BACKGROUND
the sex-determining region y  box, or sox, family consists of  <dig> highly conserved transcription factors in humans that play important roles in development  <cit> .
sox <dig> is a 47-kda protein that is encoded by a single exon gene and contains a conserved signature sequence in the high-mobility group  dna-binding domain  related to the tcf/lef family of transcription factors.
the key effector of the canonical wnt pathway, β-catenin, forms complexes with tcf/lef hmg box factors and stimulates the transcription of wnt downstream targets.
many studies have shown that β-catenin is regulated at several levels in cancer progression, but less is known about the regulation of the tcf/lef transcription factor family.
structurally related to tcf/lefs, several members of the sox family, sox <dig>  sox <dig>  sox <dig>  and sox <dig> have been implicated in regulating β-catenin activity  <cit> .
gain- and loss-of-function analyses have demonstrated that sox <dig> and sox <dig> proteins antagonize the wnt pathway by competing with tcfs for β-catenin binding while sox <dig> may function to stabilize β-catenin protein to help activate expression of target genes and promote cellular proliferation  <cit> .
although these findings have implicated how sox proteins regulate the transcriptional output of wnt pathway, the precise role of sox <dig> in the wnt pathway remains unclear.
in humans, tight regulation of the levels of transcriptional factors is crucial to maintain tissue homoeostasis and many have been found to be oncogenic when their expression is deregulated or when their activity is functionally altered  <cit> .
for example, sox <dig> is overexpressed in several cancers, such as bladder cancer, in which it is 5-fold upregulated compared with normal tissues  <cit> .
sox <dig> is also upregulated at the mrna and protein level in prostate cancer and this upregulation is correlated with higher gleason score or tumor grade  <cit> .
in addition, sox <dig> is overexpressed in leukemias, melanomas, glioblastomas, medulloblastomas  <cit> , and lung cancer  <cit> .
sox <dig> is also overexpressed in endometrial cancer through methylation-mediated silencing of mir-129- <dig>  <cit> .
consistent with the concept that sox <dig> is an oncogene, three independent studies searching for oncogenes have found sox <dig> to be one of the most common retroviral integration sites, resulting in increased mrna  <cit> .
junction plakoglobin , also known as γ-catenin, is a major component of the submembrane of adherens junctions and desmosomes in mammalian cells  <cit> .
it is closely related to the drosophila segment polarity gene armadillo, which has a role in the transduction of transmembrane signals that regulate cell fate  <cit> .
plakoglobin shares more than 76% homology with β-catenin, contains a central armadillo repeat domain flanked by the carboxyl and amino terminal domains, and functions in cell-cell junctions, along with β-catenin and α-catenin when coupled with cadherins  <cit> .
while it is known that β-catenin is essential in the wnt signaling cascade, plakoglobin also binds to tcf/lef factors, and has lower tcf/lef-dependent transcriptional activity compared to β-catenin when endogenous β-catenin is depleted  <cit> .
mass spectrometry is a highly sensitive technique that enables the rapid identification of proteins and also protein-protein interactions from a variety of biological samples.
when combined with affinity purification, whole or targeted protein interaction networks can be elucidated  <cit> .
to investigate the cellular functions of sox <dig>  we have developed a one-step affinity purification method that enables rapid purification of sox <dig> complexes in lncap cells, a prostate cancer cell line.
here, in this study, we identified proteins that interact with sox <dig> in lncap cells by liquid chromatography-tandem mass spectrometry , including plakoglobin.
this novel interaction between sox <dig> and plakoglobin may provide insights into the role of sox <dig> in key pathways in cell proliferation, development, and cancer progression.
methods
reagents and cell culture
lncap, pc3m, and mda-mb- <dig> cells were cultured as described  <cit>  by american type culture collection except using t-medium  for lncap cells.
ha-tagged sox <dig> was cloned into phr-ubq-ires-eyfp-Δu <dig> lentiviral vector , and stable cells were isolated, as previously described  <cit> .
recombinant wnt3a was purchased from r & d systems  and reconstituted in  <dig> % bsa/pbs prior to use.
nuclear export inhinbitor leptomycin b  was purchased from sigma .
cells were treated for  <dig> hrs with  <dig> ng/ml wnt3a,  <dig> μm lmb, or both unless otherwise noted.
biotinlyated ha-tagged sox <dig> expression construct
the prep4-blrpwt-ires-bira-xl <dig> plasmid was a gift from dr. jeremy boss .
the pcdna <dig> -hisa-hasox <dig> was constructed as described  <cit> .
to generate prep4-blrpwt-hasox4-ires-bira-xl <dig>  ha-tagged sox <dig> from pcdna <dig> -hisa-hasox <dig> was excised at kpni and xbai sites and overhangs were filled in with klenow fragment.
filled-in ha-sox <dig> was then blunt ligated into the filled-in noti site of prep4-blrpwt-ires-bira-xl <dig>
purification of biotinlyated ha-tagged sox4
the prep4-blrpwt-hasox4-ires-bira-xl <dig> and control empty vector were transfected into two 90% confluent  <dig> mm dishes of lncap cells respectively.
forty-eight hours post-transfection, the plates were then placed on ice and the cells were washed twice with ice-cold pbs, and lysed in  <dig> ml/plate with ice-cold ip lysis buffer  supplemented with protease inhibitors, and harvested by scraping.
biotinlyated ha-tagged sox <dig> complexes were purified by incubating with  <dig> ul slurry of dynabeads® m <dig> streptavidin  at 4°c for  <dig> hrs.
the beads ware washed  <dig> times with ip lysis buffer and eluted by boiling in laemmli sample buffer.
sample preparation for mass spectrometry
five percent of the proteins resulting from the purification were subject to 4-15% gradient sds-page and silver staining to analyze sample purity.
