treatment. This suggests a role for MAK as a cofactor participating in the assembly of the AR transcription complex.

MAK enhances the transactivation potential of AR. We next asked whether MAK functionally activates AR activity. A HA-tagged MAK expression vector and a PSA promoter-luciferase reporter were transiently cotransfected into CWR22Rv1 and LNCaP cells, and the reporter activity was assessed before and after DHT treatment. Figure 2A (top) shows that MAK can enhance androgen-dependent, endogenous AR-mediated transactivation and that the kinase activity seems important, as the MAK-KR mutant, which lacks the kinase activity, has much less potency. To generalize the finding that MAK is a coactivator of AR, we cotransfected MAK and WT AR into DU145 and PC3 cell lines. The results (Fig. 2A, bottom) confirmed the ability of MAK to enhance the transactivation ability of AR. These experiments also showed that the enhancement effect can be seen with WT AR and, thus, is not a peculiarity of the mutant ARs associated with LNCaP and CWR22Rv1 cell lines.


Figure 2
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  		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 FBS medium overnight 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.

 
The ChIP experiment described above suggested that MAK is recruited to the AR transcriptional complex, which occupies the proximal ARE sequences in the PSA promoter. We also observed that coactivator ACTR is recruited, although it is not clear whether MAK and SRC3/ACTR are in the same complex. To test whether MAK and ACTR functionally interact, we conducted transactivation assay in PC3 cells using PSA-Luc as a reporter. WT AR was cotransfected with MAK and ACTR alone or in combination, and the cells were treated with DHT. Cells transfected with AR alone and treated with DHT were used as a control. As shown in Fig. 2B, in the presence of androgen, MAK and ACTR respectively enhanced AR transactivation of PSA-Luc by 1.5- and 2.0-fold. Together, they enhanced AR activity by 5-fold, indicating a potential functional interaction between these two coactivators. To further test whether MAK, AR, and ACTR form a complex, we did coimmunoprecipitation experiment. 293T cells were cotransfected with MAK, ACTR, and AR followed by immunoprecipitation with one antibody and blotted with the other two in all three permutations. The results in Fig. 2D clearly showed that MAK, ACTR, and AR form a complex.

To define the domain(s) of AR, which functionally interacts with MAK, we used the AR truncation mutants ND and DL in the transactivation assay. These constructs were cotransfected into PC3 cells with MAK and PSA-Luc reporter gene in the presence or absence of DHT. Transfection with AR alone was used as a control. As shown in Fig. 2C, the presence of MAK enhanced the transactivation activity of the ND but not the DL domains, suggesting that the enhancing effect of MAK requires its interaction with coactivator complex residing at the NH2-terminal domain of AR. The data are consistent with the report that ATCR and p160SRC family of coactivators bind NH2-terminal domain of AR (48). The results also showed that ligand-bound AR is the target for MAK.

MAK-shRNA suppresses AR transactivation. As an additional test for the role of MAK in AR transactivation, we developed MAK-shRNAs. Of the six sets we constructed, three of them (shRNA1, shRNA2, and shRNA3) showed significant efficacies in silencing ectopic MAK expression in LNCaP, CWR22Rv1, and PC3 prostate cancer cells. Figure 3A shows an immunoblot using HA antibody to detect MAK at 48 hours after transfection. The suppression of MAK expression was nearly complete for MAK-shRNA1 and MAK-shRNA2 and close to 90% for shRNA3 compared with a control transfected with a scrambled sequence shRNA expression construct (scRNA). To define the specificity of the shRNA, we cotransfected the shRNA constructs with MRK and MOK, two related members of the MAK kinase family (49). We found that shRNA1 to be most specific, only knocking down MAK expression. This is followed by shRNA3, with shRNA2 being the least specific, which also reduced the expression of MRK.


Figure 3
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  		Figure 3. shRNA knockdown of MAK expression and activity. A, knockdown of MAK protein expression by MAK-shRNA. MAK-shRNAs were cotransfected with HA-MAK, HA-MRK, or HA-MOK into 293 cells. scRNA was used as a 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 expression as an internal control. B, suppression of AR activity by MAK-shRNA. PC3 cells were cotransfected with WT AR, PSA-Luc, and MAK-shRNA. Cells 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 as a control.

 
We then proceeded to test the effect of knocking down endogenous MAK in the AR transactivation potential. Taking advantage of the constitutive expression of MAK in PC3 cells (36), we cotransfected the shRNA constructs, a WT AR cDNA expression construct, and PSA-Luc into PC3 cells in the presence of DHT (1 nmol/L). The luciferase activities after 48 hours are shown in Fig. 3B. Knockdown of the endogenous MAK by any of the shRNA constructs significantly compromised the ability of AR to transactivate, suggesting that MAK is an intrinsic cofactor of AR activation.

