Identification of a Novel Negative Regulator of Activin/NodalSignaling in Mesendodermal Formation of Xenopus Embryos*

Received for publication, April 13, 2009 Published, JBC Papers in Press, April 22, 2009, DOI 10.1074/jbc.M109.007443

Seong-Moon Cheong, Hyunjoon Kim, and Jin-Kwan Han1

From the Department of Life Sciences, Pohang University of Science and Technology, San 31, Hyoja Dong, Pohang,Kyungbuk 790-784, Republic of Korea

Phosphotyrosine binding (PTB) domains, which are found ina large number of proteins, have been implicated in signal trans-duction mediated by growth factor receptors. However, the invivo roles of these PTB-containing proteins remain to be inves-tigated. Here, we show that Xdpcp (Xenopus dok-PTB contain-ing protein) has a pivotal role in regulating mesendoderm for-mation in Xenopus, and negatively regulates the activin/nodalsignaling pathway. We isolated cDNA for xdpcp and examinedits potential role in Xenopus embryogenesis. We found thatXdpcp is strongly expressed in the animal hemisphere at thecleavage and blastula stages. The overexpression of xdpcp RNAaffects activin/nodal signaling, which causes defects in mesend-oderm formation. In addition, loss of Xdpcp function by injec-tion of morpholino oligonucleotides leads to the expansion ofthe mesodermal territory. Moreover, we found that axis dupli-cation by ventrally forced expression of activin is recovered bycoexpression with Xdpcp. In addition, Xdpcp inhibits the phos-phorylation and nuclear translocation of Smad2. Furthermore,we also found that Xdpcp interacts with Alk4, a type I activinreceptor, and inhibits activin/nodal signaling by disturbing theinteraction between Smad2 and Alk4. Taken together, theseresults indicate that Xdpcp regulates activin/nodal signalingthat is essential for mesendoderm specification.

Phosphotyrosine binding (PTB)2 domain-containing pro-teins are implicated in signal transduction, protein trafficking,and cytoskeletal dynamics (1). The PTB domain is importantforprotein-proteininteractionsthatareeitherphosphotyrosine-dependent or -independent. As a result, PTB-containing pro-teins engage in a wide range of cellular functions (2). PTBdomain proteins play a pivotal role in signal transductionmedi-ated by various growth factors, including epidermal growth fac-tor (3), insulin (4, 5), nerve growth factor (6), and transforminggrowth factor-� (TGF-�) (7, 8). To mediate TGF-� signaling,PTB proteins such as Dab2 (7) and Dok1 (8) act as adaptors tostabilize the interaction between the receptor and Smad2/3.TGF-� signaling is implicated in a number of cellular func-

tions such as proliferation, migration, differentiation, and apo-

ptosis. Disturbance of TGF-� signaling is related to serioushuman diseases including cancer, fibrosis, and heritable disor-ders (9). TGF-� signaling is initiated by the binding of ligands totwo types of Ser/Thr kinase receptors, called type I and type IIreceptors. The type I receptor is activated by the type II recep-tor and propagates intracellular signaling to the nucleusthrough the phosphorylation of receptor-activated Smads(R-Smad) including Smad1, -2, -3, -5, and -8. Then, the activeSmad complex that is formed by the interaction of R-SmadwithSmad4 translocates into the nucleuswhere it regulates the tran-scription of target genes.TGF-� signaling is also crucial for developmental processes

including germ-layer specification and patterning duringembryogenesis. Xenopus late blastula embryo consists of threeregions with different cell fates including the animal cap, themarginal zone, and the vegetal mass (10). As development pro-ceeds, animal cap cells become ectodermal derivatives such asthe skin and nervous system, and marginal zone cells developinto mesodermal derivatives such as bone, blood, kidney, andmuscle. Vegetal cells grow into endodermal derivatives such aslung, pancreas, and digestive organs. In Xenopus early embryo,the mesodermal cell fate is established in the marginal zonebetween the animal and vegetal poles by inductive signals fromthe underlying endoderm. Several TGF-� family ligands, suchas activin, Vg1, and nodal-related proteins are responsible forthe induction of mesoderm and endoderm, as well as the sub-sequent embryonic patterning.In this study, we identified a novel PTB protein, Xdpcp

(Xenopus dok-PTB containing protein), as a negative regulatorof activin/nodal signaling, a branch of TGF-� superfamily sig-naling. Activin/nodal signaling is essential for mesoderminduction and patterning in Xenopus embryos (11). We showthat Xdpcp negatively regulates the activin/nodal signaling byinhibiting the interaction between the activin receptor andSmad2, and plays a pivotal role in the regulation of mesend-oderm formation during Xenopus embryogenesis.

