FEBS Letters 584 (2010) 3923–3928

journal homepage: www.FEBSLetters .org

Identification of ptpro as a novel target gene of Wnt signaling and its potentialrole as a receptor for Wnt

Minseong Kim 1, Hanjun Kim 1, Eek-hoon Jho ⇑Department of Life Science, The University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 4 August 2010Revised 19 August 2010Accepted 21 August 2010

Edited by Lukas Huber

Keywords:Wntprotein tyrosine phosphatase receptor typeOb-CateninT cell factor/lymphoid enhancer factorWnt receptor

0014-5793/$36.00 � 2010 Federation of European Biodoi:10.1016/j.febslet.2010.08.034

Abbreviations: PTPRO, protein tyrosine phosphadenomatous poly coli; GSK3b, glycogen synthase kinlymphoid enhancer factor; LRP5/6, low density lipopro5/6; RT-PCR, reverse transcriptase-PCR; ChIP, chromaimmunoglobulin Fc domain⇑ Corresponding author. Fax: +82 22210 2888.

E-mail address: ej70@uos.ac.kr (E.-h. Jho).1 These authors contributed equally.

Wnt/b-catenin signaling plays critical roles in embryonic development and tissue homeostasis inadults by controlling the expression of target genes. We found that expression of ptpro, whichencodes a protein tyrosine phosphatase receptor type O (PTPRO), was induced by Wnt/b-catenin sig-naling in a T cell factor/lymphoid enhancer factor dependent manner. Biochemical assays found thatPTPRO interacted with Wnt via its extracellular domain. In addition, ectopic expression of this extra-cellular domain inhibited Wnt-mediated reporter activity. These results suggest that ptpro is a tar-get gene of Wnt/b-catenin signaling and that PTPRO may function as a novel receptor for Wnt.

Structured summary:MINT-7992076: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt3a (uniprotkb:P27467)by anti tag coimmunoprecipitation (MI:0007)MINT-7992094: Ptpro (uniprotkb:Q7TSY7) physically interacts (MI:0915) with Wnt-3a (uniprotkb:P27467)by cross-linking study (MI:0030)

� 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Dishevelled, leading to inactivation of the b-catenin destruction

Wnt signaling plays pivotal roles in the regulation of cell pro-liferation and fate determination. Mis-regulation of Wnt signalingis known to be the pathogenesis of many human diseases anddevelopmental defects [1,2]. Wnts are a family of secreted glyco-proteins that can transduce signals via regulation of its keyeffector molecule, b-catenin [3]. In the absence of Wnt, cytoplas-mic b-catenin is constitutively degraded by a destruction complexcomprising Axin, adenomatous poly coli, glycogen synthase ki-nase 3b (GSK3b) and other proteins [4]. Under these conditions,T cell factor/lymphoid enhancer factor (Tcf/Lef1) acts as a tran-scriptional repressor that blocks the expression of Wnt targetgenes by forming a complex with Groucho and other proteins[5,6]. However, in the presence of Wnt, binding of Wnt to itsreceptor Frizzled and co-receptor low density lipoprotein recep-tor-related protein 5/6 leads to initiation of downstream Wnt sig-naling. Activated receptors transduce the signal through

chemical Societies. Published by E

atase receptor type O; APC,ase 3b; Tcf/Lef1, T cell factor/tein receptor-related proteintin immunoprecipitation; Fc,

complex. As a result, the level of cytoplasmic b-catenin is in-creased, and translocation to the nucleus allows b-catenin to in-duce numerous target genes by changing Tcf/Lef1 into atranscriptional activator [6,7]. To date, about 100 target genesfor Wnt have been identified, some of which enhance cellular pro-liferation or regulate differentiation, etc. (http://www.stanford.edu/~rnusse/pathways/targets.html).

