From the Department of Microbiology and Center for Microbial Pathogenesis, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214
Received for publication, July 6, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Smad proteins are essential
intracellular signal transducers of the transforming growth factor- Schistosomiasis is a parasitic disease second only to malaria in
prevalence. According to the World Health Organization records, about
300 million people in 76 countries are currently infected. More than
600 million people live in endemic areas and are at risk of contracting
the disease (1). The egg is the major cause of the pathology and
complications of the disease such as portal hypertension, liver
fibrosis hemorrhage, and death (2, 3). Female schistosomes initiate
sexual development and start laying eggs in response to a stimulus by
male worms (4). Very little is known about the nature of the signal(s)
involved or the mechanism(s) of this process. Furthermore, schistosomes
reside in portal circulation surrounded by host molecules such as
antibodies, hormones, cytokines, and growth factors. The tegument layer
covering the parasite worm body represents the interface between the
parasite and its host environment. Several studies reported the
identification of receptors for growth factors and other host molecules
on the surface of the parasite (5-7). Transforming growth factor- TGF- The signaling pathway is initiated by ligand binding to the
extracellular domain of the type II receptor kinase. The cytoplasmic domain of type II receptor catalyzes the phosphorylation and
concomitant activation of the specific type I receptor kinase. Once
activated, the type I receptor binds to and phosphorylates a member of
a subset of the Smad family, receptor-regulated Smads (R-Smads). The
phosphorylation of R-Smads destabilizes the complex with type I
receptor releasing the activated R-Smad (11) allowing it to associate
with a related protein Smad4, known as comediator Smad or co-Smad (12,
13). The heteromeric complex (R-Smad/co-Smad) then translocates into
the nucleus (14) where it associates with specific DNA-binding
partner(s) that direct it to the regulatory region of target gene(s)
(9).
Two structural motifs in the type I receptor and the corresponding
R-Smad, the L45 loop and L3 loop, respectively, contain subtype-specific residues and determine the specificity of the interaction between these two members of the signaling cascade (15,
16). R-Smads and co-Smads consist of conserved N- and C-terminal
domains and MH1 and MH2 domains. The MH1 domain possesses a DNA binding
activity (17, 18), and the MH2 domain exerts a transcriptional
activation function (19, 20). These conserved domains are linked by an
intervening, nonconserved linker region that varies both in length and
sequence (8). In the basal state, the MH1 domain of R-Smads interacts
with and inhibits the transcriptional (20) and biological (19)
activities of the MH2 domain. Upon phosphorylation by type I receptor
kinase, this auto-inhibitory MH1-MH2 interaction of R-Smad is relieved,
allowing the association of the activated R-Smad with co-Smad. The MH2
domain of R-Smads contains receptor phosphorylation sites located at
the very C-terminal end of the protein (SSXS) (11, 21).
Co-Smads lack this phosphorylation signature motif and consequently do
not serve as substrates for receptor I kinases (11). The MH2 domain
also mediates the interaction and association of R-Smads with type I
receptors (11).
We report herein the identification of S. mansoni Smad2
homologue (SmSmad2). We define its expression level in different
developmental stages and the localization of the protein and mRNA
transcripts in adult worm sections. We also demonstrate that
schistosome protein performs the biological activities exhibited by
other Smad2 homologues, including its interaction with and
phosphorylation by a schistosome homologue of T Identification of SmSmad2 cDNA--
A fragment of about 1 kb
spanning part of the MH1 domain and the linker region was coisolated
during our previous work on SmRas cDNA (22). An oligonucleotide
representing the 3'-end of the coding region of SmRas
(5'-GACGTCGACTCATTGTATACAACATTTTGC-3') amplified a 1-kb fragment. The
PCR product was cloned into TOPO-TA vector (Invitrogen) and sequenced.
Sequence analysis of this fragment showed that it is related to members
of R-Smad subclass of the Smad family. The DNA fragment was labeled
with [ Expression of SmSmad2 and Production of Specific
Antiserum--
Smad2 cDNA representing the MH1 domain was
amplified from the parent phagemid with specific 5'- primer
(Smad2-cod-5') containing KpnI (underlined) and
SalI (italicized) sites
(5'-GGTACCGTCGACCGGAATTCAAAATGAGTCTCTTTACAAGTCCAC-3') and a reverse primer representing the complementary sequence of bp
533-558. The 430-bp PCR product was subcloned in TOPO-TA vector (Invitrogen), sequenced, and then digested with KpnI at the
5'-end and HpaI at the 3'-end, a unique site present at bp
542 of the cDNA sequence. The excised DNA fragment was then
recloned into the parent phagemid digested with the same enzymes
yielding a cDNA lacking the 5'-untranslated region. The recombinant
phagemid was cleaved with SalI and NotI, and the
full-length SmSmad2 insert was subcloned in pGEX-4T-1 linearized with
the same enzymes. The recombinant pGEX-SmSmad2 plasmid was
overexpressed in TOP10 cells (Invitrogen) using 0.5 mM
isopropyl-1-thio- Western Blot and Immunocytolocalization--
S.
mansoni adult worm pairs were perfused from infected hamsters and
homogenized in an extraction 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 mixture; Sigma). The
homogenate was sonicated for 1 min and centrifuged at 35,000 rpm for
1 h at 4 °C, and the soluble fraction was aliquoted and stored
at
Schistosome extract was treated with calf intestinal alkaline
phosphatase (CIAP; Life Technologies, Inc.) at concentrations of 0.2 and 1 unit/µg extract for 30-60 min at 37 °C. The untreated extract, with and without 10× CIAP buffer, along with CIAP-treated extract were separated on 7.5% SDS-PAGE. SDS gels were transferred onto polyvinylidene difluoride membrane, and the blots were probed with
IgG fractions from preimmune rabbit and SmSmad2-GST-immunized rabbit
sera (0.5 µg/ml). Biotinylated goat anti-rabbit IgG (0.5 µg/ml;
Zymed Laboratories Inc.) was used to probe the
reacting primary antibodies, and this in turn was detected using
Z-avidin horseradish peroxidase conjugate (0.5 µg/ml;
Zymed Laboratories Inc.). The reactions were developed
with tetramethyl benzidine reagent (Zymed
Laboratories Inc.).
The IgG fractions (5 µg/ml) were also used to detect SmSmad2 native
protein in adult worm cryosections. Biotinylated goat anti-rabbit IgG
(5 µg/ml; Zymed Laboratories Inc.) and Z-avidin FITC
conjugate (5 µg/ml; Zymed Laboratories Inc.) or
streptavidin-Cy5 conjugate (10 µg/ml; Zymed Laboratories
Inc.) were used to localize the primary rabbit IgG antibodies.
Probed sections were evaluated using a Bio-Rad MRC100 confocal
microscope equipped with a krypton laser. A 674-nm filter was used to
visualize the Cy5-probed sections.
RT-PCR and in Situ Hybridization--
SmSmad2 transcripts were
detected using two approaches. RT-PCR was employed to determine the
total transcription level of SmSmad2 in different developmental stages.