the rest of the purified proteins were subjected to 4-15% gradient sds-page and concentrated on a very short distance , and the protein in those bands were excised for in-gel digestion and liquid chromatography coupled with tandem mass spectrometry based on an optimized protocol  <cit> .
co-immunoprecipitation and western blot
cells were washed twice with ice-cold pbs, lysed in  <dig> ml/plate with ice-cold ip lysis buffer supplemented with protease inhibitors, and harvested by scraping.
the whole cell lysates were pre-cleared and then incubated with  <dig> ul slurry of dynabeads® m <dig> streptavidin or protein g  at 4°c for  <dig> hrs.
the beads were washed  <dig> times with ip lysis buffer and eluted by boiling in laemmli sample buffer before running sds-page.
western blots were performed as previously described  <cit> .
antibodies to plakoglobin  and hemagglutinin  were used in immunoprecipitations .
antibodies to plakoglobin , sox <dig> , β-actin , and ha 16b <dig>  were used in western blot.
confocal microscopy
sub-confluent lncap hasox <dig> stable cells were grown on glass cover slips, and serum starved for  <dig> hrs with  <dig> % fbs  t-medium before treating with  <dig> ng/ml wnt3a,  <dig> μm lmb, or both for another  <dig> hrs.
cells were washed three times with hank's balanced salt solution  and fixed with  <dig> % paraformaldehyde for  <dig> min at room temperature.
cells were permeablized with 100% ethanol for  <dig> min at -20°c and blocked in 3% bsa in hbss+ for 1hr.
anti-ha 16b <dig>  and plakoglobin antibodies   were used as primary antibodies and were diluted in blocking buffer and incubated for  <dig> hr.
then cells were washed in hbss+ and incubated in fluorescence-labeled secondary antibodies for 1hr at room temperature.
cells were washed in hbss+ and then stained with topro  for  <dig> min at room temperature.
labeled cells were then washed in hbss+ and mounted in p-phenylene diamine antifade agent.
confocal fluorescence images were captured using a laser-scanning microscope.
cell fractionation into nuclear and cytosolic lysates
one 100-mm dish of lncap hasox <dig> stable cells was grown to 80-90% confluency and serum starved for  <dig> hrs with  <dig> % fbs t-medium before treating with  <dig> ng/ml wnt3a,  <dig> μm lmb, or both for another  <dig> hrs.
crude subcellular fractionation was performed as previously described using digitonin, np <dig>  and ripa lysis methods  <cit> .
quantitative real-time pcr
ninety-percent confluent cells were harvested using the rneasy kit , and reverse transcription was performed using iscript cdna synthesis kit .
quantitative real-time pcr  was performed using iq sybr green supermix  on a bio-rad icycler using 18s or β-actin as a control, and data were analyzed using the δct method  <cit> .
chromatin immunoprecipitation  assay
one 100-mm dish of lncap hasox <dig> stable cells was grown to 80-90% confluency.
cells were fixed with 1% formaldehyde, then lysed, and sonicated as described  <cit> .
sonicated chromatin was precleared and then immunoprecipitated with  <dig> μg of anti-ha 12ca <dig> ascites or mouse igg overnight and immunoprecipitated by dynabeads® protein g  for  <dig> hrs at 4°c.
beads were washed and eluted as described  <cit> .
chip dna was purified and then subjected to pcr amplification.
luciferase reporter assay
cells grown in twelve-well tissue culture plates were transfected with  <dig>  μg of either top-flash or fop-flash with  <dig>  μg of tk-renilla control vector .
at  <dig> hrs post-transfection, cells were placed in  <dig> % fbs medium for recovery and serum starvation.
at  <dig> hrs post-transfection, cells were treated with wnt3a and lmb, or both.
reporter gene activity was measured in a td-20/ <dig> luminometer  with the dlr luciferase assay system  after  <dig> hrs and was normalized for transfection efficiency by measuring renilla luciferase activity.
sirna transfection
the sirna sequence for plakoglobin agtcggccattgtgcatct was targeted at the 5' end of the gene lacking of homology with other catenin members  <cit>  .
lncap hasox <dig> cells were transfected using lipofectamine  <dig>  with plakoglobin or scramble sirnas at final concentration of  <dig> nm.
at  <dig> hrs post-transfection, cells were placed in  <dig> % fbs medium for recovery and serum starvation.
at  <dig> hrs post-transfection, cells were treated with wnt3a, lmb, or both for western blot analysis or chromatin immunoprecipitation assay.
RESULTS
identification of proteins that interact with sox4
to identify proteins that stably interact with sox <dig>  we developed a one-step affinity purification method that allows for rapid purification of sox <dig> complexes.
the prep4-blrpwt-ires-bira-xl <dig> plasmid contains the bira gene of e. coli that encodes a biotin holoenzyme synthetase  <cit> .
we cloned the human sox <dig> gene into this vector to generate an amino-terminal fusion to a bira recognition sequence to produce transiently expressed, intracellularly biotinylated sox <dig> protein in lncap prostate cancer cells, and purified sox <dig> complexes with streptavidin-linked magnetic beads to perform large scale proteomics analysis.
protein quantity and purity were checked by sds-page analysis and silver staining prior to mass spectrometry analysis .
plakoglobin interacts with sox4
lc-ms/ms analysis identified junction plakoglobin  as a sox <dig> binding protein via two trypsinized fragments that perfectly matched to human plakoglobin sequences .
to confirm this interaction, we first repeated the transient transfection of the prep4-blrpwt-ires-bira-xl9-hasox <dig> or vector control plasmid into lncap cells, and performed streptavidin-magnetic bead based purification followed by anti-plakoglobin  immunoblotting.
as expected, the result of the immunoprecipitation  and western blot validated the interaction between sox <dig> and plakoglobin .