Protein tyrosine phosphatase receptor type O (PTPRO) is a typeIII protein tyrosine phosphatase comprising seven repetitive fibro-nectin like III (FN III) repeats in its extracellular domain (ECD), atransmembrane domain, a juxtamembrane domain and one phos-phatase domain in its intracellular domain (ICD) (Fig. 3A) [8].PTPRO has been described as mPTP-BK, mGLEPP1 in mouse andCRYP-2 in chicken [9–11]. While receptor protein tyrosine kinasesignaling has been intensively studied, the role of protein tyrosinephosphatase, the identification of substrates/ligands or the physio-logical roles in signaling has not been examined so far. Severalstudies indicate that PTPRO is involved in axon guidance or properaxon projection [12]. Expression of ptpro is highest in embryonicday 16 of mouse embryos when axonogenesis and synapse forma-tions are most activated, and PTPRO works as a receptor to sense arepulsive guidance cue for retinal neuron growth cones [13,14].

We tried to identify novel target genes of Wnt signaling usingmicroarray analysis, which compares gene expression profiles byinducible expression of Dvl or b-catenin S37A (activated form of

lsevier B.V. All rights reserved.

3924 M. Kim et al. / FEBS Letters 584 (2010) 3923–3928

b-catenin) in the mouse fibroblast L929 cell line. We found that theexpression of ptpro was induced by the inducible expression of Dvlor b-catenin. Interestingly, we also discovered that PTPRO inter-acted with Wnt ligand through its ECD, resulting in inhibition ofWnt/b-catenin signaling. Overall, our data suggest that ptpro is atarget gene of Wnt/b-catenin signaling and that PTPRO may func-tion as a novel receptor for Wnt.

2. Materials and methods

2.1. Plasmids

Full-length mouse cDNA of PTPRO was ligated with pCS2-HA orpCMV5 vector to make a PTPRO-HA or PTPRO-Flag construct,respectively. To make PTPRO(ECD)-Flag or PTPRO(DEx)-Flag, PCRproducts containing the region from the 50 to 30 end of the extracel-lular domain or from the transmembrane domain to the 30 end ofthe mouse PTPRO cDNA were cloned into pCMV5 vector, respec-tively. For PTPRO-ECD(1-3)-Fc, a PCR product containing the regionfrom the 50 end to the third FN III repeats of the extracellular do-main was cloned into pRK-Fc vector. To make Ptpro-Luc, about2 kb from the transcription initiation site of ptpro was amplifiedby PCR and ligated into pGL3-basic vector.

2.2. Cell culture and transient transfection

HEK293T, L929 and CaCO2 cells were maintained in Dulbecco’sModified Eagle’s Medium (DMEM, Gibco BRL) supplemented with10% Fetal Bovine Serum (FBS) and antibiotics. For transient trans-fection experiments, each plasmid DNA was transfected by the cal-cium phosphate precipitation method. For L929 cells, Welfect-Ex™was used according to the manufacturer’s instruction (Welgene).The amount of DNA in each transfection was held constant bythe addition of an appropriate amount of empty expression vector.

2.3. RNA isolation and microarray analysis

Isolation of total RNAs from inducible stable cell lines express-ing EGFP, Dvl or b-catenin S37A and microarray analysis were per-formed as described elsewhere [15,16].

2.4. Reverse transcriptase-PCR and real-time PCR analysis

Total RNA was isolated using TRIzoL reagent (Invitrogen)according to the manufacturer’s protocol. cDNA was synthesizedfrom total RNA using ImProm-II™ Reverse Transcriptase (Promega)with random primer. Amplification was performed under the fol-lowing conditions: 94 �C for 2 min followed by 30 cycles at 94 �Cfor 45 s, 58 �C for 40 s and 72 �C for 40 s. Real-time PCR was carriedout using a PRISM 7000 Sequence Detector (Applied BioSystems)with SYB GREEN PCR Master Mix (Applied BioSystems). The primersused were: for b-actin, 50-AGGCCAACCGCGAGAAGATGACC-30

and 50-GAAGTCCAGGGCGACGTAGCAC-30; for Ptpro, 50-GAC-TCGTTCATCAAACCACC-30 and 50-CTGTGGGCTTCTCTGTCCTA-30.The threshold cycle (Ct) value for each gene was normalized tothe Ct value for b-actin. The relative mRNA expression was calcu-lated using the formula: 2�DDCt, where DCt = CtPtpro � Ctb-actin andDDCt = DCtDvl or b-catenin � DCtEGFP.