In situ hybridization was used to detect where SmSmad2
transcripts are localized and in which tissues the protein is
synthesized. In RT-PCR, total RNA was purified from different life
stages of S. mansoni that are related to the definitive
human host, starting with 3-h schistosomules and up to paired
45-day-old adult worms using RNA-stat 60 reagent (Tel-Test, Inc.). RNA
was copied by reverse transcription (22), and cDNA was used as
templates for subsequent PCR amplification. A primer pair corresponding
to bp 1614-1636 and the complementary sequence of bp 1902-1926 was
used to amplify a fragment that spanned part of the MH2 domain. SmRXR1
(23), a clone previously identified in our laboratory and shown to be
constitutively expressed in all human host-related stages of the
parasite, was used as an internal control for the RT-PCRs. A primer
pair representing part of the SmRXR1 AB-domain (23) was used to amplify
a 460-bp DNA fragment. To remain in the linear range of amplification,
26 cycles of PCR were performed. The amplification products were
size-separated in a 2.0% agarose gel, stained with ethidium bromide,
and quantified using the Molecular Analyst gel documentation system
(Bio-Rad). SmSmad2 PCR products were normalized to those of SmRXR to
eliminate the differences in quality and/or quantity of the input RNA
in the RT reaction.
For the in situ hybridization, an antisense oligonucleotide
corresponding to the complementary sequence of bp 695-744 was synthesized, labeled with biotin using Bright-Star biotin labeling kit
(Ambion), and used to probe paraffin-embedded sections using mRNAlocator-hyb kit (Ambion) following the
manufacturer's instructions. Two primers, one represents the sense
sequence of SmSmad2 (5'-GGAAGAGGTTCATCTTTTGGTTCACATCATTCTCG-3', bp 620-654) and the other represents the antisense sequence of Smp14 gene (a female-specific gene that produces an eggshell
protein) (24) (5'-GCCTCCACCATATCCACTATCGCCATAACCGCTATCACAATCGCTACC-3'), 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), and the sections were
lightly counterstained using nuclear fast red. The sections were
examined and photographed using Nikon microphot digital microscope.
SmSmad2 Interaction with SmT
GST pull-down analysis was performed to attempt to confirm the results
obtained from the two-hybrid interaction in yeast. In this experiment,
the four constructs of SmT In Vitro Phosphorylation of SmSmad2 by SmT
To determine more precisely the phosphorylation sites in Smad2
C-terminal motif, we constructed several mutant forms of SmSmad2-MH2 domain in which the potential phosphorylation sites were mutated 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
generated by PCR amplification using oligonucleotides encoding the
corresponding mutations. PCR products were cloned in TOPO-TA-pCR2.1
vector (Invitrogen), sequenced to confirm the presence of the specific
mutations, digested with EcoRI/XhoI, and cloned
in pCITE-4a vector (Novagen). Smad2-MH2-wt and SmT TGF- Isolation, Identification, and Computer Analyses of SmSmad2
cDNA--
The amino acid sequence predicted by the original 1-kb
DNA fragment showed strong homology to the MH1 domain of R-Smad
subfamily. This DNA fragment was used to screen Expression, Western Blot Analysis, and
Immunocytolocalization--
SmSmad2 was overexpressed in bacteria as a
GST fusion protein. The fusion protein was used to generate a specific
rabbit polyclonal antiserum. The IgG fraction of the immunized rabbit
serum detected two proteins of about 70 and 72 kDa in Western blots of
schistosome worm extracts (Fig. 2,
panel C, lanes 2 and 3). To determine
whether the slower migrating species is a phosphorylated form of Smad2, schistosome extracts were treated with calf intestinal alkaline phosphatase prior to PAGE analyses. After CIAP treatment, only the
70-kDa protein band was detected (Fig. 2, panel C, lane 1). In this particular experiment the 70-kDa band is distorted due to the
high concentration of CIAP in the sample. This result indicates that
the 72-kDa protein represents a phosphorylated form of SmSmad2 in the
parasite extract. Phosphorylation may result from receptor phosphorylation and activation of the SmSmad2 or mitogen-activated protein kinase phosphorylation of the linker region (33, 34). IgG
fraction of preimmune rabbit serum was used as a negative control (Fig.
2, panel B).
Native SmSmad2 was localized in adult worm cryosections probed with
anti-SmSmad2 antibodies. SmSmad2 was detected in the tubercles of the
tegument, in parenchymal cells of male and female worms (Fig.
3, panels A and B),
as well as in the vitellaria (Fig. 4, panels IC and IIIC), the developing embryo in egg
(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 not shown). The use of Cy5 as a detection reagent
enabled us to overcome the auto-fluorescence that occurs in female
vitellaria and unequivocally observe SmSmad2 reactivity in the
vitellaria and embryo contained within the uterine egg (Fig. 4).
RT-PCR and in Situ Hybridization--
The level of SmSmad2
mRNA was determined in different schistosome developmental stages
by RT-PCR. In the mammalian host stages, SmSmad2 mRNA was found to
be detected as early as 4 days post-infection (Fig.
5, lane 2). Compared with the
mRNA level of the internal control (SmRXR1), SmSmad2 mRNA
exhibits relatively constant levels throughout development.
SmSmad2 mRNA was also detected in paraffin sections of adult worms
by in situ hybridization. The transcripts were localized in
the subtegumental and parenchymal cells in male and female worms (Fig.
6, panels C and D)
and in sexual tissues such as vitellaria (Fig. 6, panel D),
uterus, and ovary (Fig. 6, panel C) in female worm. The
positive control (Smp14) showed strong localization in female vitelline
cells (Fig. 6, panel A) and the negative control showed
minimal background reactivity in both male (Fig. 6, panel B)
and female sections (data not shown).
