From the Department of Pathology and the ** Gene
Therapy and Viral Vector Laboratory, North Shore-Long Island
Jewish Health System and Biomedical Research Center, Manhasset,
New York 11030 and the ¶ Laboratory for Molecular Oncology, Center
for Human Genetics, University of Leuven and the Flanders
Interuniversity Institute for Biotechnology, Herestraat 49, B-3000 Leuven, Belgium
Received for publication, August 1, 2000, and in revised form, February 15, 2001
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ABSTRACT |
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Lefty polypeptides, novel members of the
transforming growth factor- Vertebrate body organization during embryogenesis is an essential
process based on the formation of three axes of asymmetry: anteroposterior, dorsoventral, and lateral (left/right). The
coordination of development of these three axes determines the
location, structure, and arrangement of different organs. From an
embryonic and evolutionary perspective, the lateral (left/right) axis
is the last one established during vertebrate development and requires
the breakage of the bilateral symmetry. This symmetry-breaking process
results in the proper positioning and formation of asymmetric organs
like liver, lung, heart, intestine, spleen, and stomach. Moreover, the
relative positioning of organs within the body cavity is conserved in
all vertebrates, suggesting that the structure and arrangement of these
organs are required for their normal function. For example, the
asymmetric positioning of the heart allows for more efficient pumping
of blood, and the asymmetric development of the digestive system
(particularly complex in vertebrates) allows for a more efficient
packing of the bowel within the peritoneal cavity. The mechanisms that
operate in the lateral symmetry-breaking process have remained
surprisingly difficult to elucidate. The current ciliary model of
left/right axis determination in vertebrates is based on the so-called
"nodal flow." This flow is produced by a specialized cluster of
monocilia present on the ventral surface of the mouse node, which is
the mammalian equivalent of the embryonic organizer region identified
in Xenopus (1-3). These monocilia, which project into the
extraembryonic space, exhibit a type of vortical motion that generates
a leftward flow of extraembryonic fluid in the node region. This,
so-called nodal flow has been proposed to function as the
initiating event in left/right axis formation by causing an initial
left/right difference in the relative distribution of extracellular
factors (4-6). Therefore, this leftward flow triggers activation of
distinct signaling pathways on the left and right sides of the embryo.
Different members of the
TGF- Members of the TGF- Materials--
The materials used in these studies included an
enhanced chemiluminescence system (Roche Molecular Biochemicals),
nitrocellulose membrane (Bio-Rad), Kodak Omat film (Sigma),
biotin-labeled goat anti-rabbit antiserum and ABC reagent (Vector
Laboratories, Inc., Burlingame, CA), and protein G Plus-agarose (Santa
Cruz Biotechnology, Santa Cruz, CA). Dephosphorylated myelin basic
protein (MBP), anti-phospho-MBP antibody, and immunoaffinity-purified
rabbit anti-MAPK (Erk1/2) bound to agarose were all obtained from
Upstate Biotechnology, Inc. (Lake Placid, NY). Unless otherwise
indicated, all other chemicals were from either Sigma or Fisher. The
mammalian cell lines were from American Type Culture Collection
(Manassas, VA). The rabbit polyclonal antibodies used in this study
were raised against a peptide at the C terminus of the lefty A/B
protein (acetyl-CASDGALVPRRLQHRP) (antibody A353) and a peptide at the N terminus of lefty A (acetyl-DRADMEKLVIPAC) (antibody A44). The monoclonal antibody to the hemagglutinin (HA) tag was obtained from
Roche Molecular Biochemicals. The monoclonal and polyclonal antibodies
directed against furin, PACE4, PC5A, PC5B, and PC7 were obtained from
Alexis Biochemicals (Laufelfingen, Switzerland). Anti-Smad2, -4, and -5 antibodies were obtained from Santa Cruz Biotechnology. Recombinant
BMP-4 was obtained from R&D Systems (Minneapolis, MN).
