Identification and Characterization of a Smad2 Homologue fromSchistosoma mansoni, a Transforming Growth Factor-b SignalTransducer*

Received for publication, July 6, 2000, and in revised form, December 5, 2000Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M005933200

Ahmed Osman, Edward G. Niles, and Philip T. LoVerde‡

From the Department of Microbiology and Center for Microbial Pathogenesis, School of Medicine and BiomedicalSciences, State University of New York, Buffalo, New York 14214

Smad proteins are essential intracellular signal trans-ducers of the transforming growth factor-b (TGF-b) su-perfamily. The TGF-b superfamily signals through phos-phorylation and activation of R-Smad proteins,receptor-regulated Smads, by heteromeric complexes ofligand-specific type I and type II serine/threonine ki-nase receptors. R-Smads receive a signal from the acti-vated receptor complex and transmit it to the nucleus. AcDNA was isolated that encodes a 649-amino acid pro-tein found to be homologous to members of R-Smadsubfamily with highest homology scored to clawed Afri-can frog and human Smad2. The Schistosoma mansonihomologue (SmSmad2) was overexpressed in bacteria asa Sj26-GST fusion protein and used to raise specific an-tibodies. The IgG fraction of the immunized rabbit se-rum identified 70- and 72-kDa protein bands in Westernanalysis of schistosome extracts. Treatment with alka-line phosphatase removed the 72-kDa band, which indi-cates that this band represents the phosphorylated formof schistosome Smad2. SmSmad2 was localized in thesubtegument, parenchymal cells, and sex organs in bothmale and female worm cryosections. Similar resultswere also obtained from the analysis of the Smad2mRNA distribution pattern revealed by in situ hybrid-ization of adult worm pair paraffin sections. SmSmad2mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian hostdevelopmental stages and found to be constitutively ex-pressed. SmSmad2 was also found to interact with apreviously identified SmTbR-I, a serine/threonine type Ikinase receptor. Furthermore, SmSmad2 was shown toundergo phosphorylation by constitutively active formsof SmTbR-I in vitro. In addition, SmSmad2 localized inthe nuclei of mink lung epithelial cells upon treatmentwith TGF-b1. These data indicate that the SmSmad2 re-sponds to the TGF-b signals by interaction with receptorI, which phosphorylates it, whereupon it translocatesinto the nucleus presumably to regulate target genetranscription and consequently elicit a specific TGF-beffect.

Schistosomiasis is a parasitic disease second only to malariain prevalence. According to the World Health Organizationrecords, about 300 million people in 76 countries are currentlyinfected. More than 600 million people live in endemic areasand are at risk of contracting the disease (1). The egg is themajor cause of the pathology and complications of the diseasesuch as portal hypertension, liver fibrosis hemorrhage, anddeath (2, 3). Female schistosomes initiate sexual developmentand start laying eggs in response to a stimulus by male worms(4). Very little is known about the nature of the signal(s) in-volved or the mechanism(s) of this process. Furthermore, schis-tosomes reside in portal circulation surrounded by host mole-cules such as antibodies, hormones, cytokines, and growthfactors. The tegument layer covering the parasite worm bodyrepresents the interface between the parasite and its hostenvironment. Several studies reported the identification of re-ceptors for growth factors and other host molecules on thesurface of the parasite (5–7). Transforming growth factor-b(TGF-b)1 serine/threonine kinase receptor I (TbRI) was re-cently identified in Schistosoma mansoni, SmTbR-I (5). Thepresence of SmTbRI on the surface of the parasite raises thepossibility of the involvement of TGF-b or a TGF-b-like ligandin the host-parasite interactions or in the stimulation of femaleworm maturation by male schistosomes.

TGF-b is a superfamily of secreted polypeptides includingvarious forms of TGF-b, bone morphogenetic proteins (BMPs),activins, inhibins, and many other structurally related factors(8). Members of this family regulate cell migration, differenti-ation, adhesion, multiplication, and death throughout the lifespan of an organism (9). These different effects result from thechanges in the expression of key target genes, which depend onthe cell type and developmental stage. Three classes of intra-cellular signal transducers are involved in the signaling cas-cade of the TGF-b family as follows: a family of plasma mem-brane serine/threonine kinase receptors (known as type I andtype II receptors); a family of cytoplasmic proteins that eithertransmit the signal from the activated receptor complex to thenucleus or down-regulate the signaling pathway, known asSmad family; and gene-specific nuclear DNA-binding partnersthat associate with Smad proteins forming transcriptional com-plexes (8, 10).

* This work was supported by National Institutes of Health GrantAI46762. The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s) AF232025.

‡ To whom correspondence should be addressed: Dept. of Microbiol-ogy, School of Medicine and Biomedical Sciences, State University ofNew York, 138 Farber Hall, Buffalo, NY 14214. Tel.: 716-829-2459; Fax:716-829-2169; E-mail: loverde@buffalo.edu.

1 The abbreviations used are: TGF-b, transforming growth factor-b;SmSmad2, S. mansoni Smad2; R-Smad, receptor-regulated Smad;BMP, bone morphogenetic protein; SmTbR-I, S. mansoni TGF-b recep-tor-I; SARA, Smad anchoring for receptor activation; MH1 and MH2,Mad homology region 1 and 2; wt, wild type; kb, kilobase pair; PCR,polymerase chain reaction; RT-PCR, reverse transcriptase-PCR; bp,base pair; CIAP, calf intestinal alkaline phosphatase; FITC, fluoresceinisothiocyanate; GST, glutathione S-transferase; PAGE, polyacrylamidegel electrophoresis; BD, binding domain; AD, activation domain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 13, Issue of March 30, pp. 10072–10082, 2001© 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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The signaling pathway is initiated by ligand binding to theextracellular domain of the type II receptor kinase. The cyto-plasmic domain of type II receptor catalyzes the phosphoryla-tion and concomitant activation of the specific type I receptorkinase. Once activated, the type I receptor binds to and phos-phorylates a member of a subset of the Smad family, receptor-regulated Smads (R-Smads). The phosphorylation of R-Smadsdestabilizes the complex with type I receptor releasing theactivated R-Smad (11) allowing it to associate with a relatedprotein Smad4, known as comediator Smad or co-Smad (12,13). The heteromeric complex (R-Smad/co-Smad) then translo-cates into the nucleus (14) where it associates with specificDNA-binding partner(s) that direct it to the regulatory regionof target gene(s) (9).

Two structural motifs in the type I receptor and the corre-sponding R-Smad, the L45 loop and L3 loop, respectively, con-tain subtype-specific residues and determine the specificity ofthe interaction between these two members of the signalingcascade (15, 16). R-Smads and co-Smads consist of conservedN- and C-terminal domains and MH1 and MH2 domains. TheMH1 domain possesses a DNA binding activity (17, 18), and theMH2 domain exerts a transcriptional activation function (19,20). These conserved domains are linked by an intervening,nonconserved linker region that varies both in length andsequence (8). In the basal state, the MH1 domain of R-Smadsinteracts with and inhibits the transcriptional (20) and biolog-ical (19) activities of the MH2 domain. Upon phosphorylationby type I receptor kinase, this auto-inhibitory MH1-MH2 in-teraction of R-Smad is relieved, allowing the association of theactivated R-Smad with co-Smad. The MH2 domain of R-Smadscontains receptor phosphorylation sites located at the veryC-terminal end of the protein (SSXS) (11, 21). Co-Smads lackthis phosphorylation signature motif and consequently do notserve as substrates for receptor I kinases (11). The MH2 do-main also mediates the interaction and association of R-Smadswith type I receptors (11).

We report herein the identification of S. mansoni Smad2homologue (SmSmad2). We define its expression level in dif-ferent developmental stages and the localization of the proteinand mRNA transcripts in adult worm sections. We also dem-onstrate that schistosome protein performs the biological ac-tivities exhibited by other Smad2 homologues, including itsinteraction with and phosphorylation by a schistosome homo-logue of TbR-I (SmTbRI) and nuclear translocation in responseto human TGF-b.

EXPERIMENTAL PROCEDURES

Identification of SmSmad2 cDNA—A fragment of about 1 kb span-ning part of the MH1 domain and the linker region was coisolatedduring our previous work on SmRas cDNA (22). An oligonucleotiderepresenting the 39-end of the coding region of SmRas (59-GACGTC-GACTCATTGTATACAACATTTTGC-39) amplified a 1-kb fragment.The PCR product was cloned into TOPO-TA vector (Invitrogen) andsequenced. Sequence analysis of this fragment showed that it is relatedto members of R-Smad subclass of the Smad family. The DNA fragmentwas labeled with [a-32P]dCTP by Megaprime labeling kit (AmershamPharmacia Biotech) and used as a probe to screen S. mansoni adultworm pair cDNA library. Eight positive clones were identified, and thesequence information showed that two contain the entire coding regionof S. mansoni homologue of Smad2 protein (SmSmad2).