2.5. Chromatin immunoprecipitation (ChIP) assay

Cells were cross-linked with 1% formaldehyde (Sigma) at roomtemperature for 10 min and then incubated with 0.125 M glycinefor 5 min. Re-suspended cells in hypotonic buffer (10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 1.5 mM MgCl2) were swollen on ice for

10 min and then passed through a 26.5 gauge needle 6 times. Aftercentrifugation at 1000�g for 5 min at 4 �C, the pellets were incu-bated with nuclei lysis buffer (1% SDS, 50 mM Tris–HCl, pH 8.0,10 mM EDTA) for 10 min on ice with occasional vortexing. Chroma-tin was sheared to an average length size of 0.2–1 kb by sonicationon ice. The appropriate volume of chromatin was diluted to 1/10 inChIP dilution buffer (0.01% SDS, 20 mM Tris–HCl, pH 8.0, 167 mMNaCl, 1.2 mM EDTA, 1.1% Triton X-100), after which preclearingwas performed at 4 �C for 2 h with 10 ll of protein A/G plus-agarosebeads (Santa cruz Biotechnology Lnc). For immunoprecipitation,goat, rabbit-IgG (Bethyl) and anti-LEF1 (Santa cruz BiotechnologyLnc, Cell Signaling) were used at 4 �C overnight. Immunoprecipi-tated chromatins were eluted, and reverse cross-linking wasachieved by the addition of 0.3 M NaCl at 65 �C overnight. Followingphenol–chloroform extraction and ethanol precipitation, DNA wasdissolved in 50 ll of TE buffer (10 mM Tris–HCl, pH 8.0, 1 mMEDTA). For PCR, 2 ll of DNA was used.

2.6. Immnunoprecipitation and Western blotting

For immnunoprecipitation, cells were lysed with RIPA buffer(20 mM Tris–Cl (pH 7.4), 120 mM NaCl, 1% Triton X-100, 0.25%Na-deoxycholate, 0.05% SDS, 50 mM sodium fluoride, 5 mM EDTA,0.5 mM sodium orthovanadate, 2 lg/ml of leupeptin, 2 mM PMSF)After 30 min on ice, the lysate was centrifuged at 12 000�g for10 min, after which the supernatant was collected. Between 300and 800 lg of lysate was then incubated with anti-Flag M2 oranti-HA antibodies and Protein A/G Plus-agarose beads (Santa CruzBiotechnology Inc.).

Immunoprecipitation under cell-free conditions was performedas follows. Conditioned media from HEK293T cells transfectedwith Wnt1-HA, PTPRO-ECD(1-3)-Fc, mFz8(CRD)-Fc or immuno-globulin Fc domain (Fc) were collected. As indicated in Fig. 3D,the conditioned media were mixed overnight and concentratedusing a centricon-20 (Millipore). The concentrated media wereincubated overnight with Protein A/G Plus-agarose beads, whichcan recognize and precipitate Fc. Immnoprecipitate was subjectedto SDS–PAGE, and Western blotting was performed using the anti-bodies indicated in the figure.

For Western blotting, the following antibodies were used:monoclonal anti-Flag-M2 antibody from Stratagene, anti-b-cateninfrom BD bioscience, anti-HA, anti-a-tubulin monoclonal antibodiesfrom Santa Cruz Biotechnology. Peroxidase-conjugated goat anti-mouse and anti-rabbit secondary antibodies (Jackson Lab) and goatanti-mouse secondary antibody (Santa Cruz Biotechnology, Inc.)were used, and the proteins were detected using an enhancedchemiluminescence reagent (ELPIS).