furthermore, we performed a reverse-ip immunoprecipitating endogenous plakoglobin and probing for transfected ha-sox <dig>  and endogenous sox <dig> in untransfected lncap cells .
to determine that the interactions were not cell-line specific or dna-dependent, we repeated the immunoprecipitations in primary human keratinocytes, mda-mb- <dig> breast cancer cells, and pc3m prostate cancer cells  following dnase i digestion of whole cell lysates.
taken together, these data demonstrate that sox <dig> directly interacts with plakoglobin and that plakoglobin is a novel sox <dig> binding partner.
interaction between sox <dig> and plakoglobin in the nucleus responds to wnt signaling
when plakoglobin is present in desmosomes, it interacts with desmoglein and desmocollin, and when in adherens junctions it interacts with e-cadherin in the cytoplasmic component  <cit> .
recently, additional evidence has suggested that plakoglobin contributes a low level of transcriptional activity to the wnt signal transduction cascade in the nucleus  <cit> .
although, it has been confirmed that sox <dig> modulates wnt signaling via interaction with β-catenin  <cit> , the role of plakoglobin in wnt signaling is still debated.
to investigate where and under what conditions sox <dig> and plakoglobin interact with each other, we used confocal microscopy to determine whether we could observe subcellular co-localization of ha-sox <dig> and plakoglobin .
after treatment of pc3m cells that stably-expressed ha-sox <dig> with recombinant human wnt3a, we observed that the interaction between ha-sox <dig> and plakoglobin was slightly increased in the nucleus.
in contrast, this was not observed in the non-transfected adjacent cells.
we observed the same phenomenon in the lncap-ha-sox <dig> stable cell line .
furthermore, when we treated with nuclear export inhibitor, leptomycin b  to inhibit nuclear export of sox <dig> and plakoglobin, the co-localization was significantly increased compared to treating the cells with either wnt3a or lmb alone .
to confirm these enhanced interactions, we performed co-immunoprecipitation under the same wnt-induced conditions in lncap-ha-sox <dig> cells .
the interaction in the whole cell lysate was quantified and significantly increased when we treated with wnt3a and lmb together .
in addition, to determine that sox <dig> and plakoglobin interact in the nucleus, we prepared cytosolic and nuclear fractions to confirm the subcellular interaction .
immunoblots against akt and nuclear lamin were used as controls to demonstrate the purity of the nuclear and cytosolic fractions .
the quantitative results showed that the interaction in the nuclear but not cytosolic fraction was significantly increased in response to the wnt3a and lmb treatment .
these results show that sox <dig> and plakoglobin physically interact in the nucleus of lncap cells.
wnt target genes and sox4-target genes are affected by sox4-plakoglobin interaction
to address the functional consequences of modulation of sox <dig> transcriptional activity by the sox4-plakoglobin complex, we tested expression of several genes including the wnt target gene axin <dig>  <cit> , as well as sox <dig> targets dicer <dig> and dhx <dig>  <cit> .
to characterize if sox <dig> dna binding activity is changed by wnt-induced interaction with plakoglobin, we performed chip assays for axin <dig>  dicer <dig>  dhx <dig>  and sox <dig> .
compared to untreated lncap hasox <dig> cells, the binding of sox <dig> to axin <dig>  dicer <dig>  and dhx <dig> promoters was increased after wnt signaling was induced.
however, sox <dig> binding was decreased after treatment with both wnt3a and lmb.
this difference indicates that increasing the interaction between sox <dig> and plakoglobin could inhibit sox <dig> binding activity to downstream targets and may inhibit sox <dig> transcriptional activity.
quantitative realtime-pcr  analysis of the lncap sox <dig> stable cell line showed reductions in axin <dig>  sox <dig>  and dhx <dig> upon co-treatment with wnt3a and lmb .
to determine whether the effects from combined wnt3a and lmb treatment were dependent on plakoglobin, we targeted plakoglobin by sirna to determine whether reduced plakoglobin levels could rescue the effects we observed on sox <dig> binding to target promoters.
transfection of lncap ha-sox <dig> cells with plakoglobin sirna or scrambled control sirna resulted in approximately 50% knockdown of endogenous plakoglobin protein levels .
furthermore, plakoglobin sirna partially rescued sox <dig> binding to target promoters by chip assay in the presence of wnt3a and lmb, while scrambled sirna had no effect .
these data suggest that sox4-plakoglobin interactions may interfere with sox4-mediated transcription in response to wnt signaling due to reduced promoter occupancy.
sox4-plakoglobin complex modulates β-catenin-mediated transcriptional activity
to evaluate whether sox4-plakoglobin complex affects the transcriptional activity of β-catenin, we performed luciferase reporter assays with t cell factor  reporter plasmids containing wild type tcf binding sites  or mutated tcf binding site   <cit>  .
as expected, the tcf/β-catenin luciferase reporter was significantly increased after we induced wnt signaling with recombinant wnt3a when compared to untreated cells.
in contrast, co-treatment with wnt3a and lmb, strongly inhibited increases in luciferase activity back to baseline unstimulated levels, suggesting that wnt3a+ lmb-induced sox4-plakoglobin complexes could compete with and inhibit the transcriptional activity of β-catenin.
in addition, we performed anti-β-catenin chip assays on the axin <dig>  c-myc, and dkk <dig> promoters, and observed that occupancy of these promoters was stimulated by wnt3a, but that stimulation was inhibited by co-treatment with wnt3a and lmb .
these results suggest that β-catenin activity was affected by the sox4-plakoglobin complex and that plakoglobin may compete with β-catenin binding to sox <dig> and/or tcf/lef in the nucleus.
discussion
while sox <dig> expression is elevated in many malignancies and is tightly correlated with prostate cancer tumor grade, little is known of the mechanism by which sox <dig> affects the progression of prostate cancer.