EXPERIMENTAL PROCEDURES

Xenopus Embryos and Microinjection—Eggs were obtainedfromXenopus laevis primed with 600 units of human chorionicgonadotropin (DAE SUNG Microbiological Labs Co.), in vitrofertilized as described previously (12). Embryos were culturedin 0.33� modified Ringer until stage 8 and then transferred to0.1� modified Ringer until they reached the appropriate stagefor the experimentation outlined below. Developmental stagesof the embryos were determined according to Nieuwkoop andFaber (13).Microinjection using aNanoliter Injector (WPI)was

* This work was supported by a grant from the National R&D Program forCancer Control, Ministry of Health & Welfare, Republic of Korea Grant0720150, and the Brain Korea 21 project.

1 To whom correspondence should be addressed. Tel.: 82-54-279-2126; Fax:82-54-279-2199; E-mail: jkh@postech.ac.kr.

2 The abbreviations used are: PTB, phosphotyrosine binding; TGF-�, trans-forming growth factor �; MO, morpholino oligonucleotide; RT, reversetranscriptase; GFP, green fluorescent protein; HA, hemagglutinin; CA, con-stitutively active; BMP, bone morphogenetic protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 25, pp. 17052–17060, June 19, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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performed in 0.33� modified Ringer containing 4% Ficoll-PM400 (GE Healthcare).Plasmids, RNA Synthesis, and Morpholino Oligonucleotides—

For expression inXenopus embryos, the entire coding region ofXdpcp was cloned into the EcoRI and XbaI sites of the pCS2�vector and GFP-pCS2� vector, and into the ClaI site of theMyc-pCS2� vector. Deletion mutants of Xdpcp were clonedinto the EcoRI and XbaI sites of the GFP-pCS2� vector.Capped mRNAs were synthesized from linearized plasmidsusing the mMessage mMachine kit (Ambion). Xdpcp, Xdpcp-Myc, and other deletionmutants of Xdpcpwere linearized withNotI, and mRNA was synthesized using SP6 RNA polymerase.Antisense morpholino oligonucleotides (MO) were obtainedfrom Gene Tools. The morpholino oligonucleotide sequenceswere as follows: xdpcp MO, 5�-TTCCAGAGTGAAA-GCCATCATGTTG-3�; control MO, 5�-CCTCTTACCT-CAGTTACAATTTATA-3�. HA-hALK4, HA-hALK4(TD),HA-hALK4(KR), and Myc-Smad2 in the pCS2� vector werelinearized with NotI. Xnr-1 in the pCS2� vector was linearizedwith Asp718 and activin-�b in the pSP64T vector was linearizedwith EcoRI.In Situ Hybridization, RT-PCR, and Luciferase Reporter

Assay—Wholemount in situ hybridizationwas performedwithdigoxigenin-labeled probes as described by Harland (14). Anti-sense in situ probes against xdpcpwere generated by linearizingthe pBSKII-xdpcp construct with XcmI and transcribing withthe T7 RNA polymerase. RT-PCR analysis was performed asdescribed (15). Primers and amplification cycles for RT-PCRanalysis were as follows: xdpcp forward, 5�-CCCAAAC-CCCAAGGAATGGT-3�; xdpcp reverse, 5�-AGGATGGCAG-GTACCTTGTG-3� (25 cycles); primers for ODC, sox17, xbra,mix2, and chordinwere as described (15). For luciferase assay inXenopus embryo, the embryos were injected with the indicatedreagents into the animal regions of the 4-cell stage, and animalcap explants isolated at stage 9 were separated into three poolsof six explants each for assay in triplicate. Luciferase activitywas measured using the Dual-Luciferase Reporter Assay Sys-tem (Promega) according to the manufacturer’s protocol.Immunoprecipitation, Western Blotting, and Antibodies—

HEK293FT cells were transfected with the indicated constructsusing Lipofectamine-Plus Reagent (Invitrogen), sonicated inradioimmunoprecipitation assay (RIPA) buffer containing 150mM NaCl, 50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 1%deoxycholate, 2 mM EDTA, 25 mM �-glycerophosphatate, 100mM sodium fluoride, 1 mM Na3VO4, 0.1 M dithiothreitol, 20�g/ml aprotinin, 40 �g/ml leupeptin, and 0.75 mM phenyl-methylsulfonyl fluoride, and then centrifuged to remove insol-uble debris. The indicated antibodies were added to the super-natants and incubated at 4 °C for 6 h. Protein-A-Sepharoseconjugates (Zymed Laboratories Inc.) were added and the mix-ture was incubated at 4 °C for 3 h. The immune complexesbound to Protein-A beads were washed four times with RIPAbuffer and then subjected to SDS-PAGE andWestern blotting.Monoclonal antibodies against GFP, Myc, and HA, and poly-clonal antibodies against Actin, Myc, and HA were from SantaCruz Biotechnology. Polyclonal anti-phospho Smad2 and -1were from Cell Signaling.