2.7. Crosslinking of proteins using BS3

HEK293T cells were transfected with plasmids as indicated inFig. 3C for 24 h. After transfection, the culture media was removed,followed by washing three times with phosphate buffered saline(PBS). The cells were then incubated in PBS containing 5 mM Bis(sul-fosuccinimidyl)suberate (BS3), which is a homobifunctional, water-soluble and membrane-impermeable cross-linker, for 30 min atroom temperature. After removal of the cross-linking solution, cellswere incubated with quenching solution (20 mM Tris–HCl, pH 7.5)in order to inactivate the residual activity of BS3. Cells were washedtwice with PBS followed by lysis with RIPA buffer for subsequentexperiments.

2.8. Preparation of conditioned media

To obtain conditioned medium (CM) for the cell-free immuno-precipitation experiments, HEK293T cells were transfected with

M. Kim et al. / FEBS Letters 584 (2010) 3923–3928 3925

the plasmids depicted in Fig. 2D. One day after transfection, theculture medium was exchanged with fresh medium containing1% FBS and 1% antibiotics. After 36 h incubation, the mediumwas collected and used as conditioned medium.

2.9. TOPFLASH luciferase assay

HEK293T cells were transfected with the following plasmids:0.5 lg of reporter plasmid (pSuperTop), 50 ng of pRL-TK, 0.5 lgof Fc, mFz(CRD)-Fc, PTPRO(ECD)-Flag or PTPRO-HA. Luciferaseactivities were measured 24 h after transfection using a Dual-Lucif-erase reporter assay kit (Promega).

Fig. 2. Tcf/Lef1 is involved in b-catenin mediated induction of ptpro promoter activity. (A)Arrows in the promoter indicates primers that were used in the ChIP assay. (B) Tcf/Lef1 bthat were isolated from L929 cells treated with MeBIO or BIO (2 lM each for overnighsequences were amplified using primers specific to the mouse ptpro promoter. (C) Exprmeasure the effect of b-catenin(S37A) on the activity of the ptpro promoter, L929 cells w(Ptpro-Luc), followed by measurement of luciferase activities.

Fig. 1. Identification of ptpro as a target gene of Wnt/b-catenin signaling. (A) Schematicpresence of Dox, activated rtTA binds to the bidirectional TRE promoter to induce expranalysis shows that the expression of ptpro was enhanced by the inducible expression o2 lM BIO) in mouse fibroblast L929 cell line. Gastrin is a known target gene of Wnt/b-cptpro or gastrin. RNAs isolated from mouse fibroblast L929 cell lines inducibly expressinenhances the level of PTPRO. L929 cells were treated with either NaCl or LiCl (30 mM forLiCl worked as expected. b-Actin was used as loading control. Asterisk indicates non-sp

3. Results

3.1. Expression of ptpro was increased by Wnt/b-catenin signaling

To identify novel target genes of Wnt/b-catenin signaling, weperformed microarray analysis using RNAs isolated from themouse fibroblast L929 cell line, which inducibly expressed mouseDvl1 (mDvl) or b-cateninS37A (constitutive active form of b-cate-nin) (Fig. 1A). Microarray analysis suggested ptpro as a candidatetarget gene. In the mDvl and b-cateninS37A stable cell line, themRNA level of ptpro was significantly increased compared to con-trol cells (Fig. 1B). In addition, the mRNA level was also increased

Schematic diagram showing conserved Tcf/Lef1 binding sites in the ptpro promoter.inds to the proximal site of the ptpro promoter. Cross-linked chromatin complexest) were immunoprecipitated with IgG or anti-Lef1 antibody. Co-precipitated DNAession of luciferase under the ptpro promoter was induced by b-catenin(S37A). Toere transfected with the indicated plasmids in the figure and a reporter construct

representation of Tet-On inducible system used in our microarray analysis. In theession of downstream genes such as EGFP, b-catenin(S37A) or mDvl1. (B) RT-PCRf mDvl1 or b-catenin(S37A), or by treatment with GSK3b inhibitors (30 mM LiCl oratenin signaling. GAPDH was used as loading control. (C) Real-time PCR analysis ofg EGFP, mDvl1 or b-catenin(S37A) were used. (D) Treatment of L929 cells with LiCl

24 h) and lysed for Western blotting. The increased level of b-catenin showed thatecific band and arrow indicates PTPRO.