using lc-ms/ms proteomic analysis, we identified a novel sox <dig> binding protein, junction plakoglobin, in lncap prostate cancer cells.
we observed a physical association between sox <dig> and plakoglobin with both epitope-tagged and endogenous sox <dig>  although the interactions of sox <dig> with plakoglobin were enhanced by co-treatment of wnt3a and lmb, we could detect this interaction using four different untransfected and unstimulated cell types  treated with dnase i. in addition, confocal microscopy and co-immunoprecipitation demonstrated co-localization of sox <dig> and plakoglobin in the nucleus when wnt signaling was induced.
chip assays showed the sox4-plakoglobin complex affected sox <dig> dna binding activity to the axin <dig>  dicer <dig>  and dhx <dig> promoters that are wnt signaling downstream genes and sox4-associated targets.
in addition, mrna expression changes were detected in axin <dig>  dicer <dig>  and dhx <dig> by realtime-pcr.
these data suggest that the sox4-plakoglobin complex may inhibit wnt signaling.
indeed, conditions that induced the increased interaction between sox <dig> and plakoglobin caused β-catenin top-flash transcriptional activity to be downregulated and reduced occupancy of the c-myc promoter by β-catenin.
it is known that sox <dig> can directly interact with β-catenin to enhance wnt signaling  <cit> , but mechanistic data remain very limited.
we found that sox <dig> interacts with plakoglobin in a wnt3a-dependent manner in our experimental cancer model.
our model not only supports the hypothesis that sox <dig> may stabilize β-catenin  <cit> , but also suggests a model in which sox <dig> can modulate wnt signaling by binding either β-catenin or plakoglobin .
in this model, transcriptional responses to wnt signaling are increased by sox4-β-catenin interactions, and subsequently reduced by sox4-plakoglobin interactions that facilitate nuclear export of sox <dig>  however, when nuclear export is inhibited, plakoglobin competes with β-catenin for binding to sox <dig> and tcf/lef factors, downregulating wnt-responsive transcription and reducing sox4-dna binding.
this model is supported by the observation that combination treatment with wnt3a and lmb enhanced sox4-plakoglobin interactions, reduced tcf/β-catenin top-flash transcription, reduced sox4-dna binding and β-catenin dna binding in chip assays, and reduced expression of sox <dig> downstream targets .
consistent with our model, plakoglobin shows little transcriptional activity compared to β-catenin in cell lines that lack β-catenin  <cit> .
our confocal data show that plakoglobin nuclear localization is strongly enhanced by lmb co-treatment, suggesting shuttling of plakoglobin into and out of the nucleus following wnt3a stimulation.
although lmb is an artificial stimulus, others have shown that plakoglobin overexpression can lead to nuclear localization  <cit> , and plakoglobin is overexpressed  <cit>  and amplified  <cit>  in several types of cancer.
moreover, plakoglobin nuclear localization was much stronger in cells expressing hasox <dig>  suggesting cytoplasmic sox <dig> may facilitate plakoglobin nuclear import.
thus, sox <dig> may induce nuclear import of plakoglobin in response to wnt3a while plakoglobin destabilizes sox <dig> from dna binding, facilitating nuclear export of sox <dig>  it is as yet unclear what signals or modifications might tip the balance between nuclear import and export of plakoglobin-sox <dig> complexes, or whether shuttling might be constitutive.
the role of plakoglobin during cancer progression is still controversial.
when plakoglobin is overexpressed, it induces cell migration and mobility in hct <dig> cells, suggesting that plakoglobin may have some oncogenic effects  <cit> .
in contrast, several reports have demonstrated that plakoglobin has a tumor-suppressive effect that inhibits tumor cell growth  <cit> .
the tumor suppressive activity of plakoglobin may be via nuclear translocation to antogonize β-catenin binding to tcf/lef proteins in keratinocytes  <cit> , supporting our model that plakoglobin competes with sox4-β-catenin interactions in the nucleus.
CONCLUSIONS
in summary, we have demonstrated that sox <dig> interacts with plakoglobin in a wnt-dependent manner in lncap cells and that this complex may function to inhibit wnt signaling.
additional studies will be required to elucidate the detailed mechanisms by which sox4-plakoglobin interactions may affect wnt signaling, but the role of the sox4-plakoglobin complex provides novel insights into the role of sox <dig> in wnt signaling and prostate cancer progression.
authors' contributions
yhl performed the immunopurifications of sox <dig>  the chip assays, co-ips, luciferase assays, and helped write the manuscript.
jc cloned sox <dig> into the prep4-blrpwt-ires-bira-xl <dig> plasmid and performed co-ips.
dc performed the proteomics analysis.
mef and kdb performed immunofluorescence confocal microscopy.
jp advised on protein purification, supervised the proteomics analysis, and edited the manuscript.
an supervised the confocal microscopy and edited the manuscript.
csm conceived the study, participated in its design and coordination, and co-wrote the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
figure.s <dig> wnt signaling induces nuclear colocalization of sox <dig> and plakoglobin.
subcellular localization of plakoglobin and sox <dig> were examined by confocal microscopy.
lncap hasox <dig> stable cell line was treated with  <dig> ng/ml human recombinant wnt3a or  <dig> μm leptomycin b , or both wnt3a+lmb for  <dig> hr.
the fields shown were visualized independently by confocal microscopy at the appropriate wavelength for plakoglobin  and sox <dig> , and topro  respectively, and then the three images were overlaid .
strong nuclear localization of plakoglobin was observed in the wnt3a+lmb treated cells that expressed hasox <dig>  representative fields from these independent repeated experiments are shown.
plakoglobin localizes to the nucleus following wnt3a treatment, and this effect is strongly enhanced by lmb co-treatment, suggesting shuttling of plakoglobin into and out of the nucleus following wnt3a stimulation.
note that plakoglobin nuclear localization is much stronger in cells expressing hasox <dig>  suggesting sox <dig> may facilitate plakoglobin nuclear import.
click here for file
acknowledgements
we thank dr. tamara caspary for advice on experimental conditions for lmb treatments, dr. chris scharer for advice on sox <dig> binding site analysis, dr. cornel phillip for advice with wnt3a treatment, and dr. andrew kowalczyk for providing primary human keratinocytes.
this work was supported by national cancer institute grants r01ca <dig>  and p30ca <dig> .