RESULTS

Cloning and Expression Patterns of Xdpcp—Because PTBdomain proteins play pivotal roles inmany biological processes,we have attempted to identify the in vivo function of novel PTBdomain proteins. Toward this goal, we cloned cDNA encodinga novel PTB domain protein using a PCR-based method andsequence information in the Xenopus EST data base (Gen-BankTM accession number BC073514) and named it xdpcp(Xenopus dok-PTB containing protein). xdpcp cDNA consistsof 1782 nucleotides encoding a protein of 593 amino acids. PTBdomains are divided into three groups, including Shc-like, IRS-like, and Dab-like (16). As implied by the name, Xdpcp has adok-PTB domain, one of the IRS-like PTB domains. To inves-tigate the function of Xdpcp in the Xenopus embryo, we firstexamined the spatial and temporal expression patterns ofxdpcp, and found that it is strongly expressed in the animalhemisphere at the cleavage and blastula stages and around theanterior border of the neural plate and paraxial mesoderm atthe neurula stages (Fig. 1). Later in development, the grossexpression of xdpcp decreases, and it is only observed in thehead region at the tailbud and tadpole stages. Consistent withthe spatial expression patterns, the temporal expression pat-terns analyzed by RT-PCR indicated that its expression isstrongest at the cleavage and blastula stages and then it gradu-ally decreases after the gastrula stages (Fig. 1G).Overexpression of Xdpcp Leads to Defects in Mesendoderm

Formation—Based on its strong localization to the animalhemisphere at the late blastula stage, we focused on the func-tion of Xdpcp in early development. Thus, we addressed theeffects of the gain-of-Xdpcp function on the early patterning of

FIGURE 1. The spatial and temporal expression patterns of xdpcp. A–D,lateral views of the cleavage, blastula, and gastrula stage embryos showingxdpcp expressed in the animal regions. E, anterior view of a neurula stageembryo with dorsal at the top. The white arrow and arrowhead indicate theparaxial mesoderm and anterior neural plate region. F, lateral view of a tail-bud stage embryo with anterior at the left. The black arrow indicates the headregion. G, RT-PCR analysis showing the temporal expression patterns of xdpcpin Xenopus early development. Stages are indicated above the lanes. Orni-thine decarboxylase (ODC) serves as a loading control. The PCR cycles con-sisted of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, andextension at 72 °C for 1 min for 25 cycles. An, animal; Vg, vegetal; D, dorsal; V,ventral; A, anterior; P, posterior.

The Role of Xdpcp in Activin/Nodal Signaling

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Xenopus embryos. Our analysis of embryos overexpressedxdpcp RNA in two dorsal blastomeres at the 4-cell stagerevealed abnormal patterning such as bending and shorteningof the anterior-posterior axis (Fig. 2A). We reasoned that thisphenotype may be due to an abnormality in mesoderm forma-tion. Therefore, we examined whether overexpression of xdpcpinhibits the expression of endogenous mesodermal markerssuch as xbra, chordin, and goosecoid. As shown in Fig. 2B, theexpression of thesemesodermalmarkers is remarkably reducedin embryos with xdpcp overexpression. These data suggest aninhibitory role of Xdpcp in mesoderm formation during Xeno-pus early development.

As mentioned previously, PTB domain proteins are alsorelated to the activin/nodal signaling pathway, which isinvolved in mesendoderm formation during Xenopus earlydevelopment. Therefore, we initially hypothesized that Xdpcpregulates mesendoderm formation by inhibiting activin/nodalsignaling. To address whether overexpression of xdpcp affectsmesendoderm formation and activin/nodal signaling, we per-formed RT-PCR analysis. Functional analysis in Xenopus ani-mal caps showed that the injection of activinRNAcould inducemesodermal markers such as xbra, chordin, andmix2 as well asendodermal markers such as sox17, endodermin, and vegT, andthe induction of thesemarkers was inhibited by coexpression ofxdpcp RNA (Fig. 2C). However, Xdpcp did not affect theexpression of BMP-induced genes such as msx-1 and xvent-1,suggesting that Xdpcp has a specific function in activin/nodalsignaling (Fig. 2D). To examine the effects of Xdpcp on activin/

nodal signaling-dependent pro-moter activity, we carried out vari-ous luciferase assays using an ARE-Luc reporter containing threecopies of an ARE (activin-responseelement of the Xenopus mix2 pro-moter) (17) or a 6DE-Luc reportercontaining six copies of a DE(activin/nodal-inducible distal ele-ment of the Xenopus gsc promoter)(18). Similar to our findings withRT-PCR analysis, we observed thatXdpcp diminished transcriptionalactivity associated with activin/nodal signaling (Fig. 2, E and F).These results suggest that Xdpcpinhibits the formation of mesend-oderm by activin/nodal signaling.Xdpcp Knockdown Leads to Ex-

pansion of Mesodermal Territory—To investigate whether Xdpcp isindispensable for regulating mesen-doderm formation, we carried outloss-of-function analysis using anti-sense MO capable of depleting theXdpcp protein (19). We designed amorpholino to target the startcodon region of the xdpcp gene todisrupt translation of the xdpcpmRNA. To confirm the efficacy and