SmSmad2 Interaction with SmT
The use of the yeast two-hybrid assay revealed that SmSmad2 interacts
in vivo with SmT
Results of the yeast two-hybrid and GST pull-down analyses clearly
demonstrated that certain constructs of SmSmad2 stably interact, both
in vivo and in vitro, with either wt or mutant forms of SmT
To confirm the phosphorylation results and to determine the specific
phosphorylation sites in SmSmad2 MH2 domain C-terminal phosphorylation
motif, we employed a different format of the in vitro
phosphorylation reaction. In this reaction we used wt and mutant
constructs of SmSmad2-MH2 domain, where the potential phosphorylation sites were mutated to alanine. Both groups of SmSmad2 and SmT
Table I summarizes the data of SmSmad2
interaction with and in vitro phosphorylation by wt and the
mutant derivatives of SmT SmSmad2 Translocates to the Nucleus in Response to
TGF- The life cycle of S. mansoni begins with penetration of
cercariae into human skin. The parasites develop into mature worms in
about 35 days when male and female worms mate and remain en copula throughout their adult life. During this period, the
parasites migrate from the skin, passing through the lungs to their
final destination in the portal circulation. There are numerous
unanswered questions regarding the development, differentiation, and
specialization of the parasite tissues, male-female worm pairing,
female worm maturation, and site finding behavior during the mammalian
phase of the parasite life cycle. Factors produced by the parasites as
well as the host must function in these processes. Our investigation of
the role of TGF- Members of TGF- The S. mansoni Smad2 cDNA encodes a protein that is
about 200 amino acids larger than other R-Smad homologues. Most of the additional amino acids are located in the linker region. The linker region contains several serine residues, which are potential
phosphorylation sites for mitogen-activated protein kinase. Such
phosphorylation results in inhibition of Smad2 nuclear translocation in
other organisms (34, 47). The MH2 domain is highly conserved in SmSmad2. The conservation of all five residues required for interaction of MH2 domain with SARA (31) suggests the presence of SARA homologue in
schistosomes. SARA would be predicted to act by presenting the SmSmad2
to the activated receptor-I to be phosphorylated and activated. L3 loop
is an important and highly conserved structural element present in
R-Smads-MH2 domains. SmSmad2 L3 loop possesses the specific subtype
residues (Arg613 and Thr616) that specify the
interaction of Smad2 and Smad3 with type I TGF- RT-PCR data indicate that SmSmad2 gene expression exhibits
relatively constant levels in the human host with mRNA detectable some time after 3 h but before 4 days post-infection. Native
SmSmad2 was localized in female sexual tissues (vitellaria, uterus, and ovary) and in male testis, as well as cells in the subtegument and
parenchyma. This pattern of distribution coincides with the pattern
observed by in situ hybridization for SmSmad2 mRNA
transcripts. This distribution profile of the mRNA and protein
indicates that SmSmad2, or TGF- To determine whether SmSmad2 could be activated by TGF- To investigate the interaction between SmSmad2 and SmT The activity of the SmT The mutation T299D was expected to yield a kinase-inactive receptor I
(25). Unexpectedly, this mutant did not interact with wild type SmSmad2
in vivo. However, it interacted with SmSmad2-3' in
vivo, and with both full-length SmSmad2-3' and the MH2 domain of
SmSmad2-wt. Surprisingly, the T299D mutant showed kinase activity, although less than Q303D mutant, in vitro. Therefore, the
T299D mutant behaved similar to the Q303D construct. An explanation for
the difference between SmT In most cases, an interaction was observed when SmSmad2 phosphorylation
was prevented, as in absence of activation when using wild type
SmT The SmT Both SmSmad2 and SmSmad2-3' fused to GAL4-BD activated the
transcription of the reporter genes (lacZ and
his3) in yeast Y190. Together with the phosphorylation data,
these results indicate that SmSmad2 possesses a transactivation
function, which does not depend on its ability to be phosphorylated at
its C-terminal end by type I receptor kinase. Consistent with this
observation, Liu et al. (20) and Hayashi et al.
(51) reported that full-length Smad1 and Smad2 and Smad1 C-terminal
domain could function as agonist-dependent and
agonist-independent transcriptional activators, respectively, when
fused to the DNA-binding domain of GAL4. Other studies also indicate
that the transactivation function may not be dependent on the ability
of the protein to be phosphorylated (49).
In conclusion, we showed that SmSmad2 interacts with SmT
(TGF-
) superfamily. The TGF-
superfamily signals through
phosphorylation and activation of R-Smad proteins, receptor-regulated
Smads, by heteromeric complexes of ligand-specific type I and type II
serine/threonine kinase receptors. R-Smads receive a signal from the
activated receptor complex and transmit it to the nucleus. A cDNA
was isolated that encodes a 649-amino acid protein found to be
homologous to members of R-Smad subfamily with highest homology scored
to clawed African frog and human Smad2. The Schistosoma
mansoni homologue (SmSmad2) was overexpressed in bacteria as a
Sj26-GST fusion protein and used to raise specific antibodies. The IgG
fraction of the immunized rabbit serum identified 70- and 72-kDa
protein bands in Western analysis of schistosome extracts. Treatment
with alkaline phosphatase removed the 72-kDa band, which indicates that
this band represents the phosphorylated form of schistosome Smad2.
SmSmad2 was localized in the subtegument, parenchymal cells, and sex
organs in both male and female worm cryosections. Similar results were
also obtained from the analysis of the Smad2 mRNA distribution
pattern revealed by in situ hybridization of adult worm
pair paraffin sections. SmSmad2 mRNA levels were determined by
reverse transcriptase-polymerase chain reaction in different
mammalian host developmental stages and found to be constitutively
expressed. SmSmad2 was also found to interact with a previously
identified SmT
R-I, a serine/threonine type I kinase receptor.
Furthermore, SmSmad2 was shown to undergo phosphorylation by
constitutively active forms of SmT
R-I in vitro. In
addition, SmSmad2 localized in the nuclei of mink lung epithelial cells
upon treatment with TGF-
1. These data indicate that the SmSmad2 responds to the TGF-
signals by interaction with receptor I,
which phosphorylates it, whereupon it translocates into the nucleus
presumably to regulate target gene transcription and consequently elicit a specific TGF-
effect.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1 serine/threonine
kinase receptor I (T
RI) was recently identified in Schistosoma
mansoni, SmT
R-I (5). The presence of SmT
RI on the surface of
the parasite raises the possibility of the involvement of TGF-
or a
TGF-
-like ligand in the host-parasite interactions or in the
stimulation of female worm maturation by male schistosomes.
is a superfamily of secreted polypeptides including various
forms of TGF-
, bone morphogenetic proteins (BMPs), activins, inhibins, and many other structurally related factors (8). Members of
this family regulate cell migration, differentiation, adhesion,
multiplication, and death throughout the life span of an organism (9).
These different effects result from the changes in the expression of
key target genes, which depend on the cell type and developmental
stage. Three classes of intracellular signal transducers are involved
in the signaling cascade of the TGF-
family as follows: a family of
plasma membrane serine/threonine kinase receptors (known as type I and
type II receptors); a family of cytoplasmic proteins that either
transmit the signal from the activated receptor complex to the nucleus
or down-regulate the signaling pathway, known as Smad family; and
gene-specific nuclear DNA-binding partners that associate with Smad
proteins forming transcriptional complexes (8, 10).
R-I (SmT
RI) and
nuclear translocation in response to human TGF-
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP by Megaprime labeling kit (Amersham
Pharmacia Biotech) and used as a probe to screen S. mansoni
adult worm pair cDNA library. Eight positive clones were
identified, and the sequence information showed that two contain the
entire coding region of S. mansoni homologue of Smad2
protein (SmSmad2).
-D-galactopyranoside at 20 °C for
5 h. The recombinant Sj26-SmSmad2 fusion protein (200 µg) was
used to immunize a New Zealand rabbit with complete Freund's adjuvant
for the primary injection and incomplete adjuvant for the following two
booster doses with 3-week intervals between each dose. The immunized
rabbit serum was then passed over protein A-Sepharose column, and the
IgG fraction was eluted and used in Western blots and
immunocytolocalization in adult worm pair cryosections.
80 °C until used.