 the sex-determining region y box, or sox, family consists of highly conserved transcription factors in humans that play important roles in development. sox is a 47-kda protein that is encoded by a single exon gene and contains a conserved signature sequence in the high-mobility group dna-binding domain related to the tcf/lef family of transcription factors. the key effector of the canonical wnt pathway, β-catenin, forms complexes with tcf/lef hmg box factors and stimulates the transcription of wnt downstream targets. many studies have shown that β-catenin is regulated at several levels in cancer progression, but less is known about the regulation of the tcf/lef transcription factor family. structurally related to tcf/lefs, several members of the sox family, sox sox sox and sox have been implicated in regulating β-catenin activity. gain- and loss-of-function analyses have demonstrated that sox and sox proteins antagonize the wnt pathway by competing with tcfs for β-catenin binding while sox may function to stabilize β-catenin protein to help activate expression of target genes and promote cellular proliferation. although these findings have implicated how sox proteins regulate the transcriptional output of wnt pathway, the precise role of sox in the wnt pathway remains unclear. in humans, tight regulation of the levels of transcriptional factors is crucial to maintain tissue homoeostasis and many have been found to be oncogenic when their expression is deregulated or when their activity is functionally altered. for example, sox is overexpressed in several cancers, such as bladder cancer, in which it is 5-fold upregulated compared with normal tissues. sox is also upregulated at the mrna and protein level in prostate cancer and this upregulation is correlated with higher gleason score or tumor grade. in addition, sox is overexpressed in leukemias, melanomas, glioblastomas, medulloblastomas, and lung cancer. sox is also overexpressed in endometrial cancer through methylation-mediated silencing of mir-129-. consistent with the concept that sox is an oncogene, three independent studies searching for oncogenes have found sox to be one of the most common retroviral integration sites, resulting in increased mrna. junction plakoglobin, also known as γ-catenin, is a major component of the submembrane of adherens junctions and desmosomes in mammalian cells. it is closely related to the drosophila segment polarity gene armadillo, which has a role in the transduction of transmembrane signals that regulate cell fate. plakoglobin shares more than 76% homology with β-catenin, contains a central armadillo repeat domain flanked by the carboxyl and amino terminal domains, and functions in cell-cell junctions, along with β-catenin and α-catenin when coupled with cadherins. while it is known that β-catenin is essential in the wnt signaling cascade, plakoglobin also binds to tcf/lef factors, and has lower tcf/lef-dependent transcriptional activity compared to β-catenin when endogenous β-catenin is depleted. mass spectrometry is a highly sensitive technique that enables the rapid identification of proteins and also protein-protein interactions from a variety of biological samples. when combined with affinity purification, whole or targeted protein interaction networks can be elucidated. to investigate the cellular functions of sox we have developed a one-step affinity purification method that enables rapid purification of sox complexes in lncap cells, a prostate cancer cell line. here, in this study, we identified proteins that interact with sox in lncap cells by liquid chromatography-tandem mass spectrometry, including plakoglobin. this novel interaction between sox and plakoglobin may provide insights into the role of sox in key pathways in cell proliferation, development, and cancer progression. methods reagents and cell culture lncap, pc3m, and mda-mb- cells were cultured as described by american type culture collection except using t-medium for lncap cells. ha-tagged sox was cloned into phr-ubq-ires-eyfp-Δu lentiviral vector, and stable cells were isolated, as previously described. recombinant wnt3a was purchased from r & d systems and reconstituted in % bsa/pbs prior to use. nuclear export inhinbitor leptomycin b was purchased from sigma. cells were treated for hrs with ng/ml wnt3a, μm lmb, or both unless otherwise noted. biotinlyated ha-tagged sox expression construct the prep4-blrpwt-ires-bira-xl plasmid was a gift from dr. jeremy boss. the pcdna -hisa-hasox was constructed as described. to generate prep4-blrpwt-hasox4-ires-bira-xl ha-tagged sox from pcdna -hisa-hasox was excised at kpni and xbai sites and overhangs were filled in with klenow fragment. filled-in ha-sox was then blunt ligated into the filled-in noti site of prep4-blrpwt-ires-bira-xl purification of biotinlyated ha-tagged sox4 the prep4-blrpwt-hasox4-ires-bira-xl and control empty vector were transfected into two 90% confluent mm dishes of lncap cells respectively. forty-eight hours post-transfection, the plates were then placed on ice and the cells were washed twice with ice-cold pbs, and lysed in ml/plate with ice-cold ip lysis buffer supplemented with protease inhibitors, and harvested by scraping. biotinlyated ha-tagged sox complexes were purified by incubating with ul slurry of dynabeads® m streptavidin at 4°c for hrs. the beads ware washed times with ip lysis buffer and eluted by boiling in laemmli sample buffer. sample preparation for mass spectrometry five percent of the proteins resulting from the purification were subject to 4-15% gradient sds-page and silver staining to analyze sample purity. the rest of the purified proteins were subjected to 4-15% gradient sds-page and concentrated on a very short distance, and the protein in those bands were excised for in-gel digestion and liquid chromatography coupled with tandem mass spectrometry based on an optimized protocol. co-immunoprecipitation and western blot cells were washed twice with ice-cold pbs, lysed in ml/plate with ice-cold ip lysis buffer supplemented with protease inhibitors, and harvested by scraping. the whole cell lysates were pre-cleared and then incubated with ul slurry of dynabeads® m streptavidin or protein g at 4°c for hrs. the beads were washed times with ip lysis buffer and eluted by boiling in laemmli sample buffer before running sds-page. western blots were performed as previously described. antibodies to plakoglobin and hemagglutinin