targeting specificity of the xdpcp MO, we coinjected the MOwithMyc-tagged xdpcpRNAswith andwithout theMO target-ing sites (untranslated region and open reading frame, respec-tively), and performed Western blot analysis with anti-Mycantibody. As shown in Fig. 3A, xdpcpMOcan specifically blockproduction of the Xdpcp protein. To examine the effects ofXdpcp depletion on embryonic patterning and mesoderm for-mation in Xenopus embryos, we injected xdpcp MO into thedorsal-animal regions of 4-cell stage embryos and thenobserved its effects on the patterning of embryos and theexpression of endogenous mesodermal markers such as xbraand goosecoid. Intriguingly, xdpcp knockdown embryosrevealed a slightly dorso-anteriorized phenotype, which istranslated into the dorso-anterior index 6 (20) (Fig. 3B). Thisdemonstrates that activin/nodal signalingmight be increased inresponse to Xdpcp depletion, because activin/nodal activityinduces dorso-anterior fates. Xdpcp-depleted embryos alsoshow an expansion in the expression of mesodermal markerstoward the ectodermal territory (Fig. 3C). To obtain more evi-dence that Xdpcp inhibits mesendoderm formation ofXenopusembryos, we performed RT-PCR analysis using mesodermalmarkers such as xbra, chordin, and mix2 and endodermalmarkers such as sox17 and endodermin. As shown in Fig. 3D,endogenous expression of these markers was decreased byxdpcp overexpression, whereas xdpcp knockdown leads to theincrease of these expression. These data suggest that the pres-ence of Xdpcp in the ectodermal region prevents the mesoder-mal territory from expanding.

FIGURE 2. Overexpression of xdpcp leads to defects in mesendoderm formation. A, xdpcp RNA (2 ng)-injected embryos show mesodermal defective phenotype. B, dorsal injection of xdpcp RNA inhibits the expres-sion of endogenous mesodermal markers such as xbra, chordin, and goosecoid (gsc). Four-cell stage embryoswere injected into the dorsal regions with 2 ng of xdpcp RNA or none, cultured until stage 10, and thensubjected to in situ hybridization. C and D, RT-PCR analysis revealing that Xdpcp inhibits the expression ofmesendodermal markers induced by activin in animal cap tissues, whereas it does not affect the expression ofthe Bmp target genes. �RT and �RT, control RT-PCR on the whole embryo RNA in the presence or absenceof reverse transcriptase; AC, uninjected animal cap cells; (�), the animal cap cells injected with activin-�B orbmp4 RNA alone. Ornithine decarboxylase (ODC) serves as a loading control. E and F, luciferase assays inXenopus embryos injected with combinations of indicated reagents. The amount of injected reagents is asfollows: activin-�B RNA, 5 pg; Xnr-1 RNA, 100 pg; xdpcp RNA, 2 ng; ARE-Luc reporter DNA, 40 pg; 6DE-Lucreporter DNA, 40 pg. Error bars represent the standard deviation.

The Role of Xdpcp in Activin/Nodal Signaling

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Xdpcp Inhibits the Activin/Nodal Signaling Pathway—Be-cause Xdpcp may inhibit activin/nodal signaling, we askedwhether activin-induced phenotypes can be rescued by forcedexpression of xdpcp. To answer this question, we testedwhether secondary axis formation by ectopic expression ofactivin into the ventral blastomeres of Xenopus 4-cell stageembryos could be rescued by coexpression of xdpcp RNA. Asshown in Fig. 4A, a partial secondary axis is formed by ectopicexpression of activin, and this phenotype is rescued by xdpcpRNA, but not by the control preprolactin RNA. These resultsconfirm that Xdpcp inhibits activin/nodal signaling.Because our combined data from RT-PCR, luciferase assay,

and ectopic axis formation reveal that Xdpcp diminishes theresponses to activin/nodal signaling, we asked whether Xdpcpinhibits phosphorylation of Smad2. To test this, we injectedactivin-�B, xdpcp RNA, and xdpcpMO into the animal regionof 4-cell stage embryos. We then isolated animal caps at theblastula stages, cultured them to stage 10, and then performed aWestern blot analysis with phospho-Smad2 antibody (Fig. 4C).

Injection of activin-�B inducedphosphorylation of Smad2, and thisphosphorylation was reduced bycoexpression of xdpcp. Interest-ingly, Xdpcp depletion by MOinduced more phosphorylation ofSmad2, which demonstrates thatthe endogenous function of Xdpcpis to negatively regulate activin/nodal signaling. However, Xdpcpdid not affect Smad1 phosphoryl-ation induced by Bmp signaling(Fig. 4D).Because Xdpcp inhibits the phos-

phorylation of Smad2, we examinedwhether it also affects the next stepin the pathway, the nuclear translo-cation of Smad2. The results ofimmunostaining analysis show thatactivin-induced nuclear accumula-tion of Smad2 is inhibited by coex-pression of xdpcp (Fig. 4E). BecauseXdpcp inhibits the phosphorylationand nuclear translocation of Smad2,we tested whether Xdpcp is incapa-ble of inhibiting the activin/nodalsignaling induced by constitutivelyactive Smad2 (CA-Smad2) (21). Asshown in Fig. 5A, the results ofRT-PCR analysis revealed thatCA-Smad2 could induce theexpression of mesendodermalmarkers, and this induction wasunaffected by xdpcp overexpres-sion. Next, we examined whetherthe morphological defects inducedby xdpcp overexpression could berescued by coexpression ofCA-Smad2. xdpcp overexpression

caused abnormal embryonic patterning such as the shorteningand bending of the anterior-posterior body axis, and these phe-notypes were partially rescued by coexpression of CA-Smad2(Fig. 5, B and C). Together, these results indicate that Xdpcpacts upstream of Smad2 in the activin/nodal signaling pathway.Xdpcp Inhibits the Interaction between Activin Receptor and