R-I--
To evaluate the
interaction of SmSmad2 with SmT
R-I, DNA encoding wild type and
mutant forms of SmSmad2 and SmT
R-I were cloned into the yeast GAL4
two-hybrid vectors (activation domain (AD) and DNA-binding domain
(BD)). SmSmad2 cDNA (wild type, wt), in which the EcoRI
site at position 1709 was mutated to GAGTTC to maintain the same
encoded amino acid sequence was amplified. Amplification was performed
using Pfu DNA polymerase (Stratagene) with Smad-5'-cod as a
forward primer and a reverse primer in which NotI site
(underlined) was inserted (NotI-Smad-3';
5'-GCGGCCGCACTGACAGATGTGCAAGGATG-3'). The PCR product
represents a SmSmad2 cDNA in which the stop codon was removed and
DNA encoding 3 amino acids (VGG) was inserted downstream of the
C-terminal motif (TSVS) of SmSmad2 (SmSmad2-3'). 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 of GAL4
DNA-binding domain and GAL4 activation domain, respectively.
SmSmad2-3' was digested with EcoRI and
SalI/XhoI and cloned in pBD-GAL4-cam linearized with EcoRI and pACT2.1 (CLONTECH) and
linearized with XhoI to encode fusion proteins of
GAL4-BD/SmSmad2-3'and GAL4-AD/SmSmad2-3', respectively. SmT
R-I
(wild type) (5) was mutated in its GS domain
(284TCSGSGSGKPLLVQRTVARQ303) to attempt to
construct both constitutively active and kinase-inactive forms (25).
Three mutants were constructed to form T299D, the expected putative
kinase
derivative, Q303D, the constitutively active form,
and the double mutant T299D/Q303D. DNA sequence analysis was performed
for the mutant forms of SmT
R-I to confirm the mutations. SmT
R-I
constructs were digested with EcoRI/SalI and
cloned into pBD-GAL4-cam and pAD-GAL4-2.1 vectors linearized with the
same enzymes, to generate fusion proteins with GAL4-BD and GAL4-AD,
respectively. The two forms of SmSmad2 and the four constructs of
SmT
R-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 genes to
activate host cell reporter gene expression. Transformed cells were
plated onto synthetic dextrose medium (SD) supplemented with amino acid dropout solutions lacking either Trp or Trp, His or Leu, or Leu, His
for BD vectors and AD vectors, respectively. LacZ filter assay was
performed according to Stratagene Two-hybrid instruction manual. To
test for an interaction, an AD vector was cotransformed with binding
domain vector, and the transformed cells were plated onto SD
supplemented with amino acid dropout solutions lacking either Leu, Trp,
or Leu, Trp, His. LacZ filter assay was also performed on this
transformation series. 3-Amino-1,2,4-triazole, a metabolic inhibitor
for histidine biosynthesis, was added to histidine-lacking medium to a
final concentration of 25 mM to inhibit the leaky expression of his3 reporter gene. Control plasmids, pGAL4,
p53, pLaminC, pSV40, and a combination of p53 + pSV40 and pLaminC + pSV40 (Stratagene) were transformed along with each transformation experiment to provide positive and negative controls.
R-I (wt and the three mutants, described
above) were subcloned into pCITE-4a vector (Novagen) using appropriate
restriction enzymes. Recombinant SmT
R-I-pCITE vectors were expressed
in vitro in rabbit reticulocyte lysates with
[35S]methionine (PerkinElmer Life Sciences) using STP-3
transcription/translation system (Novagen) following the supplier's
instructions. The regions encoding 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 5'-end,
respectively). The PCR products were cloned into TOPO-TA vector
(Invitrogen) and then subcloned into pGEX-4T-1 (Amersham Pharmacia
Biotech) and pET42a (Novagen) prokaryotic expression vectors,
respectively. DNA encoding SmSmad2-3' was also subcloned in pET42a
vector. Recombinant prokaryotic expression vectors were overexpressed
in bacteria, and the GST fusion proteins were affixed to
glutathione-Sepharose beads. Binding reactions, between SmSmad2
proteins (wild type and mutant form) and SmT
R-I in vitro
translated proteins, were performed by adding 2 µl of the translation
reactions to GST fusion protein-coupled beads equivalent to about 2 µg of the fusion protein. The final volume of the reactions was
adjusted to 100 µl by adding binding buffer (50 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 0.15%
Nonidet P-40). The reactions were incubated at room temperature for
1 h and then continued overnight at 4 °C. Glutathione-Sepharose beads were added to facilitate the recovery of the reactive beads, and
the samples were precipitated, washed four times, and boiled in 1× SDS
loading buffer, and the bound proteins were size-separated in 12%
SDS-PAGE. The gels were stained, destained, treated with Entensify
(PerkinElmer Life Sciences), dried, and exposed to x-ray film.
Developed x-ray films were scanned, and the specific reaction bands
were quantified using Molecular Analyst Gel Documentation System
(Bio-Rad). Parental pGEX-4T-1 was used to generate recombinant GST,
which was affixed to glutathione beads and used to pre-treat the
translation reactions to remove the nonspecific background. The
equivalent of 2 µg of GST-coupled beads was used to pre-clear the
amount of lysate used per reaction. GST beads were also included in the
binding reactions to serve as a negative control in the pull-down experiment.
R-I--
To
determine whether SmSmad2 can serve as a substrate for SmT
R-I, we
examined the ability of SmT
R-I to phosphorylate SmSmad2. In
principle, the phosphorylation reactions were performed as outlined
above for the pull-down experiment with few differences. Briefly, wild
type and mutant forms of SmT
RI were synthesized in vitro
using rabbit reticulocyte lysates and unlabeled methionine. The lysates
were pre-cleared with GST-coupled beads as before and then aliquoted
into four tubes (each equivalent to 4 µl of original lysate). To each
was added GST or a GST fusion protein containing SmSmad2-linker,
SmSmad2-MH2, or SmSmad2-3' coupled to the glutathione-Sepharose beads.
Phosphorylation was performed in a 100-µl volume in the presence of
10 µCi of [32P-
]ATP, for 1 h at room
temperature in a buffer similar to the binding buffer (above) except it
lacks Nonidet P-40. The beads were collected, washed four times with
the reaction buffer, boiled in 1× SDS-loading buffer, and loaded onto
12% SDS-PAGE. The gels were processed as before and subjected to autoradiography.
R-I-wt and the
mutant forms of both proteins were synthesized in vitro using unlabeled methionine. Kinase reactions were performed using 5 µl of each SmT
R-I translation reaction and 5 µl of each
SmSmad2-MH2 translation reactions in a reaction buffer containing 25 mM Tris, pH 8.0, 50 mM NaCl, 5% glycerol, and
10 µCi of [
-32P]ATP. Reactions were allowed to
proceed for 1 h at room temperature, and then 5 µg of IgG
fraction of
-SmSmad2 rabbit antiserum was added to each reaction and
incubated for 1 h at room temperature, and the antigen-antibody
complexes were precipitated by adding 20 µl of protein A-Sepharose
(Amersham Pharmacia Biotech). Immunoprecipitates were washed 4 times
with 1× phosphate-buffered saline containing 0.1% Tween 20, boiled in
1× SDS-loading buffer, and loaded onto 12% SDS-PAGE. The gels were
processed as before and subjected to autoradiography.
1 Responsiveness of SmSmad2 Protein--
To
determine the responsiveness of the SmSmad2 protein to
TGF-
1 signal, SmSmad2 cDNA was cloned into the
eukaryotic expression vector pCMV-GST (kindly provided by Dr. Randall
Reed). The pGEX-SmSmad2 vector was digested with BamHI, and
the 2.1-kb fragment was inserted in the pCMV-GST vector linearized with
the same enzyme. The recombinant transformants were checked for the
presence of the insert in the correct orientation by colony PCR using
pGEX-fwd primer (Amersham Pharmacia Biotech) as a forward primer and a
reverse primer representing the complementary sequence of bp 533-558.