were used in immunoprecipitations. antibodies to plakoglobin, sox, β-actin, and ha 16b were used in western blot. confocal microscopy sub-confluent lncap hasox stable cells were grown on glass cover slips, and serum starved for hrs with % fbs t-medium before treating with ng/ml wnt3a, μm lmb, or both for another hrs. cells were washed three times with hank's balanced salt solution and fixed with % paraformaldehyde for min at room temperature. cells were permeablized with 100% ethanol for min at -20°c and blocked in 3% bsa in hbss+ for 1hr. anti-ha 16b and plakoglobin antibodies were used as primary antibodies and were diluted in blocking buffer and incubated for hr. then cells were washed in hbss+ and incubated in fluorescence-labeled secondary antibodies for 1hr at room temperature. cells were washed in hbss+ and then stained with topro for min at room temperature. labeled cells were then washed in hbss+ and mounted in p-phenylene diamine antifade agent. confocal fluorescence images were captured using a laser-scanning microscope. cell fractionation into nuclear and cytosolic lysates one 100-mm dish of lncap hasox stable cells was grown to 80-90% confluency and serum starved for hrs with % fbs t-medium before treating with ng/ml wnt3a, μm lmb, or both for another hrs. crude subcellular fractionation was performed as previously described using digitonin, np and ripa lysis methods. quantitative real-time pcr ninety-percent confluent cells were harvested using the rneasy kit, and reverse transcription was performed using iscript cdna synthesis kit. quantitative real-time pcr was performed using iq sybr green supermix on a bio-rad icycler using 18s or β-actin as a control, and data were analyzed using the δct method. chromatin immunoprecipitation assay one 100-mm dish of lncap hasox stable cells was grown to 80-90% confluency. cells were fixed with 1% formaldehyde, then lysed, and sonicated as described. sonicated chromatin was precleared and then immunoprecipitated with μg of anti-ha 12ca ascites or mouse igg overnight and immunoprecipitated by dynabeads® protein g for hrs at 4°c. beads were washed and eluted as described. chip dna was purified and then subjected to pcr amplification. luciferase reporter assay cells grown in twelve-well tissue culture plates were transfected with μg of either top-flash or fop-flash with μg of tk-renilla control vector. at hrs post-transfection, cells were placed in % fbs medium for recovery and serum starvation. at hrs post-transfection, cells were treated with wnt3a and lmb, or both. reporter gene activity was measured in a td-20/ luminometer with the dlr luciferase assay system after hrs and was normalized for transfection efficiency by measuring renilla luciferase activity. sirna transfection the sirna sequence for plakoglobin agtcggccattgtgcatct was targeted at the 5' end of the gene lacking of homology with other catenin members. lncap hasox cells were transfected using lipofectamine with plakoglobin or scramble sirnas at final concentration of nm. at hrs post-transfection, cells were placed in % fbs medium for recovery and serum starvation. at hrs post-transfection, cells were treated with wnt3a, lmb, or both for western blot analysis or chromatin immunoprecipitation assay.  identification of proteins that interact with sox4 to identify proteins that stably interact with sox we developed a one-step affinity purification method that allows for rapid purification of sox complexes. the prep4-blrpwt-ires-bira-xl plasmid contains the bira gene of e. coli that encodes a biotin holoenzyme synthetase. we cloned the human sox gene into this vector to generate an amino-terminal fusion to a bira recognition sequence to produce transiently expressed, intracellularly biotinylated sox protein in lncap prostate cancer cells, and purified sox complexes with streptavidin-linked magnetic beads to perform large scale proteomics analysis. protein quantity and purity were checked by sds-page analysis and silver staining prior to mass spectrometry analysis. plakoglobin interacts with sox4 lc-ms/ms analysis identified junction plakoglobin as a sox binding protein via two trypsinized fragments that perfectly matched to human plakoglobin sequences. to confirm this interaction, we first repeated the transient transfection of the prep4-blrpwt-ires-bira-xl9-hasox or vector control plasmid into lncap cells, and performed streptavidin-magnetic bead based purification followed by anti-plakoglobin immunoblotting. as expected, the result of the immunoprecipitation and western blot validated the interaction between sox and plakoglobin. furthermore, we performed a reverse-ip immunoprecipitating endogenous plakoglobin and probing for transfected ha-sox and endogenous sox in untransfected lncap cells. to determine that the interactions were not cell-line specific or dna-dependent, we repeated the immunoprecipitations in primary human keratinocytes, mda-mb- breast cancer cells, and pc3m prostate cancer cells following dnase i digestion of whole cell lysates. taken together, these data demonstrate that sox directly interacts with plakoglobin and that plakoglobin is a novel sox binding partner. interaction between sox and plakoglobin in the nucleus responds to wnt signaling when plakoglobin is present in desmosomes, it interacts with desmoglein and desmocollin, and when in adherens junctions it interacts with e-cadherin in the cytoplasmic component. recently, additional evidence has suggested that plakoglobin contributes a low level of transcriptional activity to the wnt signal transduction cascade in the nucleus. although, it has been confirmed that sox modulates wnt signaling via interaction with β-catenin, the role of plakoglobin in wnt signaling is still debated. to investigate where and under what conditions sox and plakoglobin interact with each other, we used confocal microscopy to determine whether we could observe subcellular co-localization of ha-sox and plakoglobin. after treatment of pc3m cells that stably-expressed ha-sox with recombinant human wnt3a, we observed that the interaction between ha-sox and plakoglobin was slightly increased in the nucleus. in contrast, this was not observed in the non-transfected adjacent cells. we observed the same phenomenon in the lncap-ha-sox stable cell line. furthermore, when we treated with nuclear export inhibitor, leptomycin b to inhibit nuclear export of sox and plakoglobin, the co-localization was significantly increased