Smad2—Based on our finding that Xdpcp functions upstreamof Smad2 in the pathway, we asked whether Xdpcp can interactwith Alk4, a type I receptor, or Smad2. To answer this, weinjected RNAs including Myc-tagged Xdpcp and HA-taggedAlk4 into the animal regions of 2-cell stage embryos, culturedthe embryos until stage 10 and performed an immunoprecipi-tation assay. In this experiment, we found that Xdpcp interactswith Alk4 in a signal-independent manner (Fig. 6A). However,we did not observe an interaction between Xdpcp and Smad2(data not shown). In addition, we found that Xdpcp specificallyinteracts with the activin receptor, but not the Bmp receptor,indicating a specific function of Xdpcp in the activin/nodalbranch of TGF-� superfamily signaling (Fig. 6B).

FIGURE 3. xdpcp knockdown leads to expansion of mesodermal territory. A, the efficacy and targetingspecificity of xdpcp MO. Co, control morpholino; MO, xdpcp MO; ORF, Myc-tagged xdpcp RNA without morpho-lino targeting sites; UTR, Myc-tagged xdpcp RNA with morpholino targeting sites. �-Catenin serves as loadingcontrol. B, compared with control (100% normal, n � 38), xdpcp knockdown embryos showed dorso-anterior-ized phenotype (6% severe, 60% mild, 34% normal, n � 35). The embryos were injected with Co MO (40 ng) orxdpcp MO (40 ng) into the dorso-animal regions of 4-cell stage, and then cultured to the stage 34. The graphshows the percentage of the number of embryos with each phenotype. n, total number of embryos analyzed.C, loss-of-Xdpcp function affects on the expression of endogenous mesodermal markers such as xbra andgoosecoid. Compared with control (Xbra: 4% expanded, 96% normal, n � 48; goosecoid: 7% expanded, 93%normal, n � 46), Xdpcp depletion leads to expansion of these markers (xbra: 67% expanded, 33% normal, n �36; goosecoid: 59% expanded, 41% normal, n � 32). The embryos were injected with Co MO (40 ng) or xdpcpMO (40 ng) into the dorso-animal regions at the 4-cell stage, cultured until stage 10.5, and then provided for insitu hybridization. The graph shows the percentage of the number of embryos with each phenotype. n, thetotal number of embryos analyzed. D, RT-PCR analysis showing that Xdpcp inhibits the endogenous expressionof mesendodermal markers. The amount of injected reagents is as follows: xdpcp, 2 ng; Co MO, 80 ng; xdpcpMO, 80 ng. Ornithine decarboxylase (ODC) serves as a loading control.

The Role of Xdpcp in Activin/Nodal Signaling

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To address the inhibitory mechanism of Xdpcp in activin/nodal signaling, we examined whether the interaction betweenactivin receptor and Smad2 under activin signaling is affectedby coexpression of xdpcp. Thus, we injected RNAs as indicatedin Fig. 6C into the animal regions of 2-cell stage embryos, cul-tured the injected embryos until stage 10, and then performedan immunoprecipitation assay. Intriguingly, Smad2 interactswith Alk4 under activin signaling and this interaction is inhib-ited by coexpression of xdpcp (Fig. 6C).Because Xdpcp inhibits the association of Smad2 with Alk4,

we asked whether the same interface of Alk4 is involved inbinding to Xdpcp and Smad2. To test this, we cloned a mutantform of Alk4, which is deleted in the L45 region known as theSmad-binding site, then examined the capability of the mutantAlk4 to associate with Xdpcp and Smad2. As shown in Fig. 6D,wild type Alk4 interacts with Xdpcp and Smad2, whereas theL45-deletion mutant of Alk4 did not bind to both Xdpcp andSmad2. This indicates that Xdpcp and Smad2 competitivelyinteract with the same region of Alk4. To better understand themechanisms for Xdpcp action, we examined subcellular local-ization of Xdpcp in both cultured cells andXenopus embryoniccells. Consistently, the results of immunostaining analysisreveal that Xdpcp is localized in the cytoplasm, but not nucleusof HeLa cells (Fig. 7A), and it is colocalized with Alk4 in the