Recombinant TGF- Primer Design and PCR-based Cloning of Human lefty A and lefty
B--
All constructs obtained by PCR and mutagenesis were checked by
sequencing.2 The coding
regions of lefty A (ebaf) and lefty
B cDNAs were obtained by PCR using Marathon-Ready cDNA
from human pancreas (CLONTECH, Palo Alto, CA) and
cloned into pAdCMV5 (Quantum Biotechnologies Inc., Montreal, Canada).
The PCR products were separated on 1% agarose gels and purified with a
Geneclean kit (Bio 101, Inc., Vista, CA). The PCR products and the
plasmids, pcDNA3 (Invitrogen) or HA-pcDNA3, were digested with
EcoRI and XbaI (New England Biolabs Inc.,
Beverly, MA) for lefty A (ebaf) or with
BamHI and XbaI (New England Biolabs Inc.) for
lefty B. The fragments were annealed to the mammalian
expression plasmid pcDNA3 with a rapid ligation kit (Stratagene, La
Jolla, CA). The sequence of each clone was validated by restriction
enzyme digestion and sequencing using Taq DyeDeoxy
terminator cycle sequencing reactions in conjunction with an Applied
Biosystems Model 373 DNA Sequencer. The plasmid DNAs containing the
correct cDNA sequences were prepared using the Promega Wizard
miniprep method and used for transfection. Lefty A was
tagged at its C terminus with an HA tag (YPYDVPDYAG). The forward
primer used was 5'-AGC TGG AGC TGC ACA CCC TGG, and the reverse primer
used was 5'-TTT GGA TCC CTA GGC ATA GTC TGG CAC ATC ATA TGG GTA TGG CTG
GAG CCT CCT TGG CAC. The sense orientation of the lefty A
cDNA was constructed using plasmid pAdCMV5, in which lefty
A gene expression is regulated by the cytomegalovirus immediate-early promoter. A 1.2-kilobase pair
BamHI/AflIII lefty A cDNA fragment
containing minimal 5'- and 3'-untranslated regions from the plasmid
pBluescript SK Site-directed Point Mutation of Human lefty A--
Site-directed
mutagenesis was achieved with a QuikChangeTM 1-Day
site-directed mutagenesis kit (Stratagene) following the
manufacturer's protocol. The sequences of mutated clones were
determined by Taq DyeDeoxy terminator cycle sequencing
reactions in conjunction with an Applied Biosystems Model 373 DNA Sequencer.
Cells, Transfection, and Protein Preparation--
Human
embryonic kidney 293 cells, a fibroblast cell line (NIH-3T3), a Chinese
hamster ovary (CHO) cell line, and RPE40 (a variant of CHO cells
deficient in furin) cells were maintained in Dulbecco's modified
Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal
bovine serum (Life Technologies, Inc.) and 1% antibiotic/antimycotic
(Life Technologies, Inc.). For transfection, cells were seeded into
6-well plates (Falcon, Franklin lakes, NJ) at a concentration of
1.3 × 104 cells/ml and maintained in a
CO2 chamber at 37 °C for ~16 h. When 60% confluent,
cells were transfected with lefty A, lefty B, or
mutant lefty A cDNA using Superfect transfection reagent (QIAGEN Inc., Valencia, CA) or Fugene (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturers' protocol.
In transient transfections, serum-free medium was used. The medium was
collected 20-24 h after transfection and concentrated 12-fold using
Centricon YM-3 centrifugal filter devices (3-kDa protein molecular size
cutoff; Amicon, Inc., Beverly, MA). Cells were lysed by addition of 15 µl of Laemmli buffer. Protein concentration was determined with the
Bio-Rad protein assay kit.