compared to treating the cells with either wnt3a or lmb alone. to confirm these enhanced interactions, we performed co-immunoprecipitation under the same wnt-induced conditions in lncap-ha-sox cells. the interaction in the whole cell lysate was quantified and significantly increased when we treated with wnt3a and lmb together. in addition, to determine that sox and plakoglobin interact in the nucleus, we prepared cytosolic and nuclear fractions to confirm the subcellular interaction. immunoblots against akt and nuclear lamin were used as controls to demonstrate the purity of the nuclear and cytosolic fractions. the quantitative results showed that the interaction in the nuclear but not cytosolic fraction was significantly increased in response to the wnt3a and lmb treatment. these results show that sox and plakoglobin physically interact in the nucleus of lncap cells. wnt target genes and sox4-target genes are affected by sox4-plakoglobin interaction to address the functional consequences of modulation of sox transcriptional activity by the sox4-plakoglobin complex, we tested expression of several genes including the wnt target gene axin, as well as sox targets dicer and dhx. to characterize if sox dna binding activity is changed by wnt-induced interaction with plakoglobin, we performed chip assays for axin dicer dhx and sox. compared to untreated lncap hasox cells, the binding of sox to axin dicer and dhx promoters was increased after wnt signaling was induced. however, sox binding was decreased after treatment with both wnt3a and lmb. this difference indicates that increasing the interaction between sox and plakoglobin could inhibit sox binding activity to downstream targets and may inhibit sox transcriptional activity. quantitative realtime-pcr analysis of the lncap sox stable cell line showed reductions in axin sox and dhx upon co-treatment with wnt3a and lmb. to determine whether the effects from combined wnt3a and lmb treatment were dependent on plakoglobin, we targeted plakoglobin by sirna to determine whether reduced plakoglobin levels could rescue the effects we observed on sox binding to target promoters. transfection of lncap ha-sox cells with plakoglobin sirna or scrambled control sirna resulted in approximately 50% knockdown of endogenous plakoglobin protein levels. furthermore, plakoglobin sirna partially rescued sox binding to target promoters by chip assay in the presence of wnt3a and lmb, while scrambled sirna had no effect. these data suggest that sox4-plakoglobin interactions may interfere with sox4-mediated transcription in response to wnt signaling due to reduced promoter occupancy. sox4-plakoglobin complex modulates β-catenin-mediated transcriptional activity to evaluate whether sox4-plakoglobin complex affects the transcriptional activity of β-catenin, we performed luciferase reporter assays with t cell factor reporter plasmids containing wild type tcf binding sites or mutated tcf binding site. as expected, the tcf/β-catenin luciferase reporter was significantly increased after we induced wnt signaling with recombinant wnt3a when compared to untreated cells. in contrast, co-treatment with wnt3a and lmb, strongly inhibited increases in luciferase activity back to baseline unstimulated levels, suggesting that wnt3a+ lmb-induced sox4-plakoglobin complexes could compete with and inhibit the transcriptional activity of β-catenin. in addition, we performed anti-β-catenin chip assays on the axin c-myc, and dkk promoters, and observed that occupancy of these promoters was stimulated by wnt3a, but that stimulation was inhibited by co-treatment with wnt3a and lmb. these results suggest that β-catenin activity was affected by the sox4-plakoglobin complex and that plakoglobin may compete with β-catenin binding to sox and/or tcf/lef in the nucleus. discussion while sox expression is elevated in many malignancies and is tightly correlated with prostate cancer tumor grade, little is known of the mechanism by which sox affects the progression of prostate cancer. using lc-ms/ms proteomic analysis, we identified a novel sox binding protein, junction plakoglobin, in lncap prostate cancer cells. we observed a physical association between sox and plakoglobin with both epitope-tagged and endogenous sox although the interactions of sox with plakoglobin were enhanced by co-treatment of wnt3a and lmb, we could detect this interaction using four different untransfected and unstimulated cell types treated with dnase i. in addition, confocal microscopy and co-immunoprecipitation demonstrated co-localization of sox and plakoglobin in the nucleus when wnt signaling was induced. chip assays showed the sox4-plakoglobin complex affected sox dna binding activity to the axin dicer and dhx promoters that are wnt signaling downstream genes and sox4-associated targets. in addition, mrna expression changes were detected in axin dicer and dhx by realtime-pcr. these data suggest that the sox4-plakoglobin complex may inhibit wnt signaling. indeed, conditions that induced the increased interaction between sox and plakoglobin caused β-catenin top-flash transcriptional activity to be downregulated and reduced occupancy of the c-myc promoter by β-catenin. it is known that sox can directly interact with β-catenin to enhance wnt signaling, but mechanistic data remain very limited. we found that sox interacts with plakoglobin in a wnt3a-dependent manner in our experimental cancer model. our model not only supports the hypothesis that sox may stabilize β-catenin, but also suggests a model in which sox can modulate wnt signaling by binding either β-catenin or plakoglobin. in this model, transcriptional responses to wnt signaling are increased by sox4-β-catenin interactions, and subsequently reduced by sox4-plakoglobin interactions that facilitate nuclear export of sox however, when nuclear export is inhibited, plakoglobin competes with β-catenin for binding to sox and tcf/lef factors, downregulating wnt-responsive transcription and reducing sox4-dna binding. this model is supported by the observation that combination treatment with wnt3a and lmb enhanced sox4-plakoglobin interactions, reduced tcf/β-catenin top-flash transcription, reduced sox4-dna binding and β-catenin dna binding in chip assays, and reduced expression of sox downstream targets. consistent with our model, plakoglobin shows little transcriptional activity compared to β-catenin in cell lines that lack β-catenin. our confocal