DNA sequence of positive samples was determined to confirm the presence
of the start ATG codon of 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; ATCC CCL-64) employing Effectene transfection reagent (Qiagen)
following the supplier's instructions. Forty eight hours
post-transfection, cells were fixed with 2% paraformaldehyde
containing 0.1% Triton X-100. The fixed cells were probed with
anti-GST mouse monoclonal antibody (7.5 µg/ml). Anti-mouse
biotinylated IgG (7.5 µg/ml; Zymed Laboratories
Inc.) was used as a secondary antibody reagent. The biotinylated
antibodies were detected using Z-avidin-FITC conjugate (5 µg/ml;
Zymed Laboratories Inc.). A nonrelevant plasmid (pCDNA-LacZ; Invitrogen) that lacks a GST tag was used as a
negative control. Fluorescent cells were examined with Bio-Rad MRC-100 confocal microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII S. mansoni, adult worm pair cDNA library. Eight positive phages
were identified of which two were shown to contain the full-length
coding region of a S. mansoni homologue of Smad2 cDNA
(SmSmad2). SmSmad2 cDNA encodes a 649-amino acid protein
representing the largest Smad2 protein identified to date. NCBI blast
search showed that SmSmad2 exhibits a high degree of homology to Smad2
proteins of Xenopus laevis (26) and humans (27-30).
Homology is restricted to the MH2 domain (69% identity, 77%
similarity) and, to a lesser extent, 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 all R-Smads. The SmSmad2 MH2 domain also contains the L3 loop, the region that determines the
specificity of interaction with the specific type I receptor (16). The
sequence of the L3 loop is highly conserved and invariant among R-Smads
of similar signaling specificity (e.g. Smad2 and Smad3 that
are activated by T
R-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 residues Arg613 and
Thr616 (Fig. 1) which dictate the interaction of Smad2 and
Smad3 with the T
R-I and the activin type I receptors. Another
characteristic feature maintained in the MH2 domain of SmSmad2 is the
conservation of all the 5 subtype-specific residues required for the
interaction of Smad2 and Smad3 with SARA (Smad Anchoring for Receptor
Activation), Ile527, Phe532,
Tyr552, Trp554, and Asn567 (Fig. 1)
(31). SARA is an adaptor protein that interacts with both Smad2/Smad3
and type I receptor and facilitates Smad2/Smad3 MH2 domain interaction
with type I receptor and consequently its phosphorylation and
activation. The interaction of SARA with R-Smad subfamily is restricted
to Smad2 and Smad3 (31, 32).
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Fig. 1.
Pileup analysis and peptide alignment of MH1
and MH2 domains of S. mansoni Smad2 (SmSmad2;
GenBankTM accession number AF232025) with homologous
domains in other R-Smad proteins. Schematic representation of the
functional domains of SmSmad2 cDNA (top). Alignment of
SmSmad2 MH1 and MH2 domains (bottom). DmSmad2,
Drosophila melanogaster Smad2 (GenBankTM
accession number AAD11458); XlSmad2, Xenopus
laevis Smad2 (GenBankTM accession number L77885);
HsSmad2, Smad3, and Smad1 are human
Smad2, Smad3, and Smad1, respectively (GenBankTM accession
numbers NP_005892, BAA22032, and S68987, respectively). %I
and %S are identity and similarity scores. Black
boxed sequence represents the L3 loop. White boxed
sequences are R-Smad subtype-specific residues of L3 loop.
White sequence represents the C-tail, and underlined
sequences are the C-terminal phosphorylation motifs. " "
specifies Smad2/Smad3-specific recognition residues by SARA.
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Fig. 2.
Identification of native SmSmad2 in adult
worm extracts. Coomassie Blue-stained SDS gel (panel A)
and Western blot analysis of schistosome extract using IgG fraction of
preimmune (panel B) and SmSmad2-immunized (panel
C) rabbit sera. Lanes 1-3 represent schistosome
extract treated with CIAP (1 unit/µg protein) for 1 h at
37 °C, schistosome extract incubated with 1× CIAP buffer for 1 h at 37 °C, and untreated extract, respectively. Arrows
point to 72- and 70-kDa protein.
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Fig. 3.
Immunocytolocalization of SmSmad2.
S. mansoni adult worm cryosections were probed with
anti-SmSmad2 IgG (panels A-C), and preimmune rabbit IgG
(panel D) were detected using a FITC conjugate. Panel
A, male (M) and female (F) cross-sections,
showing fluorescence in tubercles and parenchymal cells. Panel
B, male section showing fluorescence in tubercles (T)
and cells of parenchyma. Panel C, female worm showing
fluorescence in ovary (O). Panel D, male worm
showing no fluorescence. G, gut.
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Fig. 4.
S. mansoni adult worm cryosections
probed with anti-SmSmad2 IgG and detected using Cy5 conjugate.
Row A shows the phase contrast fields. Row B
shows background fluorescence detected when using 520-nm filter (used
to visualize FITC-probed sections). Note the autofluorescence in
vitellaria (groups I-III) and eggshell (group
II). Row C shows the specific fluorescence of Cy5
conjugate at wavelength 674 nm. Note absence of autofluorescence in
eggshell (E) (group II) and fluorescence in ovary
(O) (group I), vitellaria (V)
(groups I-III), developing miracidium in egg (group
II) and in parenchyma and subtegumental tissues of male
(group III). G, gut; F, female; and
M, male. No specific fluorescence was observed in sections
probed with preimmune rabbit IgG.
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Fig. 5.
SmSmad2 mRNA levels in schistosome at
different stages of development. RT-PCR of SmSmad2 RNA compared
with the internal control, SmRXR1. Panel A is a diagrammatic
representation of the normalized mRNA levels of SmSmad2.
Panel B is a photograph of 2% agarose gel showing the
amplification products of SmSmad2 and SmRXR1 (arrows).
Lanes 1-4 are 3-h-, 4-day-, 7-day-, and 15-day-old
schistosomules, respectively. Lanes 5-8 are 20-, 28-, 32-, and 35-day worm pairs, respectively. Lanes 9-13, are single
sex male worms, single sex female worms, bisex male worms, bisex female
worms, and adult worm pairs, respectively.
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Fig. 6.
Localization of SmSmad2 mRNA transcripts
in tissue sections of S. mansoni adult worms by
in situ hybridization. Panel A
represents the reaction of the positive control probe (p14
antisense primer) in the vitellaria (V) of the female worm
(F). Male worms (M) were negative as expected
(data not shown). Panel B shows the negative control
reaction using SmSmad2 sense primer in male sections. Female sections
(not shown) exhibited the same level of reactivity. Panels C
and D show the specific reactions of SmSmad2 antisense probe
in male and female sections. Male sections show positive reactions in
subtegument and parenchymal cells. Female sections show reactivity in
vitellaria (V), ovary (O), developing embryo in
uterine egg (E), and subtegumental cells. G, gut;
ST, subtegument; P, parenchyma.