Expression of SmSmad2 and Production of Specific Antiserum—Smad2 cDNA representing the MH1 domain was amplified from theparent phagemid with specific 59- primer (Smad2-cod-59) containingKpnI (underlined) and SalI (italicized) sites (59-GGTACCGTCGACCG-GAATTCAAAATGAGTCTCTTTACAAGTCCAC-39) and a reverseprimer representing the complementary sequence of bp 533–558. The430-bp PCR product was subcloned in TOPO-TA vector (Invitrogen),sequenced, and then digested with KpnI at the 59-end and HpaI at the39-end, a unique site present at bp 542 of the cDNA sequence. Theexcised DNA fragment was then recloned into the parent phagemid

digested with the same enzymes yielding a cDNA lacking the 59-un-translated region. The recombinant phagemid was cleaved with SalIand NotI, and the full-length SmSmad2 insert was subcloned in pGEX-4T-1 linearized with the same enzymes. The recombinant pGEX-SmS-mad2 plasmid was overexpressed in TOP10 cells (Invitrogen) using 0.5mM isopropyl-1-thio-b-D-galactopyranoside at 20 °C for 5 h. The recom-binant Sj26-SmSmad2 fusion protein (200 mg) was used to immunize aNew Zealand rabbit with complete Freund’s adjuvant for the primaryinjection and incomplete adjuvant for the following two booster doseswith 3-week intervals between each dose. The immunized rabbit serumwas then passed over protein A-Sepharose column, and the IgG fractionwas eluted and used in Western blots and immunocytolocalization inadult worm pair cryosections.

Western Blot and Immunocytolocalization—S. mansoni adult wormpairs were perfused from infected hamsters and homogenized in anextraction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM EDTA,5% glycerol, 1% Triton X-100, and mammalian protease inhibitor mix-ture; Sigma). The homogenate was sonicated for 1 min and centrifugedat 35,000 rpm for 1 h at 4 °C, and the soluble fraction was aliquoted andstored at 280 °C until used.

Schistosome extract was treated with calf intestinal alkaline phos-phatase (CIAP; Life Technologies, Inc.) at concentrations of 0.2 and 1unit/mg extract for 30–60 min at 37 °C. The untreated extract, with andwithout 103 CIAP buffer, along with CIAP-treated extract were sepa-rated on 7.5% SDS-PAGE. SDS gels were transferred onto polyvinyli-dene difluoride membrane, and the blots were probed with IgG fractionsfrom preimmune rabbit and SmSmad2-GST-immunized rabbit sera (0.5mg/ml). Biotinylated goat anti-rabbit IgG (0.5 mg/ml; Zymed Laborato-ries Inc.) was used to probe the reacting primary antibodies, and this inturn was detected using Z-avidin horseradish peroxidase conjugate (0.5mg/ml; Zymed Laboratories Inc.). The reactions were developed withtetramethyl benzidine reagent (Zymed Laboratories Inc.).

The IgG fractions (5 mg/ml) were also used to detect SmSmad2 nativeprotein in adult worm cryosections. Biotinylated goat anti-rabbit IgG (5mg/ml; Zymed Laboratories Inc.) and Z-avidin FITC conjugate (5 mg/ml;Zymed Laboratories Inc.) or streptavidin-Cy5 conjugate (10 mg/ml;Zymed Laboratories Inc.) were used to localize the primary rabbit IgGantibodies. Probed sections were evaluated using a Bio-Rad MRC100confocal microscope equipped with a krypton laser. A 674-nm filter wasused to visualize the Cy5-probed sections.

RT-PCR and in Situ Hybridization—SmSmad2 transcripts were de-tected using two approaches. RT-PCR was employed to determine thetotal transcription level of SmSmad2 in different developmental stages.In situ hybridization was used to detect where SmSmad2 transcriptsare localized and in which tissues the protein is synthesized. In RT-PCR, total RNA was purified from different life stages of S. mansonithat are related to the definitive human host, starting with 3-h schis-tosomules and up to paired 45-day-old adult worms using RNA-stat 60reagent (Tel-Test, Inc.). RNA was copied by reverse transcription (22),and cDNA was used as templates for subsequent PCR amplification. Aprimer pair corresponding to bp 1614–1636 and the complementarysequence of bp 1902–1926 was used to amplify a fragment that spannedpart of the MH2 domain. SmRXR1 (23), a clone previously identified inour laboratory and shown to be constitutively expressed in all humanhost-related stages of the parasite, was used as an internal control forthe RT-PCRs. A primer pair representing part of the SmRXR1 AB-domain (23) was used to amplify a 460-bp DNA fragment. To remain inthe linear range of amplification, 26 cycles of PCR were performed. Theamplification products were size-separated in a 2.0% agarose gel,stained with ethidium bromide, and quantified using the MolecularAnalyst gel documentation system (Bio-Rad). SmSmad2 PCR productswere normalized to those of SmRXR to eliminate the differences inquality and/or quantity of the input RNA in the RT reaction.

For the in situ hybridization, an antisense oligonucleotide corre-sponding to the complementary sequence of bp 695–744 was synthe-sized, labeled with biotin using Bright-Star biotin labeling kit (Am-bion), and used to probe paraffin-embedded sections usingmRNAlocator-hyb kit (Ambion) following the manufacturer’s instruc-tions. Two primers, one represents the sense sequence of SmSmad2(59-GGAAGAGGTTCATCTTTTGGTTCACATCATTCTCG-39, bp 620–654) and the other represents the antisense sequence of Smp14 gene (afemale-specific gene that produces an eggshell protein) (24) (59-GCCTC-CACCATATCCACTATCGCCATAACCGCTATCACAATCGCTACC-39),were similarly processed and used as a negative and positive controls,respectively. The probed sections were developed using streptavidin-alkaline phosphatase conjugate (mRNAlocator-Biotin kit; Ambion), andthe sections were lightly counterstained using nuclear fast red. The

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sections were examined and photographed using Nikon microphot dig-ital microscope.

SmSmad2 Interaction with SmTbR-I—To evaluate the interaction ofSmSmad2 with SmTbR-I, DNA encoding wild type and mutant forms ofSmSmad2 and SmTbR-I were cloned into the yeast GAL4 two-hybridvectors (activation domain (AD) and DNA-binding domain (BD)). SmS-mad2 cDNA (wild type, wt), in which the EcoRI site at position 1709was mutated to GAGTTC to maintain the same encoded amino acidsequence was amplified. Amplification was performed using Pfu DNApolymerase (Stratagene) with Smad-59-cod as a forward primer and areverse primer in which NotI site (underlined) was inserted (NotI-Smad-39; 59-GCGGCCGCACTGACAGATGTGCAAGGATG-39). ThePCR product represents a SmSmad2 cDNA in which the stop codon wasremoved and DNA encoding 3 amino acids (VGG) was inserted down-stream of the C-terminal motif (TSVS) of SmSmad2 (SmSmad2–39).The PCR product was cloned in pCRII-Blunt-TOPO vector (Invitrogen).The wt SmSmad2 was digested with EcoRI/SalI and cloned in pBD-GAL4-cam and pAD-GAL4–2.1 vectors (Stratagene) downstream ofGAL4 DNA-binding domain and GAL4 activation domain, respectively.SmSmad2–39 was digested with EcoRI and SalI/XhoI and cloned inpBD-GAL4-cam linearized with EcoRI and pACT2.1 (CLONTECH) andlinearized with XhoI to encode fusion proteins of GAL4-BD/SmSmad2–39and GAL4-AD/SmSmad2–39, respectively. SmTbR-I (wild type) (5)was mutated in its GS domain (284TCSGSGSGKPLLVQRTVARQ303) toattempt to construct both constitutively active and kinase-inactiveforms (25). Three mutants were constructed to form T299D, the ex-pected putative kinase2 derivative, Q303D, the constitutively activeform, and the double mutant T299D/Q303D. DNA sequence analysiswas performed for the mutant forms of SmTbR-I to confirm the muta-tions. SmTbR-I constructs were digested with EcoRI/SalI and clonedinto pBD-GAL4-cam and pAD-GAL4–2.1 vectors linearized with thesame enzymes, to generate fusion proteins with GAL4-BD and GAL4-AD, respectively. The two forms of SmSmad2 and the four constructs ofSmTbR-I in both BD and AD vectors were transformed individually,into Y190 competent cells using Fast Track Yeast Transformation Kit(Geno Technology, Inc.) as controls to evaluate the ability of these genesto activate host cell reporter gene expression. Transformed cells wereplated onto synthetic dextrose medium (SD) supplemented with aminoacid dropout solutions lacking either Trp or Trp, His or Leu, or Leu, Hisfor BD vectors and AD vectors, respectively. LacZ filter assay wasperformed according to Stratagene Two-hybrid instruction manual. Totest for an interaction, an AD vector was cotransformed with bindingdomain vector, and the transformed cells were plated onto SD supple-mented with amino acid dropout solutions lacking either Leu, Trp, orLeu, Trp, His. LacZ filter assay was also performed on this transforma-tion series. 3-Amino-1,2,4-triazole, a metabolic inhibitor for histidinebiosynthesis, was added to histidine-lacking medium to a final concen-tration of 25 mM to inhibit the leaky expression of his3 reporter gene.Control plasmids, pGAL4, p53, pLaminC, pSV40, and a combination ofp53 1 pSV40 and pLaminC 1 pSV40 (Stratagene) were transformedalong with each transformation experiment to provide positive andnegative controls.