data show that plakoglobin nuclear localization is strongly enhanced by lmb co-treatment, suggesting shuttling of plakoglobin into and out of the nucleus following wnt3a stimulation. although lmb is an artificial stimulus, others have shown that plakoglobin overexpression can lead to nuclear localization, and plakoglobin is overexpressed and amplified in several types of cancer. moreover, plakoglobin nuclear localization was much stronger in cells expressing hasox suggesting cytoplasmic sox may facilitate plakoglobin nuclear import. thus, sox may induce nuclear import of plakoglobin in response to wnt3a while plakoglobin destabilizes sox from dna binding, facilitating nuclear export of sox it is as yet unclear what signals or modifications might tip the balance between nuclear import and export of plakoglobin-sox complexes, or whether shuttling might be constitutive. the role of plakoglobin during cancer progression is still controversial. when plakoglobin is overexpressed, it induces cell migration and mobility in hct cells, suggesting that plakoglobin may have some oncogenic effects. in contrast, several reports have demonstrated that plakoglobin has a tumor-suppressive effect that inhibits tumor cell growth. the tumor suppressive activity of plakoglobin may be via nuclear translocation to antogonize β-catenin binding to tcf/lef proteins in keratinocytes, supporting our model that plakoglobin competes with sox4-β-catenin interactions in the nucleus.  in summary, we have demonstrated that sox interacts with plakoglobin in a wnt-dependent manner in lncap cells and that this complex may function to inhibit wnt signaling. additional studies will be required to elucidate the detailed mechanisms by which sox4-plakoglobin interactions may affect wnt signaling, but the role of the sox4-plakoglobin complex provides novel insights into the role of sox in wnt signaling and prostate cancer progression. authors' contributions yhl performed the immunopurifications of sox the chip assays, co-ips, luciferase assays, and helped write the manuscript. jc cloned sox into the prep4-blrpwt-ires-bira-xl plasmid and performed co-ips. dc performed the proteomics analysis. mef and kdb performed immunofluorescence confocal microscopy. jp advised on protein purification, supervised the proteomics analysis, and edited the manuscript. an supervised the confocal microscopy and edited the manuscript. csm conceived the study, participated in its design and coordination, and co-wrote the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 figure.s wnt signaling induces nuclear colocalization of sox and plakoglobin. subcellular localization of plakoglobin and sox were examined by confocal microscopy. lncap hasox stable cell line was treated with ng/ml human recombinant wnt3a or μm leptomycin b, or both wnt3a+lmb for hr. the fields shown were visualized independently by confocal microscopy at the appropriate wavelength for plakoglobin and sox, and topro respectively, and then the three images were overlaid. strong nuclear localization of plakoglobin was observed in the wnt3a+lmb treated cells that expressed hasox representative fields from these independent repeated experiments are shown. plakoglobin localizes to the nucleus following wnt3a treatment, and this effect is strongly enhanced by lmb co-treatment, suggesting shuttling of plakoglobin into and out of the nucleus following wnt3a stimulation. note that plakoglobin nuclear localization is much stronger in cells expressing hasox suggesting sox may facilitate plakoglobin nuclear import. click here for file acknowledgements we thank dr. tamara caspary for advice on experimental conditions for lmb treatments, dr. chris scharer for advice on sox binding site analysis, dr. cornel phillip for advice with wnt3a treatment, and dr. andrew kowalczyk for providing primary human keratinocytes. this work was supported by national cancer institute grants r01ca and p30ca.
conditions that enhanced sox4-plakoglobin interactions resulted in reduced transcriptional activity of β-catenin luciferase reporters. these data suggest that this newly identified interaction between sox and plakoglobin is inhibitory and provides new insights into the role of sox in key pathways in cell proliferation, development, and cancer progression. here, we have adapted a one-step affinity purification method that enables rapid purification of sox complexes via intracellular biotinylation of the amino-terminus of sox to perform large-scale proteomics analysis. sox is overexpressed in many human malignancies, but the precise role of sox in cancer progression is still not well understood. thus, the identification of additional sox binding partners is essential for elucidating the mechanism of sox4-mediated effects in cancer progression.
 sox is a developmental transcription factor that is required for differentiation and proliferation in multiple tissues. sox is overexpressed in many human malignancies, but the precise role of sox in cancer progression is still not well understood. thus, the identification of additional sox binding partners is essential for elucidating the mechanism of sox4-mediated effects in cancer progression.  here, we have adapted a one-step affinity purification method that enables rapid purification of sox complexes via intracellular biotinylation of the amino-terminus of sox to perform large-scale proteomics analysis. we have discovered that junction plakoglobin interacts with sox in both the cytosol and the nucleus and the interaction between sox and plakoglobin is significantly increased when prostate and breast cancer cells are stimulated with wnt3a. interactions between sox and plakoglobin were further enhanced by the nuclear export inhibitor leptomycin b, suggesting that plakoglobin promotes nuclear export of sox the sox4-plakoglobin complex affected the expression of wnt pathway target genes and sox downstream targets, such as axin dicer and dhx in addition, sox dna binding activity to the promoters of dicer axin dhx and sox itself was reduced by conditions that promote sox4-plakoglobin complex formation. conditions that enhanced sox4-plakoglobin interactions resulted in reduced transcriptional activity of β-catenin luciferase reporters.  these data suggest that this newly identified interaction between sox and plakoglobin is inhibitory and provides new insights into the role of sox in key pathways in cell proliferation, development, and cancer progression. 