R-I--
Initially, the yeast
two-hybrid system was employed to evaluate the direct interaction
between SmSmad2 and SmT
R-I. Several mutant constructs for both
SmSmad2 and SmT
R-I were evaluated. SmSmad2-3', a SmSmad2 mutant in
which the stop codon was removed to extend the reading frame beyond the
TSVS phosphorylation motif, was expected to retain SmT
R-I binding
but not to undergo receptor-mediated phosphorylation at its C-terminal
end (35). Mutations in the downstream sequence of the GS region of
SmT
R-I corresponding to Q303D and T299D were expected to yield a
constitutively active and a kinase-negative version of type I receptor,
respectively (21, 25, 36, 37). Both SmSmad2 (wt) and SmSmad2-3' when fused downstream to GAL4 BD strongly activated transcription of the
reporter genes (lacZ and his3) in the yeast host cells Y190. This resulted in the production of blue color in the LacZ filter lift
assay and the growth of the transformed cells on histidine-lacking medium (Fig. 7, panel A).
Neither of these two SmSmad2 constructs activated transcription when
fused to the GAL4 AD (data not shown). Based on these data, we tested
the interaction of SmSmad2-AD with SmT
R-I fused to GAL4-BD. In
vivo, SmSmad2 interacted with SmT
R-I under certain conditions.
SmSmad2-AD (wt) strongly interacted with SmT
R-I-BD (wt) (Fig. 7,
panel B), whereas no interaction was observed between
SmSmad2-AD (wt) and the 3 mutants of SmT
R-I fused to GAL4-BD (not
shown). On the other hand, when SmSmad2-3'-AD was used, a stronger
interaction was observed with SmT
R-I (Q303D) and SmT
R-I
(T299D/Q303D) (Fig. 7, panel B), whereas the wild type
SmT
R-I and T299D mutant showed weaker interaction. In general, the
results of the LacZ assay were obtained after 3-4 days, while weak
interaction required incubation periods of up to 10 days.
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Fig. 7.
Interaction of SmSmad2 and
SmT R-I in the yeast two-hybrid system.
Panel A, transactivation by SmSmad2 and SmSmad2-3' fused to
GAL4-BD, positive control (pGAL4), and negative controls (p53 or
pLaminC). Panel B, two-hybrid interactions between
SmSmad2-AD and SmT
R-I-BD. Combinations of pSV40 with either p53 or
pLaminC served as positive and negative controls, respectively.
Diagrammatic representations of the experiments appear above the LacZ
filter lift assay results.
R-I. To evaluate this interaction
in vitro, we examined the ability of GST-SmSmad2 fusion
constructs (linker region and MH2 domain of SmSmad2-wt and full-length
SmSmad2-3'), coupled to glutathione-Sepharose beads, to bind
[35S]methionine-labeled SmT
R-I proteins (wt and the
three mutant forms). SmSmad2-MH2 domain as well as the full-length
protein (SmSmad2-3') bound the four forms of SmT
R-I (Fig.
8, panels A-D, lanes 3 and
4). In this experiment, GST domain was used to evaluate the
background interaction. Neither GST nor GST-linker fusion showed
significant interaction with SmT
R-I proteins (Fig. 8, panels
A-D, lanes 1 and 2).
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Fig. 8.
Pull-down experiment. GST or GST fusion
proteins coupled to glutathione beads were incubated with in
vitro translated, 35S-labeled SmT R-I constructs
(labeled on the sides of panels A-D). Bound proteins were
separated in 12% SDS-PAGE, processed for fluorography, and exposed to
x-ray films. Lanes 1-4 represent the reaction of the
radiolabeled protein with resin-bound GST, GST-SmSmad2-Linker,
GST-SmSmad2-MH2, and GST-SmSmad2-3' (full length), respectively.
Lane 5 represents 50% of the input radiolabeled protein per
binding reaction run on the same gels. Arrows point to the
specific precipitated products.
R-I. To determine whether this interaction is
functional, in vitro phosphorylation of SmSmad2 by wt and
mutant constructs of SmT
R-I proteins was tested. In vitro
synthesized SmT
R-I derivatives were incubated in the presence of
[
-32P]ATP with GST or GST-SmSmad2 fusion proteins
(linker, MH2 domain, and SmSmad2-3') coupled to the
glutathione-Sepharose beads. After incubation for 1 h at room
temperature, samples were centrifuged, the pellets washed for 4 times,
resuspended in PAGE sample buffer, and denatured, and the denatured
proteins were separated by electrophoresis. Neither GST nor any of the
GST-SmSmad2 fusion proteins incorporated the 32P label when
incubated with wild type SmT
R-I (Fig.
9 panel A, panel
I). [32P]Phosphate was only incorporated into the
MH2 domain of wt SmSmad2 by SmT
R-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
SmT
R-I-Q303D was expected since corresponding mutations in other
type I receptors resulted in constitutive phosphorylation activity
in vitro (21) and ligand-independent signaling in
vivo (25, 36, 37). Surprisingly, the SmT
R-I-T299D mutant form showed kinase activity, although less than other mutant forms. This
result is in contrast to previous work, which demonstrated that a
corresponding mutant to T299D in human T
R-I yielded an inactive
version of the receptor kinase (25). GST, GST linker, and
GST-SmSmad2-3' were not phosphorylated by any of the SmT
R-I constructs (Fig. 9, panel A, lanes 1, 2, and
4). Indeed, the lack of phosphorylation of SmSmad2-3' by
any of the SmT
R-I constructs was expected since this modification of
the C-terminal end of SmSmad2 may result in masking the phosphorylation
sites and blocking SmT
R-I-mediated phosphorylation of the
protein.
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Fig. 9.
Panel A, in vitro phosphorylation of
SmSmad2-MH2 domain by SmT R-I. Different SmT
R-I derivatives
synthesized in a rabbit reticulocyte lysate by coupled transcription
and translation (shown at the top of the figure) were
incubated with GST or GST fusion derivatives of wt and mutant SmSmad2,
in the presence of 10 µCi of [
-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. Lanes 1-4
represent GST, GST-SmSmad2-Linker, GST-SmSmad2-MH2, and GST-SmSmad2-3'
(full length), respectively. Labeled arrows on the
side of the figure point to the expected size of these
products. Panel B, in vitro
phosphorylation of wild type or mutant SmSmad2-MH2 by wild type or
mutant forms of SmT
R-I. SmSmad2-MH2 and SmT
R-I derivatives were
synthesized in vitro by coupled transcription and
translation using rabbit reticulocyte lysate with unlabeled methionine.
SmSmad2-MH2 proteins were incubated with the translation mix of either
a negative control (no DNA transcription reaction) or one of the
following SmT
R-I derivatives: wild type, T299D, Q303D, and
T299D/Q303D, in the presence of 10 µCi [
-32P]ATP
(panels I-V, respectively). Anti-SmSmad2 rabbit IgG was
added to each reaction, and the IgG-SmSmad2-MH2 complexes were
precipitated by protein A-Sepharose, washed 4 times, denatured in SDS,
separated in 12% SDS gels, and subjected to autoradiography.