GST pull-down analysis was performed to attempt to confirm theresults obtained from the two-hybrid interaction in yeast. In this ex-periment, the four constructs of SmTbR-I (wt and the three mutants,described above) were subcloned into pCITE-4a vector (Novagen) usingappropriate restriction enzymes. Recombinant SmTbR-I-pCITE vectorswere expressed in vitro in rabbit reticulocyte lysates with [35S]methi-onine (PerkinElmer Life Sciences) using STP-3 transcription/transla-tion system (Novagen) following the supplier’s instructions. The regionsencoding the SmSmad2 linker and MH2 domain were PCR-amplified(primer pairs bp 488–512 and a complementary sequence of bp 1455–1480, and bp 1424–1448 and a complementary sequence of bp 2045–2068 with SalI site at the 59-end, respectively). The PCR products werecloned into TOPO-TA vector (Invitrogen) and then subcloned intopGEX-4T-1 (Amersham Pharmacia Biotech) and pET42a (Novagen)prokaryotic expression vectors, respectively. DNA encodingSmSmad2–39 was also subcloned in pET42a vector. Recombinant pro-karyotic expression vectors were overexpressed in bacteria, and theGST fusion proteins were affixed to glutathione-Sepharose beads. Bind-ing reactions, between SmSmad2 proteins (wild type and mutant form)and SmTbR-I in vitro translated proteins, were performed by adding 2ml of the translation reactions to GST fusion protein-coupled beadsequivalent to about 2 mg of the fusion protein. The final volume of thereactions was adjusted to 100 ml by adding binding buffer (50 mM

Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 0.15% Nonidet P-40). Thereactions were incubated at room temperature for 1 h and then contin-ued overnight at 4 °C. Glutathione-Sepharose beads were added to

facilitate the recovery of the reactive beads, and the samples wereprecipitated, washed four times, and boiled in 13 SDS loading buffer,and the bound proteins were size-separated in 12% SDS-PAGE. Thegels were stained, destained, treated with Entensify (PerkinElmer LifeSciences), dried, and exposed to x-ray film. Developed x-ray films werescanned, and the specific reaction bands were quantified using Molec-ular Analyst Gel Documentation System (Bio-Rad). Parental pGEX-4T-1 was used to generate recombinant GST, which was affixed toglutathione beads and used to pre-treat the translation reactions toremove the nonspecific background. The equivalent of 2 mg of GST-coupled beads was used to pre-clear the amount of lysate used perreaction. GST beads were also included in the binding reactions to serveas a negative control in the pull-down experiment.

In Vitro Phosphorylation of SmSmad2 by SmTbR-I—To determinewhether SmSmad2 can serve as a substrate for SmTbR-I, we examinedthe ability of SmTbR-I to phosphorylate SmSmad2. In principle, thephosphorylation reactions were performed as outlined above for thepull-down experiment with few differences. Briefly, wild type and mu-tant forms of SmTbRI were synthesized in vitro using rabbit reticulo-cyte lysates and unlabeled methionine. The lysates were pre-clearedwith GST-coupled beads as before and then aliquoted into four tubes(each equivalent to 4 ml of original lysate). To each was added GST or aGST fusion protein containing SmSmad2-linker, SmSmad2-MH2, orSmSmad2–39 coupled to the glutathione-Sepharose beads. Phosphoryl-ation was performed in a 100-ml volume in the presence of 10 mCi of[32P-g]ATP, for 1 h at room temperature in a buffer similar to thebinding buffer (above) except it lacks Nonidet P-40. The beads werecollected, washed four times with the reaction buffer, boiled in 13SDS-loading buffer, and loaded onto 12% SDS-PAGE. The gels wereprocessed as before and subjected to autoradiography.

To determine more precisely the phosphorylation sites in Smad2C-terminal motif, we constructed several mutant forms of SmSmad2-MH2 domain in which the potential phosphorylation sites were mu-tated to alanine. Four mutant forms were constructed in which Ser649,Ser647, Ser647,649, and Thr646-Ser647,649 were mutated to alanine (TSVA,TAVS, TAVA, and AAVA, respectively). The mutant forms were gener-ated by PCR amplification using oligonucleotides encoding the corre-sponding mutations. PCR products were cloned in TOPO-TA-pCR2.1vector (Invitrogen), sequenced to confirm the presence of the specificmutations, digested with EcoRI/XhoI, and cloned in pCITE-4a vector(Novagen). Smad2-MH2-wt and SmTbR-I-wt and the mutant forms ofboth proteins were synthesized in vitro using unlabeled methionine.Kinase reactions were performed using 5 ml of each SmTbR-I transla-tion reaction and 5 ml of each SmSmad2-MH2 translation reactions ina reaction buffer containing 25 mM Tris, pH 8.0, 50 mM NaCl, 5%glycerol, and 10 mCi of [g-32P]ATP. Reactions were allowed to proceedfor 1 h at room temperature, and then 5 mg of IgG fraction of a-SmS-mad2 rabbit antiserum was added to each reaction and incubated for1 h at room temperature, and the antigen-antibody complexes wereprecipitated by adding 20 ml of protein A-Sepharose (Amersham Phar-macia Biotech). Immunoprecipitates were washed 4 times with 13phosphate-buffered saline containing 0.1% Tween 20, boiled in 13SDS-loading buffer, and loaded onto 12% SDS-PAGE. The gels wereprocessed as before and subjected to autoradiography.

TGF-b1 Responsiveness of SmSmad2 Protein—To determine the re-sponsiveness of the SmSmad2 protein to TGF-b1 signal, SmSmad2cDNA was cloned into the eukaryotic expression vector pCMV-GST(kindly provided by Dr. Randall Reed). The pGEX-SmSmad2 vector wasdigested with BamHI, and the 2.1-kb fragment was inserted in thepCMV-GST vector linearized with the same enzyme. The recombinanttransformants were checked for the presence of the insert in the correctorientation by colony PCR using pGEX-fwd primer (Amersham Phar-macia Biotech) as a forward primer and a reverse primer representingthe complementary sequence of bp 533–558. DNA sequence of positivesamples was determined to confirm the presence of the start ATG codonof SmSmad2 cDNA in frame with the GST sequence. Double CsCl-purified pCMV-GST-SmSmad2, as well as the parent vector pCMV-GST, were used to transfect mink lung epithelial cells (MV1Lu; ATCCCCL-64) employing Effectene transfection reagent (Qiagen) followingthe supplier’s instructions. Forty eight hours post-transfection, cellswere fixed with 2% paraformaldehyde containing 0.1% Triton X-100.The fixed cells were probed with anti-GST mouse monoclonal antibody(7.5 mg/ml). Anti-mouse biotinylated IgG (7.5 mg/ml; Zymed Laborato-ries Inc.) was used as a secondary antibody reagent. The biotinylatedantibodies were detected using Z-avidin-FITC conjugate (5 mg/ml;Zymed Laboratories Inc.). A nonrelevant plasmid (pCDNA-LacZ; In-vitrogen) that lacks a GST tag was used as a negative control. Fluores-cent cells were examined with Bio-Rad MRC-100 confocal microscope.