3926 M. Kim et al. / FEBS Letters 584 (2010) 3923–3928

upon treatment with LiCl or 6-bromoindirubin-30-oxime (BIO)(specific GSK3b inhibitors that activate Wnt/b-catenin signaling)but not with control NaCl or MeBIO treatment (Fig. 1B, upper pa-nel). These patterns were very similar to the expression patternof gastrin, a well-known Wnt/b-catenin target gene (Fig. 1B, middlepanel). Real-time PCR analysis confirmed that expression of ptprowas induced by the inducible expression of mDvl and b-cate-ninS37A (Fig. 1C). Moreover, the protein level of PTPRO was alsoincreased by treatment of LiCl (Fig. 1D, arrow). Taken together,these data suggest that ptpro is a novel target gene of Wnt/b-cate-nin signaling.

3.2. Tcf/Lef1 transcription factor is involved in regulation of the Wntmediated induction of ptpro

Since most of the target genes of the Wnt/b-catenin signalingpathway are mediated by Tcf/Lef1, we investigated whether ornot Tcf/Lef1 transcription factor could bind directly to putativeTcf/Lef1 binding sites. We identified three TCF binding sites (50-A/T A/T CAAAG-30 in Fig. 2A) in the promoter region of mouseand rat ptpro using the Blat program at UCSC Genome Bioinfor-

Fig. 3. PTPRO interacts with Wnt via its extracellular domain. (A) Schematic diagram of coJD, juxtamembrane domain; PTP, protein tyrosine phosphatase domain. Flag or Fc (immlight gray, respectively. PTPRO(ECD)-Flag or PTPRO-ECD(1-3)-Fc encodes proteins that canwas immunoprecipitated with Wnt3a-HA but not with PTPRO(DEx)-Flag. HEK293T cellsindicated in the figure. After transfection, lysates were immunoprecipitated with anti-Flinked with PTPRO-Flag, but not with PTPRO(DEx)-Flag. After transfection with the indicusing BS3, followed by immunoprecipitation with anti-Flag antibody and blotting with a(bottom panel). (D) Extracellular domain of PTPRO interacts with Wnt1 under cell-free coCM was collected and immunoprecipitated with A/G Plus agarose and then blotted withPTPRO-ECD(1-3)-Fc in cell lysates were detected with anti-human IgG antibody or anti-HFc or PTPRO-ECD(1-3)-Fc.

matics Site (http://genome.ucsc.edu/, data not shown). ChIP as-say was performed to determine whether or not Lef1 binds tothe conserved Tcf/Lef1 binding site of the ptpro promoter. Asshown in Fig. 2B, ChIP with anti-Lef1 antibodies showed a posi-tive signal. Although treatment of cells with BIO enhanced theexpression of ptpro, it did not change the amount of Lef1 boundto the promoter (Fig. 2B). This may have been due to activationof target gene expression caused by Tcf/Lef1 changing its rolefrom a repressor to an activator via interaction with b-catenin[6].

To determine whether or not the transcription of ptpro is con-trolled at the transcriptional level, 2 kb of the putative promoterregion of ptpro was cloned upstream of luciferase cDNA and lucif-erase assays were performed. Co-transfection of b-cateninS37A en-hanced ptpro promoter-driven luciferase activity (Fig. 2C). Inaddition, this increase was blocked by exogenous expression ofDN-Tcf4 (dominant negative form of Tcf4 that binds to conservedDNA but cannot interact with b-catenin) in a dose-dependent man-ner (Fig. 2C). In summary, these data suggest that the level ofPTPRO was regulated at the transcriptional level by the activationof Tcf/Lef1 transcription factor.