Panel VI represents the results of immunoprecipitation of
wild type and mutant [35S]Met-labeled SmSmad2-MH2
derivatives, demonstrating that the anti-SmSmad2 antibody was capable
of precipitating each SmSmad2-MH2, with similar efficiency. Lanes
1-5 represent SmSmad2-MH2 with the following terminal amino
acids: TSVS (wild type), TSVA, TAVS, TAVA, and AAVA, respectively.
Lane 6 (panel VI) represents the
immunoprecipitation background reaction using
[35S]Met-labeled negative control lysate.
R-I (wt
and mutant forms) were synthesized in vitro using unlabeled methionine and then the phosphorylation of SmSmad2-MH2 constructs by
SmT
R-I forms, in the presence of [
-32P]-ATP, was
evaluated. Fig. 9, panel B, shows that the
[32P]phosphate was incorporated into the wt MH2 domain
and to a lesser extent into the TSVA and TAVS mutant forms when using
any of the SmT
R-I mutant derivatives (panels III-V, lanes
1-3). The highest incorporation level was obtained using
SmT
R-I-Q303D followed by SmT
R-I-T299D/Q303D mutants. Neither the
negative control (no DNA-transcription reaction) nor SmT
R-I-wt
translation mix showed significant incorporation of the 32P
label in any of the SmSmad2-MH2 constructs (panels I and
II). However, minor phosphoproteins derived from the
reticulocyte lysate that migrate at the same approximate size of
SmSmad2-MH2, independent of SmT
R-I and SmSmad2, were observed in
some experiments. The mutant forms of SmT
R-I did not show
significant incorporation of the radioactive label into either TAVA or
AAVA mutants of SmSmad2-MH2 domain (panels III-V, lanes 4 and 5) indicating that Thr646 is not a phosphate
acceptor. Fig. 9, panel B, panel VI, shows that
the anti-SmSmad2 antibody efficiently precipitated the wt as well as
the mutant forms of the SmSmad2-MH2 domain.
R-I.
Interaction of SmSmad2 derivatives with SmTR-I
, weak positive; and
,
negative.
1--
The above data indicate that SmSmad2
stably interacts with SmT
R-I in vitro and in
yeast. The ability of SmSmad2 to interact productively with T
R-I and
translocate to the nucleus in a ligand-dependent way was
tested in Mv1Lu cells, which are known to be responsive to induction by
mammalian TGF-
(38). Mv1Lu cells that were transfected with
pCMV-GST-SmSmad2 and treated with TGF-
1 showed nuclear
fluorescence when probed with an anti-GST monoclonal antibody (Fig.
10, panels E and
F), whereas non-TGF-
1-treated cells showed cytoplasmic 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,
(negative control), a non-GST tag expressing, showed no fluorescence
(Fig. 10, panel B).
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Fig. 10.
Nuclear translocation of SmSmad2 upon
stimulation by TGF- 1.
Transfected mink lung epithelial cells (Mv1Lu) were fixed and probed
with anti-GST monoclonal antibody. Panel A shows the
parental pCMV-GST vector-transfected Mv1Lu cells (positive control)
expressing GST, and panel B shows typical cells from
pcDNA-LacZ-transfected cells (negative control). Mv1Lu cells were
transfected with pCMV-GST-Smad2 vector untreated (panel D)
and stimulated with TGF-
1 (panels E and
F). Panel C shows the phase contrast field of the
examined cells in panel D. Note that there is only
cytoplasmic staining in panel A and nuclear staining in
panels E and F.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in parasite development may provide insight into
some of these issues.
superfamily regulate many important developmental
processes, such as mesenchymal differentiation, skeletal morphogenesis,
and skin formation (39). In both vertebrate and invertebrate model
systems, TGF-
family members can serve as morphogens, acting across
developing tissues in a graded fashion to specify a patterned array of
cell fates (40, 41). Defects in TGF-
signaling have been implicated
in multiple developmental disorders and in various human diseases,
including cancer, fibrosis, and autoimmune diseases (8, 10,
42-46).
and activin
receptors (16). Another characteristic structural feature of SmSmad2 is
its unique C-terminal phosphorylation signature. Schistosome Smad2 is
the only member of R-Smad subfamily with TSVS phosphorylation motif
instead of the conserved SSXS motif present in all other
members of the subfamily. The significance of this difference is
unknown and needs further investigation. However, this difference does
not appear to interfere with the biological functions of the protein.
In this regard, studies on the C-terminal phosphorylation motif in
other Smad proteins showed that threonine could substitute for serines
(21).
signaling, plays multiple roles in
the development of the parasite in general, and the sexual maturation
of both male and female worms. As vitelline cell development (gene
expression) is regulated by a signal from the male, the finding of
SmSmad2 expression in vitelline cells implicates the TGF-
signaling
cascade in female reproductive development.
, the ability
of SmSmad2 to translocate to the nucleus upon induction by TGF-
was
evaluated. Mink lung epithelial cells (Mv1Lu) transfected with
GST-tagged SmSmad2 showed some nuclear fluorescence with most of the
fluorescence localized in the cytoplasm. This observation is similar to
the results of previous studies on Smad3 (48) and Xenopus
Mad-like protein (XenMLP) (49). Treatment of transfected Mv1Lu cells
with rhTGF-
1 resulted in predominant nuclear
localization of SmSmad2, demonstrating that SmSmad2 responded to the
signals initiated by the human ligand. These data are consistent with Nakao et al. (35) who reported that the endogenous Smad2,
Smad3, and Smad4 exhibited predominant fluorescence in the cytoplasm of
uninduced Mv1Lu cells, whereas the fluorescence was mainly localized in
the nuclei in case of cells stimulated with TGF-
1. These
results not only demonstrate that SmSmad2 is biologically active but
also that the mechanism employed in nuclear transport in higher
eukaryotes recognizes a schistosome protein.
R-I, we used
wild type and mutant constructs of both proteins in yeast two-hybrid
and GST pull-down assays. SmSmad2-3', a mutant in which the stop codon
was removed to extend the reading frame beyond the "TSVS"
phosphorylation motif, was constructed. This modification of the
C-terminal end of the protein was expected to block type I
receptor-mediated phosphorylation of SmSmad2. Such a modification was
demonstrated to abolish the ability of the C-terminal serine residues
to become phosphorylated (35). For SmT
R-I, three mutants were
constructed in which either Thr299, Gln303, or
both were mutated to Asp. These constructs were expected to yield a
kinase-deficient form, a constitutively active derivative (25), and a
construct that contains both kinase-activating and -inactivating
mutations, respectively. Wild type SmT
R-I interacted with wild type
SmSmad2 and to a lesser extent with SmSmad2-3' in vivo in
yeast two-hybrid assay, with MH2 domain of SmSmad2, and full-length
SmSmad2-3' in GST pull-down experiment, but failed to phosphorylate
the MH2 domain in vitro. The interaction between wild type
SmSmad2 and SmT
R-I in yeast was unexpected since other authors have
reported that the interaction between wild type human Smad2 and either
wild type T
R-I (11, 35) or the constitutively active form of the
receptor (11) could not be detected in COS cells. Perhaps the
difference in results is due to the different systems utilized in each
study. Transfected COS cells followed by immunoprecipitation of the
interacting proteins may not be sensitive enough to detect a weak
interaction compared with transformed yeast cells, where the
interaction is allowed to proceed over a relatively longer period.