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RESULTS

Isolation, Identification, and Computer Analyses of SmS-mad2 cDNA—The amino acid sequence predicted by the orig-inal 1-kb DNA fragment showed strong homology to the MH1domain of R-Smad subfamily. This DNA fragment was used toscreen lZAPII S. mansoni, adult worm pair cDNA library.Eight positive phages were identified of which two were shownto contain the full-length coding region of a S. mansoni homo-logue of Smad2 cDNA (SmSmad2). SmSmad2 cDNA encodes a649-amino acid protein representing the largest Smad2 proteinidentified to date. NCBI blast search showed that SmSmad2exhibits a high degree of homology to Smad2 proteins of Xeno-pus laevis (26) and humans (27–30). Homology is restricted tothe MH2 domain (69% identity, 77% similarity) and, to a lesserextent, the MH1 domain (61% identity and 70% similarity, Fig.1). SmSmad2 C-terminal phosphorylation motif is “TSVS”

which is different from the standard SSXS motif, present in allR-Smads. The SmSmad2 MH2 domain also contains the L3loop, the region that determines the specificity of interactionwith the specific type I receptor (16). The sequence of the L3loop is highly conserved and invariant among R-Smads of sim-ilar signaling specificity (e.g. Smad2 and Smad3 that are acti-vated by TbR-I and activin type I receptor, or Smad1, Smad5,and Smad8 activated by BMP or BMP-like type I receptors)(16). L3 loop in SmSmad2 has the specific subtype residuesArg613 and Thr616 (Fig. 1) which dictate the interaction ofSmad2 and Smad3 with the TbR-I and the activin type Ireceptors. Another characteristic feature maintained in theMH2 domain of SmSmad2 is the conservation of all the 5subtype-specific residues required for the interaction of Smad2and Smad3 with SARA (Smad Anchoring for Receptor Activa-tion), Ile527, Phe532, Tyr552, Trp554, and Asn567 (Fig. 1) (31).

FIG. 1. Pileup analysis and peptidealignment of MH1 and MH2 domainsof S. mansoni Smad2 (SmSmad2; Gen-BankTM accession number AF232025)with homologous domains in other R-Smad proteins. Schematic representa-tion of the functional domains of SmS-mad2 cDNA (top). Alignment ofSmSmad2 MH1 and MH2 domains (bot-tom). DmSmad2, Drosophila melano-gaster Smad2 (GenBankTM accessionnumber AAD11458); XlSmad2, Xenopuslaevis Smad2 (GenBankTM accessionnumber L77885); HsSmad2, Smad3, andSmad1 are human Smad2, Smad3, andSmad1, respectively (GenBankTM acces-sion numbers NP_005892, BAA22032,and S68987, respectively). %I and %S areidentity and similarity scores. Blackboxed sequence represents the L3 loop.White boxed sequences are R-Smad sub-type-specific residues of L3 loop. Whitesequence represents the C-tail, and un-derlined sequences are the C-terminalphosphorylation motifs. “●” specifiesSmad2/Smad3-specific recognition resi-dues by SARA.

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SARA is an adaptor protein that interacts with both Smad2/Smad3 and type I receptor and facilitates Smad2/Smad3 MH2domain interaction with type I receptor and consequently itsphosphorylation and activation. The interaction of SARA withR-Smad subfamily is restricted to Smad2 and Smad3 (31, 32).

Expression, Western Blot Analysis, and Immunocytolocaliza-tion—SmSmad2 was overexpressed in bacteria as a GST fusionprotein. The fusion protein was used to generate a specificrabbit polyclonal antiserum. The IgG fraction of the immunizedrabbit serum detected two proteins of about 70 and 72 kDa inWestern blots of schistosome worm extracts (Fig. 2, panel C,lanes 2 and 3). To determine whether the slower migratingspecies is a phosphorylated form of Smad2, schistosome ex-tracts were treated with calf intestinal alkaline phosphataseprior to PAGE analyses. After CIAP treatment, only the 70-kDa protein band was detected (Fig. 2, panel C, lane 1). In thisparticular experiment the 70-kDa band is distorted due to thehigh concentration of CIAP in the sample. This result indicatesthat the 72-kDa protein represents a phosphorylated form ofSmSmad2 in the parasite extract. Phosphorylation may resultfrom receptor phosphorylation and activation of the SmSmad2or mitogen-activated protein kinase phosphorylation of thelinker region (33, 34). IgG fraction of preimmune rabbit serumwas used as a negative control (Fig. 2, panel B).

Native SmSmad2 was localized in adult worm cryosectionsprobed with anti-SmSmad2 antibodies. SmSmad2 was detectedin the tubercles of the tegument, in parenchymal cells of maleand female worms (Fig. 3, panels A and B), as well as in thevitellaria (Fig. 4, panels IC and IIIC), the developing embryo inegg (Fig. 4, panel IIC) and the ovary (Fig. 3, panel C, and Fig.4, panel IC) of the female, and the testis of the male (data notshown). The use of Cy5 as a detection reagent enabled us toovercome the auto-fluorescence that occurs in female vitellariaand unequivocally observe SmSmad2 reactivity in the vitel-laria and embryo contained within the uterine egg (Fig. 4).

RT-PCR and in Situ Hybridization—The level of SmSmad2mRNA was determined in different schistosome developmentalstages by RT-PCR. In the mammalian host stages, SmSmad2mRNA was found to be detected as early as 4 days post-infection (Fig. 5, lane 2). Compared with the mRNA level of theinternal control (SmRXR1), SmSmad2 mRNA exhibits rela-tively constant levels throughout development.

SmSmad2 mRNA was also detected in paraffin sections ofadult worms by in situ hybridization. The transcripts werelocalized in the subtegumental and parenchymal cells in maleand female worms (Fig. 6, panels C and D) and in sexual tissuessuch as vitellaria (Fig. 6, panel D), uterus, and ovary (Fig. 6,panel C) in female worm. The positive control (Smp14) showedstrong localization in female vitelline cells (Fig. 6, panel A) andthe negative control showed minimal background reactivity inboth male (Fig. 6, panel B) and female sections (data notshown).

SmSmad2 Interaction with SmTbR-I—Initially, the yeast

two-hybrid system was employed to evaluate the direct inter-action between SmSmad2 and SmTbR-I. Several mutant con-structs for both SmSmad2 and SmTbR-I were evaluated.SmSmad2–39, a SmSmad2 mutant in which the stop codon wasremoved to extend the reading frame beyond the TSVS phos-phorylation motif, was expected to retain SmTbR-I binding butnot to undergo receptor-mediated phosphorylation at its C-terminal end (35). Mutations in the downstream sequence ofthe GS region of SmTbR-I corresponding to Q303D and T299Dwere expected to yield a constitutively active and a kinase-negative version of type I receptor, respectively (21, 25, 36, 37).Both SmSmad2 (wt) and SmSmad2–39 when fused downstreamto GAL4 BD strongly activated transcription of the reportergenes (lacZ and his3) in the yeast host cells Y190. This resultedin the production of blue color in the LacZ filter lift assay andthe growth of the transformed cells on histidine-lacking me-dium (Fig. 7, panel A). Neither of these two SmSmad2 con-structs activated transcription when fused to the GAL4 AD(data not shown). Based on these data, we tested the interac-tion of SmSmad2-AD with SmTbR-I fused to GAL4-BD. In vivo,SmSmad2 interacted with SmTbR-I under certain conditions.SmSmad2-AD (wt) strongly interacted with SmTbR-I-BD (wt)(Fig. 7, panel B), whereas no interaction was observed betweenSmSmad2-AD (wt) and the 3 mutants of SmTbR-I fused toGAL4-BD (not shown). On the other hand, when SmSmad2–39-AD was used, a stronger interaction was observed withSmTbR-I (Q303D) and SmTbR-I (T299D/Q303D) (Fig. 7, panel

FIG. 3. Immunocytolocalization of SmSmad2. S. mansoni adultworm cryosections were probed with anti-SmSmad2 IgG (panels A–C),and preimmune rabbit IgG (panel D) were detected using a FITCconjugate. Panel A, male (M) and female (F) cross-sections, showingfluorescence in tubercles and parenchymal cells. Panel B, male sectionshowing fluorescence in tubercles (T) and cells of parenchyma. Panel C,female worm showing fluorescence in ovary (O). Panel D, male wormshowing no fluorescence. G, gut.

FIG. 2. Identification of native SmSmad2 in adult worm extracts. Coomassie Blue-stained SDS gel (panel A) and Western blot analysisof schistosome extract using IgG fraction of preimmune (panel B) and SmSmad2-immunized (panel C) rabbit sera. Lanes 1–3 represent schistosomeextract treated with CIAP (1 unit/mg protein) for 1 h at 37 °C, schistosome extract incubated with 13 CIAP buffer for 1 h at 37 °C, and untreatedextract, respectively. Arrows point to 72- and 70-kDa protein.