MAK-shRNA adenovirus infection inhibits the growth of LNCaP cells. If MAK is a coactivator of AR, we surmised knocking down MAK should compromise the androgen response of LNCaP, thereby reducing the androgen-dependent growth. Because of the high specificity of shRNA1 and shRNA3, we structured the constructs into an adenovirus vector to generate ad-MAKi-1 (Mi1) and ad-MAKi-3 (Mi3) vectors. An adenovirus carrying shRNA targeting GFP (Gi) was used as a control. To show the efficacies of the viruses, LNCaP cells were infected with either Mi or Gi viruses and the MAK expression was determined by RT-PCR. The Mi1 and Mi3 viruses effectively reduced the MAK expression in infected cells, whereas Gi virus had no effect, confirming the specificity of the approach (Fig. 4A ). Importantly, both Mi1 and Mi3 viruses significantly slowed down the growth of LNCaP, whereas the Gi virus-infected LNCaP exhibited a similar growth rate as uninfected cells (Fig. 4B). These data together suggest that knockdown of MAK expression has a significant effect on the androgen-dependent growth of LNCaP cells, implicating MAK in AR signaling.


Figure 4
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  		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.

 
MAK-defective LNCaP exhibited reduced growth and impeded androgen response. The adenovirus shRNA experiments described above provide preliminary indication that MAK expression is involved in androgen-dependent growth of LNCaP. Due to the transient nature of the experiment, it was difficult to conduct detailed molecular analysis. To this end, we generated LNCaP clones ectopically expressing the kinase-dead MAK-KR as an alternative means to stably knock down the MAK activity. LNCaP cells ectopically expressing WT MAK were also obtained to serve as a positive control. Although we had no problem in attaining LNCaP-MAK clones with a high level of MAK expression, we experienced difficulty in isolating MAK-KR clones perhaps due to the deleterious effect of MAK knockdown in LNCaP cells. We, nevertheless, were able to isolate one LNCaP/MAK-KR clone with a moderate level of MAK-KR expression (Fig. 5A, inset ). In vitro kinase assay confirmed the kinase-negative nature of MAK-KR (Fig. 5A, inset). When the growth curves are compared with LNCaP/vector cells, LNCaP/MAK-WT exhibited a similar, if not slightly increased, growth rate that is dependent on androgen. The LNCaP/MAK-KR clone, on the other hand, grew at a much reduced rate (Fig. 5A). The availability of LNCaP/MAK-KR subline also afforded us an opportunity to study the influence of endogenous MAK on global gene expression using Affymetrix HG-U133A GeneChip oligonucleotide arrays. The microarray analysis yielded an expression signature that was indeed consistent with the observed phenotype for MAK knockdown, thereby imparting credibility to our data. A representative subset of the results is presented in Fig. 5B and Table 1 . Consistent with our findings that MAK-shRNA decreased AR transactivation in reporter assays, a diminished AR response was clearly evident as indicated by the decreased expression of classically androgen-induced genes (e.g., PSA/KLK3, NKX3.1, FKBP5, and VEGF) and restored expression of androgen-repressed genes (clusterin/TRPM-2, CHES1, and T-cell receptor {gamma} transcript). Growth inhibition was indicated by an induction of p53 target gene p21Cip1 and decreased expression of genes driving progression through various phases of the cell cycle, such as CDC2 and CDC6. Notably, several genes encoding critical components of the DNA replication machinery (MCM2/MCM4/MCM7) and DNA metabolism (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.


Figure 5
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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.

 

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

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 expression of androgen-responsive genes in LNCaP cells. This conclusion was drawn not only by a functional assay on PSA promoter but also based on results of global gene expression profiling. We also showed that MAK physically associates with AR and enhances the transcriptional potential of AR. Although we do not know whether AR and MAK directly contact each other, our evidence suggests a strong interaction between them. Their 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 androgen transmits signals to induce cell growth or differentiation, the discovery of a protein kinase, which is a direct target gene of AR and which modulates the AR activity, is thus significant. Furthermore, the male tissue-selective expression of MAK makes it a potential target for therapeutic attenuation of AR activities.