Affinity Purification of Lefty A Proteins--
Lefty A proteins
were purified by affinity purification from culture media of
cells transfected with lefty A. Affinity columns for lefty
were prepared by binding rabbit anti-lefty peptide antibody A353 or A44
to cyanogen-activated Sepharose 4B (referred to as A353 and A44
affinity columns). Cell-free conditioned media of lefty-transfected cells were transferred to fresh tubes and
mixed with 250 µl of anti-lefty A antibody A353 or A44-Sepharose 4B suspension/10 ml of medium. To each 2.5 ml of urine or 250 µl of
serum 25 µl of the antibody-Sepharose suspension was added. These
mixtures were incubated at room temperature for 1 h with gentle
shaking. The suspension of bound lefty protein(s) with Sepharose 4B was
then poured into a 0.7 × 15-cm column. The column was washed with
20 column volumes of 50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, and 5 mM EDTA. The bound lefty
protein(s) was eluted with 0.5 column volumes of 2.5 M
guanidine HCl in 10 mM Tris-HCl (pH 8.1) and 5 mM EDTA, followed by 0.25 column volumes of 50 mM Tris-HCl (pH 7.5) and 5 mM EDTA. The eluted
lefty protein was then dialyzed against three changes of 10 mM sodium phosphate buffer (pH 7.4) at 4 °C and stored
at 4 °C. Culture media were first applied to the A353 affinity
column. The proteins eluted from the A353 column were then applied to
the A44 affinity column and eluted. Eluted and flow-through materials
were subjected to Western blotting with antibody A353 to identify the
affinity-purified proteins. The affinity column made with antibody A353
bound all lefty proteins (42, 34, and 28 kDa). On the other hand, the
affinity column made with antibody A44 bound only the 42-kDa protein,
allowing the 34- and 28-kDa proteins to flow through this column (data not shown).The amount of purified lefty from large-scale purification procedures was determined with the Bio-Rad protein assay kit.
SDS-Polyacrylamide Gel Electrophoresis and Western
Blotting--
Conditioned media or cell lysates (12-15 µg of
protein/lane) were fractionated on a 12% denaturing gel together with
a prestained protein ladder (Life Technologies, Inc.) and subsequently
blotted onto polyvinylidene difluoride membrane in a Mini-Trans-Blot
apparatus (Bio-Rad). The blot was stained using either
affinity-purified rabbit anti-C-terminal lefty A antibody A353 (1-2
µg/ml) or a mouse monoclonal anti-HA antibody (1-2 µg/ml). The
secondary antibodies used were horseradish peroxidase-conjugated mouse
anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse
IgG (Santa Cruz Biotechnology). The specific bands were detected with the chemiluminescence system as described by the manufacturer.
Immunoprecipitation--
Immunoprecipitations of the transfected
convertases were performed as described (30). Briefly, for
immunoprecipitation, 2-5 µl of specific antibody was added to each
milliliter of cell lysate (~106 cells). The
immunoprecipitates were subjected to gel electrophoresis, followed by autoradiography.
Determination of the Effect of Lefty Proteins on the MAPK
Pathway--
Regulation of the MAPK and JNK pathways was analyzed
using the PathDetectTM in vivo signal
transduction reporting system (Stratagene). P19 mouse embryonic
carcinoma cells were transfected with the respective pathway-specific
fusion activator vectors (pFA-Elk for MAPK and pFA-Jun for JNK pathway;
3 µg/ml of each plasmid) and the luciferase reporter plasmid
(pFR-Luc) according to the manufacturer's instruction. These cells
were maintained in
The induction of MAPK activity by lefty was also examined by showing
the kinase activity of Erk1/2 as previously described (31, 32).
Briefly, P19 cells were treated with affinity-purified 42/34/28-kDa
protein mixture, 34/28-kDa protein mixture, 42-kDa lefty A protein, or
26-kDa recombinant E. coli lefty A for the durations shown
below. After washing cells with ice-cold phosphate-buffered saline,
cells were lysed in cell lysis buffer (50 mM Tris-HCl (pH
7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM
NaCl, 1 mM EGTA, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 1 mM Na3VO4, and 1 mM NaF), and the concentration of protein in the cell
lysate was adjusted to 1 µg/µl protein. Five microliters of
anti-MAPK antibody-agarose gel slurry was added to 500 µg of cell
lysate protein, and the mixture was gently rocked overnight at 4 °C.