cytoplasmic vesicles and plasma membrane of both HeLa cellsand Xenopus animal cap cells (Fig. 7B). These subcellular local-ization data support the actionmechanism of Xdpcp that inter-acts with Alk4 and inhibits the association of Alk4 with Smad2in the cytoplasmic vesicles and plasma membrane. Takentogether, these results suggest that Xdpcp may inhibit activin/nodal signaling by masking the receptor, thereby inhibiting theinteraction between activin receptor and Smad2.To test our hypothesis that Xdpcp inhibits the activin/nodal

signaling by masking the activin receptor, we examinedwhether interaction between Xdpcp and the receptor is essen-tial for the inhibitory roles of Xdpcp in activin/nodal signaling.To test this, we designed several deletion mutants of Xdpcp(Fig. 8A), transfected them into HEK293FT cells, and then car-ried out an immunoprecipitation assay. As shown in Fig. 8B, thePTB-deletion mutant (Xdpcp-�N2) cannot bind to Alk4,whereas wild type (Xdpcp-fl) and PTB-including mutants(Xdpcp-�N1 and -�C) interact with the activin receptor. Thisindicates that the PTB domain is essential for interactionbetween Alk4 and Xdpcp. Next, to test whether the PTB-dele-tion mutant lacks the inhibitory function in activin/nodal sig-naling, we analyzed the effects of PTBdeletion on the transcrip-tional activity of luciferase reporters driven by the goosecoidpromoter responding to the activin/nodal signals. As shown in

FIGURE 4. Xdpcp inhibits activin/nodal signaling. A, the secondary axis formation by ectopic expression of activin RNA is rescued by coexpression of xdpcpRNA. Xenopus 4-cell stage embryos were injected with the indicated RNAs into the ventral regions, and then cultured until stage 35. The amount of injectedRNAs is as follows: activin-�B, 2.5 pg; xdpcp, 2 ng; preprolactin (PPRL), 2 ng. B, the graph showing the results from the axis duplication assay. C, Smad2phosphorylation induced by activin was reduced by coexpression of xdpcp, whereas Xdpcp depletion by using MO increases phosphorylation of Smad2. Theamount of injected reagents is as follows: activin-�B RNA, 5 pg; xdpcp RNA, 2 ng; CoMO, 40 ng; xdpcp MO, 40 ng. Actin serves as a loading control.D, Xdpcp does not inhibit the phosphorylation of Smad1, an effector of Bmp signaling. Animal cap tissues injected with bmp4 RNA (200 pg) and xdpcpRNA (2 ng) as indicated were subjected to immunoblotting analysis with phospho-Smad1 antibody. E, Xdpcp inhibits nuclear translocation of Smad2 inHeLa cells. HeLa cells were transfected with Myc-tagged Smad2 in combinations with GFP or GFP-tagged Xdpcp, treated with activin protein (50 ng/ml)for 10 min after 36 h of transfection, fixed with 4% paraformaldehyde, and then subjected to the immunostaining analysis with anti-Myc antibody todetect localization of Smad2.

The Role of Xdpcp in Activin/Nodal Signaling

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Fig. 8C, the transcriptional response stimulated by Xnr-1(Xenopus nodal-related-1) is down-regulated by coexpressionof xdpcp. However, coexpression of the PTB-deletion mutant

(Xdpcp-�N2), which is incapable ofinteracting with Alk4, did not affectthat response and coexpressionof the PTB-containing mutants(Xdpcp-�N1 and -�C), which arecapable of interacting with thereceptor, resulted in down-regula-tion of Xnr-1. These data suggestthat interaction of Xdpcp with Alk4via its PTB domain is essential fornegative regulation of activin/nodalsignaling. Taken together, theresults suggest a model in whichXdpcp inhibits activin/nodal signal-ing by masking the receptor,thereby protecting the ectodermalfate fromactivin/nodal signals capa-ble of inducing mesoderm duringearly embryogenesis (Fig. 9).

DISCUSSION

In this study, we have providedevidence that a novel PTB protein,Xdpcp, negatively regulates theactivin/nodal signaling pathwayin germ-layer specification duringXenopus development. First, xdpcpis expressed in the prospective ecto-dermal region in the Xenopus earlyembryo. Second, gain-of- and loss-of-Xdpcp function lead to abnormalmesoderm formation and embry-onic patterning in the Xenopusembryo. Third, using RT-PCR,luciferase assay, and axis duplica-tion assay, we found that Xdpcpinhibits responses to the activin/nodal signaling. In addition, Xdpcpinhibits phosphorylation and nu-clear translocation of Smad2.Finally, Xdpcp binds to Alk4, a typeI receptor and interferes with theinteraction between Alk4 andSmad2. Together, these results indi-cate that the Xdpcp-Alk4 interac-tion mediates inhibition of Smad2binding and phosphorylation, thusregulating activin/nodal signaling.This Xdpcp-mediated regulationof the activin/nodal signalingpathway is pivotal for correctgerm-layer specification in Xeno-pus embryogenesis.Germ-layer Specification and

Activin/Nodal Signaling—Regula-tion of germ-layer specification is one of the most critical andclassical problems in vertebrate development. As mentionedpreviously,mesoderm is formed bymesoderm-inducing signals

FIGURE 5. Xdpcp acts upstream of Smad2. A, RT-PCR analysis revealing that Xdpcp cannot inhibit the expression ofmesendodermal markers induced by CA-Smad2 in animal cap tissues. WE and �RT, control RT-PCR on the wholeembryo RNA in the presence or absence of reverse transcriptase; AC, uninjected animal cap cells; (�), the animal capcells injected with CA-Smad2 RNA alone. Ornithine decarboxylase (ODC) serves as a loading control. B, the morpho-logical defects by xdpcp overexpression are partially rescued by CA-Smad2. C, the graph showing the results of panel B.