The coactivator role of MAK was defined first by transactivation assay on PSA promoter in a variety of prostate cancer cell lines, indicating the generality of the phenomenon. It is further enforced by the expression profile analysis using a kinase-dead MAK mutant, which blunts the expression of a large number, but not all, of androgen-induced genes. We interpret this to mean that MAK is part of a general transcriptional complex of AR, which recognizes a large subset of the ARE-containing promoters. One consequence of the significant attenuation of androgen responsiveness in LNCaP would be the attenuation of the growth responses. This is indeed what was observed. This conclusion was further confirmed by the adenovirus-shRNA experiment. We selected shRNA species, which target only MAK but not its related kinases MOK and MRK (49). The growth of LNCaP in the presence of at least two different shRNAs against MAK is significantly slowed down. This suggests that MAK is an important component in the AR complex in LNCaP cells. MAK endogenous protein expression level is generally low as assessed by the presently available antibody, and reliable quantitation comes primarily from RT-PCR (36). It is interesting that overexpression of MAK in LNCaP cells only marginally increased the growth rate and did not overcome the androgen dependence of the cells. This is, however, not entirely surprising, as most of the coactivators, while significantly enhancing the action of AR, are not sufficient to cause androgen-independent growth of the cells. Thus, as a modulator of AR, MAK is required for full androgen response but in itself is not sufficient for causing androgen independence.

Numerous coactivators and corepressors for AR have been identified. Very few are protein kinases. Before this study, five kinases, SPAK (31), ANPK (30), PAK6 (34, 35), CDK6 (32), and CDK7 (TFIIH; ref. 33) were identified as cofactors, which bind AR. Their modes of action differ significantly. SPAK, ANPK, CDK6, and CDK7 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 DNA-binding/hinge region of AR, whereas CDK7 binds the NH2-terminal domain of AR. Interestingly, thus far, none of these kinases have been shown to directly phosphorylate AR, presumably the kinase activity, if required, is directed toward cofactors or other chromatin proteins.

MAK shares some similarities with these kinases but also has distinct features. First, it seems to be the only one shown to be a direct transcriptional target of AR. We previously showed that androgen augments the transcription of MAK in the presence of cycloheximide and indicated the presence of three AREs in MAK promoter, which respond to androgen stimulation (36). In a recent publication, AR was found to be recruited to the major ARE site of the MAK promoter we defined (39). By contrast, SPAK was shown to be transcriptionally activated by androgen but likely to be a secondary target (31). Second, the highly restricted tissue expression only in testis and prostate among the tissues tested is rivaled only by SPAK (31) and ANPK (30). Third, transactivation potential of MAK largely depends on its kinase activity, whereas the kinase activity of CDK6 is apparently not required for its augmentation of AR activity (32). It is noteworthy, however, that the kinase-dead mutant of MAK is not completely null for AR activation, suggesting some kinase-independent function. Finally, direct in vitro or in vivo phosphorylation assays and transactivation assays with AR phosphorylation site mutants (29) failed to show AR as a direct phosphorylation target of MAK nor do we see the effect of MAK on the stability of AR and AR dimerization.4 This, together with the observation that MAK and AR are primarily localized in the nucleus and that they are both recruited to the PSA promoter, suggests that MAK first assembles into an AR complex and is then carried to the chromosomal site where MAK phosphorylates other coactivators and chromatin proteins. The conclusive demonstration of this model awaits the identification of physiologic relevant substrate of MAK in the future. In this regard, it is interesting that MAK and ACTR seem to be in proximity and synergize with each other in the transactivation of AR. In addition, MAK and ACTR seem to be assembled in the same AR complex based on coimmunoprecipitations.

The kinase domain of MAK contains TDY, a phosphorylation motif recognized by MAPK kinase. We showed that it is active, capable of autophosphorylation and phosphorylation of exogenous substrate. However, the unstimulated MAK activity is relatively low. Our effort to show its activation and phosphorylation by MAPK/ERK kinase, MAPK kinase 3, and MAPK kinase 6 has not yielded positive results.4 An intriguing recent report by Fu et al. (50) revealed that ICK (also called MRK), a related family member of MAK, is a substrate of CDK7. Although we have not tested whether CDK7 is an activator of MAK, the nuclear localization of MAK and the association of both CDK7 and MAK with AR make it an appealing hypothesis that MAK is activated by CDK7 in situ.

Being a protein kinase transcriptionally regulated by AR, MAK is likely to be a feedback modulator of AR or a downstream effector for androgen signaling or both. Our data presented in this article showed that MAK is a part of the AR transcriptional complex and a positive modulator of the transactivation activity of AR. We think likely that MAK also serves as a downstream effector of AR by phosphorylating critical substrates involved in proliferation and differentiation of prostate cells. This awaits the identification of cellular substrates of MAK. The possibility that MAK may require an additional agonist to fully activate its kinase activity suggests that MAK may be an integrator of multiple signal pathways.


   Acknowledgments
 
Grant support: NIH grants RO1CA114575-01 and RO1DK52659 and Department of Defense grant W81XWH0410835 (H-J. Kung). The University of California Davis Cancer Center Gene Expression Resource is supported by National Cancer Institute Cancer Center grant P30 CA93373-01 (R.W. deVere White).

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.

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 before publication.


   Footnotes
 
4 Unpublished data. Back

Received 5/ 4/06. Revised 6/19/06. Accepted 7/ 7/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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