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B), whereas the wild type SmTbR-I and T299D mutant showedweaker interaction. In general, the results of the LacZ assaywere obtained after 3–4 days, while weak interaction requiredincubation periods of up to 10 days.

The use of the yeast two-hybrid assay revealed that SmS-mad2 interacts in vivo with SmTbR-I. To evaluate this inter-action in vitro, we examined the ability of GST-SmSmad2 fu-sion constructs (linker region and MH2 domain ofSmSmad2-wt and full-length SmSmad2–39), coupled to gluta-thione-Sepharose beads, to bind [35S]methionine-labeledSmTbR-I proteins (wt and the three mutant forms). SmSmad2-MH2 domain as well as the full-length protein (SmSmad2–39)bound the four forms of SmTbR-I (Fig. 8, panels A–D, lanes 3

and 4). In this experiment, GST domain was used to evaluatethe background interaction. Neither GST nor GST-linker fu-sion showed significant interaction with SmTbR-I proteins(Fig. 8, panels A–D, lanes 1 and 2).

Results of the yeast two-hybrid and GST pull-down analysesclearly demonstrated that certain constructs of SmSmad2 sta-bly interact, both in vivo and in vitro, with either wt or mutantforms of SmTbR-I. To determine whether this interaction is

FIG. 4. S. mansoni adult worm cryo-sections probed with anti-SmSmad2IgG and detected using Cy5 conju-gate. Row A shows the phase contrastfields. Row B shows background fluores-cence detected when using 520-nm filter(used to visualize FITC-probed sections).Note the autofluorescence in vitellaria(groups I–III) and eggshell (group II). RowC shows the specific fluorescence of Cy5conjugate at wavelength 674 nm. Note ab-sence of autofluorescence in eggshell (E)(group II) and fluorescence in ovary (O)(group I), vitellaria (V) (groups I–III), de-veloping miracidium in egg (group II) andin parenchyma and subtegumental tis-sues of male (group III). G, gut; F, female;and M, male. No specific fluorescence wasobserved in sections probed with preim-mune rabbit IgG.

FIG. 5. SmSmad2 mRNA levels in schistosome at differentstages of development. RT-PCR of SmSmad2 RNA compared withthe internal control, SmRXR1. Panel A is a diagrammatic representa-tion of the normalized mRNA levels of SmSmad2. Panel B is a photo-graph of 2% agarose gel showing the amplification products of SmS-mad2 and SmRXR1 (arrows). Lanes 1–4 are 3-h-, 4-day-, 7-day-, and15-day-old schistosomules, respectively. Lanes 5–8 are 20-, 28-, 32-, and35-day worm pairs, respectively. Lanes 9–13, are single sex maleworms, single sex female worms, bisex male worms, bisex femaleworms, and adult worm pairs, respectively.

FIG. 6. Localization of SmSmad2 mRNA transcripts in tissuesections of S. mansoni adult worms by in situ hybridization.Panel A represents the reaction of the positive control probe (p14antisense primer) in the vitellaria (V) of the female worm (F). Maleworms (M) were negative as expected (data not shown). Panel B showsthe negative control reaction using SmSmad2 sense primer in malesections. Female sections (not shown) exhibited the same level of reac-tivity. Panels C and D show the specific reactions of SmSmad2 anti-sense probe in male and female sections. Male sections show positivereactions in subtegument and parenchymal cells. Female sections showreactivity in vitellaria (V), ovary (O), developing embryo in uterine egg(E), and subtegumental cells. G, gut; ST, subtegument; P, parenchyma.

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functional, in vitro phosphorylation of SmSmad2 by wt andmutant constructs of SmTbR-I proteins was tested. In vitrosynthesized SmTbR-I derivatives were incubated in the pres-ence of [g-32P]ATP with GST or GST-SmSmad2 fusion proteins(linker, MH2 domain, and SmSmad2–39) coupled to the gluta-thione-Sepharose beads. After incubation for 1 h at room tem-perature, samples were centrifuged, the pellets washed for 4times, resuspended in PAGE sample buffer, and denatured,and the denatured proteins were separated by electrophoresis.Neither GST nor any of the GST-SmSmad2 fusion proteinsincorporated the 32P label when incubated with wild typeSmTbR-I (Fig. 9 panel A, panel I). [32P]Phosphate was onlyincorporated into the MH2 domain of wt SmSmad2 by SmTbR-I-Q303D, T299D/Q303D, and T299D mutants (Fig. 9, panel A,lane 3, panels II–IV). The phosphorylation of the SmSmad2-MH2 domain by the mutant form SmTbR-I-Q303D was ex-pected since corresponding mutations in other type I receptorsresulted in constitutive phosphorylation activity in vitro (21)and ligand-independent signaling in vivo (25, 36, 37). Surpris-ingly, the SmTbR-I-T299D mutant form showed kinase activ-ity, although less than other mutant forms. This result is incontrast to previous work, which demonstrated that a corre-sponding mutant to T299D in human TbR-I yielded an inactiveversion of the receptor kinase (25). GST, GST linker, and GST-SmSmad2–39 were not phosphorylated by any of the SmTbR-Iconstructs (Fig. 9, panel A, lanes 1, 2, and 4). Indeed, the lack

of phosphorylation of SmSmad2–39 by any of the SmTbR-Iconstructs was expected since this modification of the C-termi-nal end of SmSmad2 may result in masking the phosphoryla-tion sites and blocking SmTbR-I-mediated phosphorylation ofthe protein.

To confirm the phosphorylation results and to determine thespecific phosphorylation sites in SmSmad2 MH2 domain C-terminal phosphorylation motif, we employed a different for-mat of the in vitro phosphorylation reaction. In this reaction weused wt and mutant constructs of SmSmad2-MH2 domain,where the potential phosphorylation sites were mutated toalanine. Both groups of SmSmad2 and SmTbR-I (wt and mu-tant forms) were synthesized in vitro using unlabeled methio-nine and then the phosphorylation of SmSmad2-MH2 con-structs by SmTbR-I forms, in the presence of [g-32P]-ATP, wasevaluated. Fig. 9, panel B, shows that the [32P]phosphate wasincorporated into the wt MH2 domain and to a lesser extentinto the TSVA and TAVS mutant forms when using any of theSmTbR-I mutant derivatives (panels III–V, lanes 1–3). Thehighest incorporation level was obtained using SmTbR-I-Q303D followed by SmTbR-I-T299D/Q303D mutants. Neitherthe negative control (no DNA-transcription reaction) norSmTbR-I-wt translation mix showed significant incorporationof the 32P label in any of the SmSmad2-MH2 constructs (panelsI and II). However, minor phosphoproteins derived from thereticulocyte lysate that migrate at the same approximate sizeof SmSmad2-MH2, independent of SmTbR-I and SmSmad2,were observed in some experiments. The mutant forms ofSmTbR-I did not show significant incorporation of the radioac-tive label into either TAVA or AAVA mutants of SmSmad2-MH2 domain (panels III–V, lanes 4 and 5) indicating thatThr646 is not a phosphate acceptor. Fig. 9, panel B, panel VI,shows that the anti-SmSmad2 antibody efficiently precipitatedthe wt as well as the mutant forms of the SmSmad2-MH2domain.

Table I summarizes the data of SmSmad2 interaction withand in vitro phosphorylation by wt and the mutant derivativesof SmTbR-I.

FIG. 7. Interaction of SmSmad2 and SmTbR-I in the yeast two-hybrid system. Panel A, transactivation by SmSmad2 andSmSmad2–39 fused to GAL4-BD, positive control (pGAL4), and nega-tive controls (p53 or pLaminC). Panel B, two-hybrid interactions be-tween SmSmad2-AD and SmTbR-I-BD. Combinations of pSV40 witheither p53 or pLaminC served as positive and negative controls, respec-tively. Diagrammatic representations of the experiments appear abovethe LacZ filter lift assay results.

FIG. 8. Pull-down experiment. GST or GST fusion proteins coupledto glutathione beads were incubated with in vitro translated, 35S-la-beled SmTbR-I constructs (labeled on the sides of panels A–D). Boundproteins were separated in 12% SDS-PAGE, processed for fluorography,and exposed to x-ray films. Lanes 1–4 represent the reaction of theradiolabeled protein with resin-bound GST, GST-SmSmad2-Linker,GST-SmSmad2-MH2, and GST-SmSmad2–39 (full length), respectively.Lane 5 represents 50% of the input radiolabeled protein per bindingreaction run on the same gels. Arrows point to the specific precipitatedproducts.