FIGURE 6. Xdpcp inhibits the interaction between activin receptor and Smad2. A, Xdpcp interacts with type Iactivin receptor, Alk4. Xenopus embryo lysates injected with combinations of Myc-tagged xdpcp RNA (1 ng) andHA-tagged Alk4 RNA (1 ng) as indicated were immunoprecipitated with anti-HA antibody and then subjected toimmunoblotting (IB) analysis. Asterisk represents the constitutively active form of Alk4. B, Xdpcp does not bind toAlk3, the Bmp receptor. HEK293FT cell lysates transfected with combinations of the indicated expression plasmidsfor GFP-tagged Xdpcp, HA-tagged Alk4, and HA-tagged Alk3 protein were immunoprecipitated with anti-HA anti-body, and subsequently subjected to immunoblotting analysis with anti-GFP antibody to detect their association.C, the association between Alk4 and Smad2 is inhibited by coexpression of xdpcp in Xenopus embryos. The amountof injected reagents is as follows: activin-�B, 5 pg; GFP-tagged Xdpcp, 2 ng; HA-tagged Alk4, 1 ng; Myc-taggedSmad2, 1 ng. D, the L45 region of Alk4 is required for binding to Xdpcp. The cells expressing the indicated reagentswere subjected to immunoprecipitation (IP) and Western blot analysis after a 1-h TGF-� treatment.

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such as activin/nodal signaling from the underlying endodermin the Xenopus embryo. In addition, mesoderm-restriction fac-tors from the overlying ectoderm are also required for deter-mining the proper territory of the mesoderm. Recently, severalmaternal proteins including Coco (22), Ectodermin (23), Sox3(24), and serum-response factor (SRF) (25) and zygotic proteinsincluding Xema (26) and XFDL156 (27) have been identified asmesoderm-restriction factors in Xenopus germ-layer specifica-tion and most of these factors are involved in regulating theactivin/nodal signaling pathway. Briefly, Coco is a secretedantagonist of BMP, TGF-�, andWnt ligands, and Sox3 inhibitsnodal expression and induces other regulators such as Ectoder-min, Xema, and Coco. In addition, Ectodermin controls thelevel of Smad4 as an ubiquitin ligase, and serum response factorinhibits the formation of the Smad2-Fast-1 complex. Finally,XFDL156 inhibits the function of p53 cooperating with Smad2.These findings provide evidence that the tight regulation ofactivin/nodal signaling at multiple points is involved in correctgerm-layer formation. Consistent with this concept, we haveshown that Xdpcp is involved in the proper formation ofembryonic germ-layers by inhibiting activin/nodal signalingthrough disruption of the association betweenAlk4 and Smad2.Negative Regulation of Activin/Nodal Signaling—TGF-�

family growth factors including activin and nodal-related pro-teins are pivotal for controlling diverse cellular processes suchas cell proliferation, apoptosis, migration, and differentiationand disturbances of these signals can cause many serioushumandiseases anddisorders. Therefore, it is very important to

clarify the fine molecular mechanisms regulating these signals.Many molecules have been identified as negative regulators toachieve tight regulation of the TGF-� signaling pathway (28).Especially when it comes to the regulation at the level of type Ireceptor, which has three regulatory points including dephos-phorylation, degradation of type I receptor, and interference ofR-Smad binding and phosphorylation. The inhibitory Smads(Smad6 and -7), known as core players in the negative regula-tion of TGF-� signaling, are involved in regulation of receptors,including the competitive inhibition of R-Smad binding (29),degradation of active receptors by interacting with Smurfs(30), dephosphorylation of active receptors (31), and inhibi-tion of Smad-dependent promoter activation (32). In addi-tion, phosphatases such as PP1c (31) and PP1� (33) de-phosphorylate the type I receptor and HECT domainE3-ubiquitin ligases such as Smurf1/2 (30, 34), NEDD4–2(35), and Tiul1/WWP1 (36, 37) are involved in degradationof the type I receptor. Moreover, Akt has been suggested tobind to Smad3, thereby sequestering it from the type I recep-tor in an Akt kinase-independent manner (38, 39). However,there is still controversy over whether Akt inhibits Smad3phosphorylation via mTOR in an Akt kinase-dependentmanner (40). We suggest that Xdpcp inhibits activin/nodalsignaling by interacting with Alk4, a type I receptor, andmasking it from Smad2. Xdpcp is a novel regulator that com-petes with Smad2 for binding to Alk4. Like Xdpcp, Smad7was originally reported as a negative regulator inhibiting theassociation between type I receptor and R-Smad. In addition,Smad7 is also involved in the dephosphorylation and degra-dation of the type I receptor by protein phosphatases andubiquitin ligases. However, we did not observe an associa-tion between Xdpcp and Smad7 (data not shown), and therelationship of Xdpcp with other molecules implicated in theregulation of type I receptor and other mechanisms of Xdpcpfunction remains to be further elucidated.TheRole of PTBProtein inActivin/Nodal Signaling—Herewe