nstructs used in this figure. ECD, extracellular domain; TD, transmembrane domain;unoglobulin Fc domain) epitope tags at the c-terminal end were marked as black or

be secreted into the media due to lack of a transmembrane domain. (B) PTPRO-Flagwere transfected for 24 h with PTPRO-Flag or PTPRO(DEx)-Flag with Wnt3a-HA aslag antibody and blotted with the indicated antibodies. (C) Wnt3a-HA was cross-

ated plasmids, PTPRO-Flag and PTPRO(DEx)-Flag were cross-linked with Wnt3a-HAnti-HA antibody (top panel). Total cell lysates were blotted with anti-HA antibodynditions. HEK293T cells were transfected with the plasmids indicated in the figure.anti-human IgG or anti-HA antibody (right panel). Wnt1-HA, Fc, mFz8(CRD)-Fc or

A antibody (left panel). Asterisks indicate expected sizes of bands for Fc, mFz8(CRD)-

Fig. 4. Negative role for PTPRO in Wnt-mediated reporter activity. (A) Ectopic expression of PTPRO(ECD)-Flag inhibited Wnt-mediated reporter activity. HEK293T cells weretransfected for 24 h with the indicated plasmids in the figure with reporter constructs (pSuperTopflash and pRL-TK). After transfection, luciferase activities were measured.Luciferase activities were normalized based on the activity of Renilla luciferase (pRL-TK). (B) Reporter constructs were transfected into CaCO2 colorectal cancer cells withmFz8(CRD)-Fc or PTP-BK-HA, followed by dual luciferase assay.

M. Kim et al. / FEBS Letters 584 (2010) 3923–3928 3927

3.3. PTPRO is a novel receptor of Wnt

PTPRO is a transmembrane protein that is mainly expressed inthe brain and kidney, and is shown to be involved in axon guidance[12,17]. Since Wnt is also known to be a morphogen for axon guid-ance, we examined whether or not PTPRO works as a novel recep-tor for Wnt. Immunoprecipitation experiments showed thatPTPRO-Flag, but not PTPRO(DEx)-Flag (a deletion construct lackingan extracellular domain (Fig. 3A)), was co-immunoprecipitatedwith HA-tagged Wnt3a (Fig. 3B and C). To determine if this inter-action occurs at the plasma membrane, a cell membrane non-per-meable cross-linking chemical, BS3, was used. We assumed that ifWnt and PTPRO interact with each other at the plasma membrane,treatment with a cross-linking reagent would cause a mobilityshift during SDS–PAGE. Consistent with our expectation, HA-Wnt3a, which was detected as a 40 kDa band, was shifted to thesize of PTPRO (about 220 kDa) after cross-linking (compare lanes3 and 4 in Fig. 3C). When total cell lysates were examined, we alsodetected a modest shift for Wnt3a-HA after cross-linking inPTPRO(DEx)-Flag transfected cells (lane 8 of the lower panel inFig. 3C). This shift might have been caused by interaction withendogenous receptor proteins such as Frizzled, LRP6 or PTPRO,since immunoprecipitation with anti-Flag antibody in lysates ofPTPRO(DEx)-Flag transfected cells did not produce any signalindicative of Wnt3a-HA (upper panel in Fig. 3C). Overall, these re-sults suggest that Wnt3a interacted with PTPRO at the plasmamembrane.

To eliminate the possibility that the interaction between PTPROand Wnt3a was an artifact caused by overexpression, we performedimmunoprecipitation under cell-free conditions. Conditioned med-ia collected from the culture media of HEK293T cells transfectedwith Wnt1-HA or PTPRO-ECD(1-3)-Fc (a construct containing threeextracellular FN3-like domains each fused with Fc) were used, sincethese proteins were secreted more than Wnt3a-HA or PTPRO(ECD)-Flag, respectively. The conditioned media indicated in Fig. 3D weremixed and immunoprecipitated with A/G Plus agarose, followed byWestern blotting with either anti-human IgG or anti-HA antibodies(Fig. 3D). mFz8(CRD)-Fc, which contains a cyteine rich domain ofmouse Frizzled8 that interacts with Wnt, was used as a positive con-trol. Both mFz8(CRD)-Fc and PTPRO-ECD(1-3)-Fc strongly inter-acted with Wnt1-HA, but Fc that was used as a negative controldid not (Fig. 3A and D). To confirm the interaction between Wntand PTPRO, we tested whether or not the ectopic expression ofPTPRO(ECD)-Flag inhibits Wnt-mediated reporter activity, similarto how ectopic expression of mFz8(CRD)-Fc inhibits Wnt/b-cateninsignaling by preventing Wnt from interacting with Frizzled [18]