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SmSmad2 Translocates to the Nucleus in Response to TGF-b1—The above data indicate that SmSmad2 stably interactswith SmTb R-I in vitro and in yeast. The ability of SmSmad2 tointeract productively with TbR-I and translocate to the nucleusin a ligand-dependent way was tested in Mv1Lu cells, whichare known to be responsive to induction by mammalian TGF-b(38). Mv1Lu cells that were transfected with pCMV-GST-SmS-mad2 and treated with TGF-b1 showed nuclear fluorescencewhen probed with an anti-GST monoclonal antibody (Fig. 10,panels E and F), whereas non-TGF-b1-treated cells showedcytoplasmic fluorescence with limited nuclear staining (Fig. 10,panel D). Cells transfected with parental pCMV-GST vector(positive control) showed cytoplasmic fluorescence only (Fig.10, panel A). Mv1Lu cells transfected with pcDNA-LacZ, (neg-ative control), a non-GST tag expressing, showed no fluores-cence (Fig. 10, panel B).

DISCUSSION

The life cycle of S. mansoni begins with penetration of cer-cariae into human skin. The parasites develop into matureworms in about 35 days when male and female worms mateand remain en copula throughout their adult life. During thisperiod, the parasites migrate from the skin, passing throughthe lungs to their final destination in the portal circulation.There are numerous unanswered questions regarding the de-velopment, differentiation, and specialization of the parasitetissues, male-female worm pairing, female worm maturation,and site finding behavior during the mammalian phase of theparasite life cycle. Factors produced by the parasites as well asthe host must function in these processes. Our investigation ofthe role of TGF-b in parasite development may provide insightinto some of these issues.

Members of TGF-b superfamily regulate many importantdevelopmental processes, such as mesenchymal differentiation,skeletal morphogenesis, and skin formation (39). In both ver-tebrate and invertebrate model systems, TGF-b family mem-bers can serve as morphogens, acting across developing tissuesin a graded fashion to specify a patterned array of cell fates (40,41). Defects in TGF-b signaling have been implicated in mul-tiple developmental disorders and in various human diseases,including cancer, fibrosis, and autoimmune diseases (8, 10,42–46).

The S. mansoni Smad2 cDNA encodes a protein that is about200 amino acids larger than other R-Smad homologues. Most ofthe additional amino acids are located in the linker region. Thelinker region contains several serine residues, which are po-tential phosphorylation sites for mitogen-activated protein ki-nase. Such phosphorylation results in inhibition of Smad2 nu-clear translocation in other organisms (34, 47). The MH2domain is highly conserved in SmSmad2. The conservation ofall five residues required for interaction of MH2 domain withSARA (31) suggests the presence of SARA homologue in schis-tosomes. SARA would be predicted to act by presenting theSmSmad2 to the activated receptor-I to be phosphorylated andactivated. L3 loop is an important and highly conserved struc-tural element present in R-Smads-MH2 domains. SmSmad2 L3loop possesses the specific subtype residues (Arg613 and Thr616)that specify the interaction of Smad2 and Smad3 with type ITGF-b and activin receptors (16). Another characteristic struc-tural feature of SmSmad2 is its unique C-terminal phosphoryl-ation signature. Schistosome Smad2 is the only member ofR-Smad subfamily with TSVS phosphorylation motif instead ofthe conserved SSXS motif present in all other members of thesubfamily. The significance of this difference is unknown andneeds further investigation. However, this difference does notappear to interfere with the biological functions of the protein.In this regard, studies on the C-terminal phosphorylation motif

FIG. 9. Panel A, in vitro phosphorylation of SmSmad2-MH2 domainby SmTbR-I. Different SmTbR-I derivatives synthesized in a rabbitreticulocyte lysate by coupled transcription and translation (shown atthe top of the figure) were incubated with GST or GST fusion deriva-tives of wt and mutant SmSmad2, in the presence of 10 mCi of[g-32P]ATP. Reaction products, bound to glutathione-Sepharose beads,were pelleted by centrifugation, washed four times, denatured in SDS,separated in 12% SDS gels, and subjected to autoradiography. Lanes1–4 represent GST, GST-SmSmad2-Linker, GST-SmSmad2-MH2, andGST-SmSmad2–39 (full length), respectively. Labeled arrows on theside of the figure point to the expected size of these products. Panel B,in vitro phosphorylation of wild type or mutant SmSmad2-MH2 by wildtype or mutant forms of SmTbR-I. SmSmad2-MH2 and SmTbR-I de-rivatives were synthesized in vitro by coupled transcription and trans-lation using rabbit reticulocyte lysate with unlabeled methionine. SmS-mad2-MH2 proteins were incubated with the translation mix of eithera negative control (no DNA transcription reaction) or one of the follow-ing SmTbR-I derivatives: wild type, T299D, Q303D, and T299D/Q303D,in the presence of 10 mCi [g-32P]ATP (panels I–V, respectively). Anti-SmSmad2 rabbit IgG was added to each reaction, and the IgG-SmS-mad2-MH2 complexes were precipitated by protein A-Sepharose,washed 4 times, denatured in SDS, separated in 12% SDS gels, andsubjected to autoradiography. Panel VI represents the results of immu-noprecipitation of wild type and mutant [35S]Met-labeled SmSmad2-MH2 derivatives, demonstrating that the anti-SmSmad2 antibody wascapable of precipitating each SmSmad2-MH2, with similar efficiency.Lanes 1–5 represent SmSmad2-MH2 with the following terminal aminoacids: TSVS (wild type), TSVA, TAVS, TAVA, and AAVA, respectively.Lane 6 (panel VI) represents the immunoprecipitation background re-action using [35S]Met-labeled negative control lysate.

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in other Smad proteins showed that threonine could substitutefor serines (21).

RT-PCR data indicate that SmSmad2 gene expression ex-hibits relatively constant levels in the human host with mRNAdetectable some time after 3 h but before 4 days post-infection.Native SmSmad2 was localized in female sexual tissues (vitel-laria, uterus, and ovary) and in male testis, as well as cells inthe subtegument and parenchyma. This pattern of distributioncoincides with the pattern observed by in situ hybridization forSmSmad2 mRNA transcripts. This distribution profile of themRNA and protein indicates that SmSmad2, or TGF-b signal-ing, plays multiple roles in the development of the parasite ingeneral, and the sexual maturation of both male and femaleworms. As vitelline cell development (gene expression) is reg-ulated by a signal from the male, the finding of SmSmad2expression in vitelline cells implicates the TGF-b signalingcascade in female reproductive development.

To determine whether SmSmad2 could be activated by

TGF-b, the ability of SmSmad2 to translocate to the nucleusupon induction by TGF-b was evaluated. Mink lung epithelialcells (Mv1Lu) transfected with GST-tagged SmSmad2 showedsome nuclear fluorescence with most of the fluorescence local-ized in the cytoplasm. This observation is similar to the resultsof previous studies on Smad3 (48) and Xenopus Mad-like pro-tein (XenMLP) (49). Treatment of transfected Mv1Lu cells withrhTGF-b1 resulted in predominant nuclear localization of SmS-mad2, demonstrating that SmSmad2 responded to the signalsinitiated by the human ligand. These data are consistent withNakao et al. (35) who reported that the endogenous Smad2,Smad3, and Smad4 exhibited predominant fluorescence in thecytoplasm of uninduced Mv1Lu cells, whereas the fluorescencewas mainly localized in the nuclei in case of cells stimulatedwith TGF-b1. These results not only demonstrate that SmS-mad2 is biologically active but also that the mechanism em-ployed in nuclear transport in higher eukaryotes recognizes aschistosome protein.

To investigate the interaction between SmSmad2 andSmTbR-I, we used wild type and mutant constructs of bothproteins in yeast two-hybrid and GST pull-down assays.SmSmad2–39, a mutant in which the stop codon was removedto extend the reading frame beyond the “TSVS” phosphoryla-tion motif, was constructed. This modification of the C-terminalend of the protein was expected to block type I receptor-medi-ated phosphorylation of SmSmad2. Such a modification wasdemonstrated to abolish the ability of the C-terminal serineresidues to become phosphorylated (35). For SmTbR-I, threemutants were constructed in which either Thr299, Gln303, orboth were mutated to Asp. These constructs were expected toyield a kinase-deficient form, a constitutively active derivative(25), and a construct that contains both kinase-activating and-inactivating mutations, respectively. Wild type SmTbR-I in-teracted with wild type SmSmad2 and to a lesser extent withSmSmad2–39 in vivo in yeast two-hybrid assay, with MH2domain of SmSmad2, and full-length SmSmad2–39 in GSTpull-down experiment, but failed to phosphorylate the MH2domain in vitro. The interaction between wild type SmSmad2and SmTbR-I in yeast was unexpected since other authors havereported that the interaction between wild type human Smad2and either wild type TbR-I (11, 35) or the constitutively activeform of the receptor (11) could not be detected in COS cells.Perhaps the difference in results is due to the different systemsutilized in each study. Transfected COS cells followed by im-munoprecipitation of the interacting proteins may not be sen-sitive enough to detect a weak interaction compared withtransformed yeast cells, where the interaction is allowed toproceed over a relatively longer period. Consistent with ourresults, Zhang et al. (29) reported that the heteromeric inter-actions between human MAD-3 and-4 could only be detected in

TABLE IInteraction of SmSmad2 derivatives with SmTbR-I

The abbreviation and symbols used are as follows: ND, not determined; 11, strong positive; 1, positive; 1/2, weak positive; and 2, negative.