report on a novel PTB protein, Xdpcp, that is implicated inactivin/nodal signaling. Growth factor receptors generallyemploy adaptor proteins for stabilization and amplification ofsignaling (41). PTB domains, which include IRS-like, Shc-like,andDab-like domains, are found inmany adaptor proteins (16).In particular, in the activin/nodal pathway of TGF-� signaling,two PTB adaptor proteins, Dab2 and Dok1, play similar rolespromoting the signals between receptors and R-Smads (7, 8).Dok1 has an IRS-like PTB domain similar to Xdpcp, whereasDab2 has a Dab-like PTB. Regardless of the similarity betweenPTB domains, our data show that Xdpcp possesses an oppositerole to that of Dok1 in activin/nodal signaling. Unlike Dok1,Xdpcp lacks a PH domain and does not interact with Smad2 or-4 proteins that bind to theN-terminal PH and PTB domains ofDok1. This finding suggests that there are structural differencesin the PTB region between Dok1 and Xdpcp. Moreover, ourdata using deletion mutants show that the association of Alk4with the PTB domain of Xdpcp is essential for negative regula-tion of activin/nodal signaling (Fig. 8). In addition, Yamakawaand colleagues (8) reported that a deletion mutant (Dok PP) ofDok1 composed of only PH and PTB domains down-regulatesactivin A- and TGF-�-induced promoter activity, indicating

FIGURE 7. The subcellular localization of Xdpcp. A, Xdpcp is localized at thecytoplasm, but not nucleus in HeLa cells. HeLa cells were transfected withGFP-tagged Xdpcp, treated with or without activin protein (50 ng/ml) for 10min after 36 h of transfection. B, Xdpcp is co-localized with Alk4 in both HeLacells (top) and Xenopus animal cap cells (bottom). Scale bars are 20 (HeLa cells)and 50 �m (animal cap cells).

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that another region of Dok1 is related to up-regulation of thesignaling. We reason that these differences between Dok1 andXdpcp lead to their opposite roles in activin signaling.In summary, our study reveals that Xdpcp plays a crucial role

in correct germ-layer formation during vertebrate embryogen-esis by disrupting the interaction between Alk4 and Smad2,resulting in the negative regulation of activin/nodal signaling.The detailed mechanisms by which Xdpcp regulates activin/nodal signaling in embryo development remain to be furtherelucidated.

Acknowledgments—We thank Douglas Melton, Christopher Wright,Sergei Sokol, Kohei Miyazono, Malcolm Whitman, Ken Cho, Chang-Yeol Yeo, andMakoto Asashima for generous gifts of reagents. We arealso grateful to the other members of our laboratory for helpful dis-cussion and support.

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FIGURE 8. PTB domain of Xdpcp is essential for interaction with Alk4. A, mapping of deletion mutants. B, HEK293FT cells were transfected with HA-taggedAlk4 alone or in combination with each deletion mutant as indicated. Cell lysates were immunoprecipitated (IP) with anti-HA antibody, followed by immuno-blotting (IB) with anti-GFP antibody to observe their association. C, luciferase assays in Xenopus embryos injected with combinations of the indicated reagents.The amount of injected reagents is as follows: xnr-1 RNA, 100 pg; RNAs of Xdpcp full-length and deletion mutants, 2 ng; 6DE-Luc reporter DNA, 40 pg. Error barsrepresent the standard deviation.

FIGURE 9. A model for the function of Xdpcp to regulate activin/nodal signalingduring embryogenesis. A, in the presence of Xdpcp, it inhibits activin/nodal signal-ing by masking the receptor to inhibit the contact of Smad2 to the receptor, therebyinhibiting the phosphorylation and nuclear translocation of Smad2 protein. B, in theXenopusembryo,Xdpcpintheanimalregioninhibitsthemesoderminducingsignalssuch as activin/nodal signaling from the vegetal region.

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The Role of Xdpcp in Activin/Nodal Signaling

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Seong-Moon Cheong, Hyunjoon Kim and Jin-Kwan Han EmbryosXenopusMesendodermal Formation of

Identification of a Novel Negative Regulator of Activin/Nodal Signaling in

doi: 10.1074/jbc.M109.007443 originally published online April 22, 20092009, 284:17052-17060.J. Biol. Chem. 

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