Consistent with our results, Zhang et al. (29) reported that
the heteromeric interactions between human MAD-3 and-4 could only be
detected in yeast two-hybrid assays but not by coimmunoprecipitation of
transfected cultured cell lysates. The interaction of wt-SmT
R-I with
SmSmad2-3' in vivo and in vitro was expected
since the modification of the C-terminal end of SmSmad2 was likely to
block the phosphorylation mediated by SmT
R-I resulting in
stabilizing SmSmad2-SmT
R-I complex (11). The interaction with
SmSmad2-MH2 domain in vitro was also expected as Smad2 was
shown to serve as a direct substrate of the activated T
R complex
(T
R-I activated by a ligand-bound T
R-II) (11). Therefore, in the
absence of T
R-II, SmSmad2-MH2 would not be expected to be
phosphorylated by wt-SmT
R-I, and thus, as shown by our results,
allowed SmSmad2-MH2 to form a stable complex with the wt receptor. Our
interpretation of this interaction is supported by the lack of
phosphorylation of SmSmad2-MH2 by wt-SmT
R-I.
R-I constitutive mutant Q303D showed results
consistent with those obtained in other systems (25, 36, 37). This
mutant was found to interact stably in vivo and in
vitro with SmSmad2-3', and in vitro, although to a
lesser extent, with the MH2 domain of SmSmad2. However, the in
vivo interaction in yeast with wt-SmSmad2 was undetectable.
SmT
R-I-Q303D mutant was also able to phosphorylate the MH2 domain of
SmSmad2-wt in vitro. This mutant construct may exert its
constitutive signaling activity due to the replacement of uncharged
polar residue (Gln303) by a negatively charged residue
(Asp). This would favor the initial apposition between type I receptor
and SmSmad2, which should facilitate subsequent binding and
phosphorylation of the substrate (31). The stable interaction between
Q303D mutant and SmSmad2-3' was observed because the ability of
SmT
R-I-Q303D to phosphorylate SmSmad2-3' was blocked by the
C-terminal extension. However, in case of wt SmSmad2, which was
susceptible to phosphorylation by the constitutive mutant Q303D, the
interaction in vivo was only transient due to the
phosphorylation of SmSmad2 and the dissociation of SmT
R-I-SmSmad2 complex.
R-I-T299D and the corresponding mutants in
other type I receptors may be provided by a direct comparison of the GS
domain and its downstream region in these receptors. SmT
R-I has one
amino acid substitution (Lys292) instead of the conserved
"L" at this position for all other known type I receptors.
This basic amino acid located just after the GS region may participate
in stabilizing the T299D mutant interaction with SmSmad2 and provide
the net charge equilibrium required for phosphorylating its substrate,
mimicking the effect of the Q303D mutant. Therefore, the use of
"less-active kinase" term rather than "kinase-inactive" in case
of T299D-SmT
R-I mutant would be more applicable for this system.
R-I, or the use of a nonphosphorylatable form of SmSmad2, as in
case of SmSmad2-3'. These data are in agreement with the hypothesis
proposed by Macias-Silva et al. (11) who reported that this
transient interaction can be stabilized by preventing phosphorylation
of R-Smad either by using a kinase-deficient version of type I receptor
or a mutant version of Smad2 in which the C-terminal phosphorylation is
blocked. Furthermore, previous results also showed that C-terminal
FLAG-tagged Smad3 stably associated with wild type T
R-I (35). Our
data revealed that the MH2 domain interacted in vitro with
the mutant forms of SmT
R-I, whereas this interaction was not
detected in the two-hybrid analysis using full-length SmSmad2. The
interaction may have been undetectable in vivo due to its
transient nature due to phosphorylation and dissociation of the
complex. This would be true for Q303D and T299D in vitro in
the absence of ATP.
R-I-catalyzed phosphorylation of in vitro
translated SmSmad2-MH2-wt and mutant forms showed results consistent
with the two-hybrid and pull-down assays, as well as the in
vitro phosphorylation reaction of the bacterially expressed
SmSmad2-GST fusion proteins. The lack of phosphorylation of wild type
or mutant SmSmad2-MH2 by wild type SmT
R-I confirms the inability of
the wt-SmT
R-I to phosphorylate SmSmad2 in absence of ligand,
TGF-
. Alternatively, each mutant form of SmT
R-I phosphorylated
the wt-MH2 domain of SmSmad2 with varied efficiencies, demonstrating
that each mutant receptor I is constitutively active. SmSmad2-MH2
mutants TSVA and TAVS were phosphorylated by the mutant forms of
receptor I demonstrating that neither serine is the sole phosphate
acceptor. Phosphorylation of double TAVA and triple AAVA mutants was
blocked, implicating both Ser647 and Ser649 as
the receptor-mediated phosphorylation sites, as in 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.
Furthermore, these results indicate that Thr646 is not a
phosphate acceptor.
R-I in
vivo and in vitro. We also showed that the
constitutively active mutant versions of SmT
R-I could phosphorylate
SmSmad2 MH2 domain in vitro, although with varied
efficiencies. We also showed that Ser647 and
Ser649 are likely to serve as receptor-mediated
phosphorylation sites of the SmSmad2. In addition, in a heterologous
system, SmSmad2 was translocated to the nucleus in response to
rhTGF-
1. Our results also showed that SmSmad2 mRNA
transcript and its encoded protein localized in the subtegumental layer
of the parasite. This may represent an intracellular link between the
specific receptor located on the surface of the parasite (SmT
R-I)
(5) and specific effects elicited by TGF-
or TGF-
-like signaling.
Whether ligands are derived from the host, representing a new chapter
in host-parasite interaction, or are produced by the parasite itself to
elicit the development, differentiation, and specialization of its
tissues, further investigations are needed. In either case, this study argues that SmSmad2 plays an important role in various stages in
various tissues of the mammalian (human) phase of schistosome development. The identification of schistosome Smad2 provides a
molecular tool to investigate the role of TGF-
signaling in schistosomes. The identification of the ligand(s) as well as the cooperative partners and the responsive genes are current targets to
help in understanding the role of this signaling pathway in the
development of the parasite and its interaction with the human host.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Wade Sigurdson, Head of the Confocal Microscopy and Three-dimensional Imaging Laboratory, for help with microscopy and Dr. Randall Reed, Johns Hopkins University, for providing the pCMV-GST vector.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI46762.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF232025.
To whom correspondence should be addressed: Dept. of Microbiology,
School of Medicine and Biomedical Sciences, State University of New
York, 138 Farber Hall, Buffalo, NY 14214. Tel.: 716-829-2459; Fax:
716-829-2169; E-mail: loverde@buffalo.edu.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M005933200
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
SmSmad2, S. mansoni Smad2;
R-Smad, receptor-regulated Smad;
BMP, bone morphogenetic protein;
SmT
R-I, S. mansoni TGF-
receptor-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, fluorescein isothiocyanate;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
BD, binding domain;
AD, activation domain.
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