SmTbR-I SmSmad2 Yeast two-hybrid GST-pull down In vitro

phosphorylation

wt wt 11 ND NDMH2 ND 1 2SmSmad2–39 1/2 11 2

T299D wt 2 ND NDMH2 ND 1 1/2SmSmad2–39 1/2 11 2

Q303D wt 2 ND NDMH2 ND 1 11SmSmad2–39 11 1 2

T299D/Q303D wt 2 ND NDMH2 ND 1 1SmSmad2–39 1 11 2

FIG. 10. Nuclear translocation of SmSmad2 upon stimulationby TGF-b1. Transfected mink lung epithelial cells (Mv1Lu) were fixedand probed with anti-GST monoclonal antibody. Panel A shows theparental pCMV-GST vector-transfected Mv1Lu cells (positive control)expressing GST, and panel B shows typical cells frompcDNA-LacZ-transfected cells (negative control). Mv1Lu cells weretransfected with pCMV-GST-Smad2 vector untreated (panel D) andstimulated with TGF-b1 (panels E and F). Panel C shows the phasecontrast field of the examined cells in panel D. Note that there is onlycytoplasmic staining in panel A and nuclear staining in panels E and F.

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yeast two-hybrid assays but not by coimmunoprecipitation oftransfected cultured cell lysates. The interaction of wt-SmTbR-I with SmSmad2–39 in vivo and in vitro was expectedsince the modification of the C-terminal end of SmSmad2 waslikely to block the phosphorylation mediated by SmTbR-I re-sulting in stabilizing SmSmad2-SmTbR-I complex (11). Theinteraction with SmSmad2-MH2 domain in vitro was also ex-pected as Smad2 was shown to serve as a direct substrate of theactivated TbR complex (TbR-I activated by a ligand-boundTbR-II) (11). Therefore, in the absence of TbR-II, SmSmad2-MH2 would not be expected to be phosphorylated by wt-SmTbR-I, and thus, as shown by our results, allowed SmS-mad2-MH2 to form a stable complex with the wt receptor. Ourinterpretation of this interaction is supported by the lack ofphosphorylation of SmSmad2-MH2 by wt-SmTbR-I.

The activity of the SmTbR-I constitutive mutant Q303Dshowed results consistent with those obtained in other systems(25, 36, 37). This mutant was found to interact stably in vivoand in vitro with SmSmad2–39, and in vitro, although to alesser extent, with the MH2 domain of SmSmad2. However, thein vivo interaction in yeast with wt-SmSmad2 was undetect-able. SmTbR-I-Q303D mutant was also able to phosphorylatethe MH2 domain of SmSmad2-wt in vitro. This mutant con-struct may exert its constitutive signaling activity due to thereplacement of uncharged polar residue (Gln303) by a nega-tively charged residue (Asp). This would favor the initial appo-sition between type I receptor and SmSmad2, which shouldfacilitate subsequent binding and phosphorylation of the sub-strate (31). The stable interaction between Q303D mutant andSmSmad2–39 was observed because the ability of SmTbR-I-Q303D to phosphorylate SmSmad2–39 was blocked by the C-terminal extension. However, in case of wt SmSmad2, whichwas susceptible to phosphorylation by the constitutive mutantQ303D, the interaction in vivo was only transient due to thephosphorylation of SmSmad2 and the dissociation of SmTbR-I-SmSmad2 complex.

The mutation T299D was expected to yield a kinase-inactivereceptor I (25). Unexpectedly, this mutant did not interact withwild type SmSmad2 in vivo. However, it interacted withSmSmad2–39 in vivo, and with both full-length SmSmad2–39and the MH2 domain of SmSmad2-wt. Surprisingly, the T299Dmutant showed kinase activity, although less than Q303D mu-tant, in vitro. Therefore, the T299D mutant behaved similar tothe Q303D construct. An explanation for the difference be-tween SmTbR-I-T299D and the corresponding mutants inother type I receptors may be provided by a direct comparisonof the GS domain and its downstream region in these receptors.SmTbR-I has one amino acid substitution (Lys292) instead ofthe conserved “L” at this position for all other known type Ireceptors. This basic amino acid located just after the GS re-gion may participate in stabilizing the T299D mutant interac-tion with SmSmad2 and provide the net charge equilibriumrequired for phosphorylating its substrate, mimicking the ef-fect of the Q303D mutant. Therefore, the use of “less-activekinase” term rather than “kinase-inactive” in case of T299D-SmTbR-I mutant would be more applicable for this system.

In most cases, an interaction was observed when SmSmad2phosphorylation was prevented, as in absence of activationwhen using wild type SmTbR-I, or the use of a nonphosphory-latable form of SmSmad2, as in case of SmSmad2–39. Thesedata are in agreement with the hypothesis proposed by Macias-Silva et al. (11) who reported that this transient interaction canbe stabilized by preventing phosphorylation of R-Smad eitherby using a kinase-deficient version of type I receptor or amutant version of Smad2 in which the C-terminal phosphoryl-ation is blocked. Furthermore, previous results also showed

that C-terminal FLAG-tagged Smad3 stably associated withwild type TbR-I (35). Our data revealed that the MH2 domaininteracted in vitro with the mutant forms of SmTbR-I, whereasthis interaction was not detected in the two-hybrid analysisusing full-length SmSmad2. The interaction may have beenundetectable in vivo due to its transient nature due to phos-phorylation and dissociation of the complex. This would be truefor Q303D and T299D in vitro in the absence of ATP.

The SmTbR-I-catalyzed phosphorylation of in vitro trans-lated SmSmad2-MH2-wt and mutant forms showed resultsconsistent with the two-hybrid and pull-down assays, as well asthe in vitro phosphorylation reaction of the bacterially ex-pressed SmSmad2-GST fusion proteins. The lack of phospho-rylation of wild type or mutant SmSmad2-MH2 by wild typeSmTbR-I confirms the inability of the wt-SmTbR-I to phospho-rylate SmSmad2 in absence of ligand, TGF-b. Alternatively,each mutant form of SmTbR-I phosphorylated the wt-MH2domain of SmSmad2 with varied efficiencies, demonstratingthat each mutant receptor I is constitutively active. SmSmad2-MH2 mutants TSVA and TAVS were phosphorylated by themutant forms of receptor I demonstrating that neither serine isthe sole phosphate acceptor. Phosphorylation of double TAVAand triple AAVA mutants was blocked, implicating both Ser647

and Ser649 as the receptor-mediated phosphorylation sites, asin the case of the human Smad2 homologue (50). In addition,the preferential phosphorylation of Ser647 suggests that Ser647

may enhance the efficient phosphorylation of Ser649. Further-more, these results indicate that Thr646 is not a phosphateacceptor.

Both SmSmad2 and SmSmad2–39 fused to GAL4-BD acti-vated the transcription of the reporter genes (lacZ and his3) inyeast Y190. Together with the phosphorylation data, theseresults indicate that SmSmad2 possesses a transactivationfunction, which does not depend on its ability to be phospho-rylated at its C-terminal end by type I receptor kinase. Con-sistent with this observation, Liu et al. (20) and Hayashi et al.(51) reported that full-length Smad1 and Smad2 and Smad1C-terminal domain could function as agonist-dependent andagonist-independent transcriptional activators, respectively,when fused to the DNA-binding domain of GAL4. Other studiesalso indicate that the transactivation function may not be de-pendent on the ability of the protein to be phosphorylated (49).