(Fig. 4). Ectopic expression of PTPRO(ECD)-Flag inhibited Wnt-med-iated reporter activity while Fc did not. Overall, these results sug-gest that Wnt bound to the extracellular domain of PTPRO, whichserved as a novel receptor of Wnt.

Next, we examined whether or not the ectopic expression offull-length PTPRO has any effect on Wnt signaling. When plasmidencoding PTPRO-HA was transfected with reporter plasmids intoCaCO2 colorectal cancer cells, within which most Wnt proteinsare expressed and autocrine Wnt/b-catenin signaling can beblocked by the expression of secreted frizzled-related proteins(SFRPs) [19], the reporter activity was significantly reduced similarto the expression of mFz8(CRD)-Fc (Fig. 4D). This result suggeststhat the increased level of PTPRO due to Wnt signaling may playa role in negative feedback.

4. Discussion

In this report, we present evidence that PTPRO is a new target ofthe Wnt signaling pathway. We showed that the levels of both themRNA and protein of ptpro were increased by Wnt/b-catenin sig-naling. Consistent with this finding, it has been shown that theexpression of ptpro is reduced 12-fold in the kidney of Wnt4knockout mice [20]. In addition, ChIP and reporter assays sug-gested that the increased expression of ptpro was mediated byTcf/Lef1. Interestingly, we found that PTPRO interacted with Wntvia its extracellular domain and that ectopic expression of PTPROinhibited autocrine Wnt/b-catenin signaling in CaCO2 cells. Overall,these data strongly suggest that Wnt bound to PTPRO at the plas-ma membrane where it serves as a novel receptor for Wntsignaling.

Since hypermethylation at the promoter of ptpro in human lungcancer leads to reduced expression of ptpro, PTPRO is thought as atumor suppressor protein [21]. Consistent with this finding, it hasbeen shown that the expression of mir 17–92 cluster, which tar-gets the 30 UTR of ptpro, is increased in human lung cancer cells,which leads to reduction of PTPRO [22,23]. The reduction of Wntreporter activity by ectopic expression of PTPRO in CaCO2 cells(Fig. 4B) seems to be consistent with previous findings. Therefore,our data suggest that PTPRO may act as a tumor suppressor byblocking autocrine Wnt signaling.

No ligands of receptor type protein tyrosine phosphatase (RPTP)have been found thus far, besides the fact that pleiotrophin is a li-gand of RPTPb/f [24]. Although we could not identify any sub-strates of activated PTPRO, we provided evidence that Wnt was aligand for PTPRO and that PTPRO may have inhibited Wnt signaling(Fig 3). PTPRO may inhibit Wnt signaling not only by sequestration

3928 M. Kim et al. / FEBS Letters 584 (2010) 3923–3928

of Wnt ligand but also by regulation of other Wnt signaling compo-nents. For example, Drosophila homolog of PTPRO, ptp4e or ptp10d,plays an inhibitory role in EGF signaling by genetically interactingwith epidermal growth factor receptor (EGFR) [25]. It may be pos-sible that activation of PTPRO by binding with Wnt regulates EGFsignaling as well. Therefore, identification of substrate(s) that spe-cifically interact with PTPRO upon binding of Wnt will allow us tomine novel signaling pathway(s) that can crosstalk with Wntsignaling.

Acknowledgements

We appreciate Dr. Roger Wiggins (anti-PTPRO antibody), Dr.Takuji Shirasawa (PTP-BK-Flag plasmid) and Dr. Xi He (pRK-mFz8(CRD)-Fc and pRK-Fc vector) for sending reagents that wereused in this work. This work was supported by the Korea ResearchFoundation (Grant C00711) and Brain Korea 21 program.

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