In conclusion, we showed that SmSmad2 interacts withSmTbR-I in vivo and in vitro. We also showed that the consti-tutively active mutant versions of SmTbR-I could phosphoryl-ate SmSmad2 MH2 domain in vitro, although with variedefficiencies. We also showed that Ser647 and Ser649 are likely toserve as receptor-mediated phosphorylation sites of the SmS-mad2. In addition, in a heterologous system, SmSmad2 wastranslocated to the nucleus in response to rhTGF-b1. Our re-sults also showed that SmSmad2 mRNA transcript and itsencoded protein localized in the subtegumental layer of theparasite. This may represent an intracellular link between thespecific receptor located on the surface of the parasite(SmTbR-I) (5) and specific effects elicited by TGF-b or TGF-b-like signaling. Whether ligands are derived from the host,representing a new chapter in host-parasite interaction, or areproduced by the parasite itself to elicit the development, dif-ferentiation, and specialization of its tissues, further investi-gations are needed. In either case, this study argues that SmS-mad2 plays an important role in various stages in varioustissues of the mammalian (human) phase of schistosome devel-opment. The identification of schistosome Smad2 provides amolecular tool to investigate the role of TGF-b signaling inschistosomes. The identification of the ligand(s) as well as thecooperative partners and the responsive genes are current tar-

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gets to help in understanding the role of this signaling pathwayin the development of the parasite and its interaction with thehuman host.

Acknowledgments—We thank Dr. Wade Sigurdson, Head of the Con-focal Microscopy and Three-dimensional Imaging Laboratory, for helpwith microscopy and Dr. Randall Reed, Johns Hopkins University, forproviding the pCMV-GST vector.

REFERENCES

1. Engers, H. D., Bergquist, R., and Modabber, F. (1996) Dev. Biol. Stand. 87,73–84

2. Boros, D. L. (1989) Clin. Microbiol. Rev. 2, 250–2693. Smithers, S. R., and Doenhoff, M. J. (1982) in Immunology of Parasitic Dis-

eases (S. Cohen and K. S. Warren) pp. 527–607, Blackwell Scientific, Ox-ford, UK

4. LoVerde, P. T., and Chen, L. (1991) Parasitol. Today 7, 303–3085. Davies, S. J., Shoemaker, C. B., and Pearce, E. J. (1998) J. Biol. Chem. 273,

11234–112406. Ramachandran, H., Skelly, P. J., and Shoemaker, C. B. (1996) Mol. Biochem.

Parasitol. 83, 1–107. Silva, E. E., Clarke, M. W., and Podesta, R. B. (1993) J. Immunol. 151,

7057–70668. Massague, J. (1998) Annu. Rev. Biochem. 67, 753–7919. Massague, J., and Wotton, D. (2000) EMBO J. 19, 1745–1754

10. Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465–47111. Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and

Wrana, J. L. (1996) Cell 87, 1215–122412. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massague, J. (1996) Nature

383, 832–83613. Zhang, Y., Musci, T., and Derynck, R. (1997) Curr. Biol. 7, 270–27614. Liu, F., Pouponnot, C., and Massague, J. (1997) Genes Dev. 11, 3157–316715. Feng, X. H., and Derynck, R. (1997) EMBO J. 16, 3912–392316. Lo, R. S., Chen, Y. G., Shi, Y., Pavletich, N. P., and Massague, J. (1998) EMBO

J. 17, 996–100517. Kim, J., Johnson, K., Chen, H. J., Carroll, S., and Laughon, A. (1997) Nature

388, 304–30818. Shi, Y., Wang, Y. F., Jayaraman, L., Yang, H., Massague, J., and Pavletich,

N. P. (1998) Cell 94, 585–59419. Baker, J. C., and Harland, R. M. (1996) Genes Dev. 10, 1880–188920. Liu, F., Hata, A., Baker, J. C., Doody, J., Carcamo, J., Harland, R. M., and

Massague, J. (1996) Nature 381, 620–62321. Kretzschmar, M., Liu, F., Hata, A., Doody, J., and Massague, J. (1997) Genes

Dev. 11, 984–99522. Osman, A., Niles, E. G., and LoVerde, P. T. (1999) Mol. Biochem. Parasitol.

100, 27–4123. Freebern, W. J., Osman, A., Niles, E. G., Christen, L., and LoVerde, P. T.

(1999) J. Biol. Chem. 274, 4577–458524. Bobek, L., Rekosh, D. M., van Keulen, H., and LoVerde, P. T. (1986) Proc. Natl.

Acad. Sci. U. S. A. 83, 5544–5548

25. Wieser, R., Wrana, J. L., and Massague, J. (1995) EMBO J. 14, 2199–220826. Graff, J. M., Bansal, A., and Melton, D. A. (1996) Cell 85, 479–48727. Riggins, G. J., Thiagalingam, S., Rozenblum, E., Weinstein, C. L., Kern, S. E.,

Hamilton, S. R., Willson, J. K., Markowitz, S. D., Kinzler, K. W., andVogelstein, B. (1996) Nat. Genet. 13, 347–349

28. Eppert, K., Scherer, S. W., Ozcelik, H., Pirone, R., Hoodless, P., Kim, H., Tsui,L. C., Bapat, B., Gallinger, S., Andrulis, I. L., Thomsen, G. H., Wrana, J. L.,and Attisano, L. (1996) Cell 86, 543–552

29. Zhang, Y., Feng, X., We, R., and Derynck, R. (1996) Nature 383, 168–17230. Nakao, A., Roijer, E., Imamura, T., Souchelnytskyi, S., Stenman, G., Heldin,

C. H., and ten Dijke, P. (1997) J. Biol. Chem. 272, 2896–290031. Wu, G., Chen, Y. G., Ozdamar, B., Gyuricza, C. A., Chong, P. A., Wrana, J. L.,

Massague, J., and Shi, Y. (2000) Science 287, 92–9732. Tsukazaki, T., Chiang, T. A., Davison, A. F., Attisano, L., and Wrana, J. L.

(1998) Cell 95, 779–79133. Kretzschmar, M., Doody, J., and Massague, J. (1997) Nature 389, 618–62234. Calonge, M. J., and Massague, J. (1999) J. Biol. Chem. 274, 33637–3364335. Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda,

E., Tamaki, K., Hanai, J., Heldin, C. H., Miyazono, K., and ten Dijke, P.(1997) EMBO J. 16, 5353–5362

36. Willis, S. A., Zimmerman, C. M., Li, L. I., and Mathews, L. S. (1996) Mol.Endocrinol. 10, 367–379

37. Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O’Connor, M. B.,Attisano, L., and Wrana, J. L. (1996) Cell 85, 489–500

38. Laiho, M., DeCaprio, J. A., Ludlow, J. W., Livingston, D. M., and Massague, J.(1990) Cell 62, 175–185

39. Derynck, R. (1998) Nature 393, 737–73940. Nellen, D., Burke, R., Struhl, G., and Basler, K. (1996) Cell 85, 357–36841. Neumann, C., and Cohen, S. (1997) BioEssays 19, 721–72942. Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H., and Gelbart,

W. M. (1995) Genetics 139, 1347–135843. Chen, T., Carter, D., Garrigue-Antar, L., and Reiss, M. (1998) Cancer Res. 58,

4805–481044. Nomura, M., and Li, E. (1998) Nature 393, 786–79045. Waldrip, W. R., Bikoff, E. K., Hoodless, P. A., Wrana, J. L., and Robertson, E. J.

(1998) Cell 92, 797–80846. Sirard, C., de la Pompa, J. L., Elia, A., Itie, A., Mirtsos, C., Cheung, A., Hahn,

S., Wakeham, A., Schwartz, L., Kern, S. E., Rossant, J., and Mak, T. W.(1998) Genes Dev. 12, 107–119

47. Kretzschmar, M., Doody, J., Timokhina, I., and Massague, J. (1999) Genes Dev.13, 804–816

48. Chen, Y., Lebrun, J. J., and Vale, W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,12992–12927

49. Meersseman, G., Verschueren, K., Nelles, L., Blumenstock, C., Kraft, H.,Wuytens, G., Remacle, J., Kozak, C. A., Tylzanowski, P., Niehrs, C., andHuylebroeck, D. (1997) Mech. Dev. 61, 127–140

50. Abdollah, S., Macias-Silva, M., Tsukazaki, T., Hayashi, H., Attisano, L., andWrana, J. L. (1997) J. Biol. Chem. 272, 27678–27685

51. Hayashi, H., Abdollah, S., Qiu, Y., Cai, J., Xu, Y. Y., Grinnell, B. W., Richard-son, M. A., Topper, J. N., Gimbrone, M. A., Jr., Wrana, J. L., and Falb, D.(1997) Cell 89, 1165–1173

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Ahmed Osman, Edward G. Niles and Philip T. LoVerde Signal Transducer β, a Transforming Growth Factor-mansoni

SchistosomaIdentification and Characterization of a Smad2 Homologue from

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