The beads were collected and washed three times with ice-cold
phosphate-buffered saline and mixed with 100 µl of assay
dilution buffer (20 mM MOPS (pH 7.2), 25 mM
In Vivo Phosphorylation of Smad Proteins--
P19 cells were
grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 for
24 h. Cells were then washed and incubated for 2 h in
Dulbecco's modified Eagle's medium without glutamine and phosphate.
Cells were incubated for another 2 h in the same medium
supplemented with 15 µCi/ml [32P]orthophosphate. After
cytokine treatment, cells were lysed in 1 ml of radioimmune
precipitation assay buffer, and the samples were subjected to
immunoprecipitation with Smad2- or Smad5-specific antibodies. The
immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis, followed by blotting and autoradiography.
Identification of the Secreted Lefty Forms--
Lefty polypeptides
were identified as new members of the TGF-
Polyclonal anti-lefty antibody A353 was raised against the peptide to
amino acids 353-366 of lefty A (13, 14). The antibody was purified on
a peptide affinity column and characterized by Western blotting and
immunoprecipitation of the culture media of control and transfected
cells. Antibody A353 showed a specific immunoreactivity against lefty A
protein in the culture media of different transfected cell lines,
including human embryonic kidney 293 cells and the CHO cell line.
Antibody A353 recognized three polypeptides of 42, 34, and 28 kDa in
the conditioned media of the transfected cells in the same way that
anti-HA antibody reacted with the protein released into the media of
both 293 (Fig. 1B) and CHO (Fig. 1C) cells.
Blocking experiments with the immunizing peptide also proved the
specificity of this antibody. Incubation of the antibody with the
immunizing peptide prevents the immunoreactivity of these three
polypeptides in a peptide concentration-dependent fashion
(33). We did not find immunoreactivity with any endogenous protein in
the different cell lines tested. Based on the fact that antibody A353
did not show any reaction with endogenous proteins, we believe that
these cells do not express a lefty homolog or that their endogenous
expression is very weak. Human 293 cells were transfected with lefty B,
and the immunoreactivity of the culture media was probed by Western
blotting using antibody A353 (Fig. 1D). In agreement with
the 95% identity between lefty A and lefty B, antibody A353
also bound lefty B in the same fashion as it reacted with lefty A. These two proteins exhibit a complete identity in the carboxyl-terminal
region used for raising antibody A353. Antibody A353 recognized three
polypeptides of 42, 34, and 28 kDa, suggesting than lefty A and lefty B
are subjected to the same endoproteolytic processing in cells (Fig.
1D).
Endoproteolytic Processing of Lefty A Polypeptides by Members of
the PC Family of Endoproteases--
To prove that the polypeptides of
34 and 28 kDa are produced by endoproteolytic processing of the 42-kDa
precursor, we studied the proteases involved in the processing of lefty
polypeptides. First, we attempted to inhibit lefty processing by using
the polypeptide
Because of the implication of the PCs in the processing of other
cytokines of the TGF- Identification of the Endoproteolytic Cleavage Sites of Lefty
A--
To validate that the PC family of endoproteases is involved in
the in vivo endoproteolytic processing of lefty
polypeptides, we identified the cleavage sites according to the
consensus sequences required for convertase cleavage. We suggested the
sequences RGKR (aa 74-77) and RHGR (aa 132-135) as potential cleavage
sites for the processing of lefty proteins by the convertases (Fig.
3A) (13). These sequences are
conserved in both lefty A and lefty B, suggesting the same processing
for these two proteins. To prove these sequences to be cleavage sites
in vivo, we analyzed the effect of the mutations of RGKR to
GGKG (aa 74-77) and of RHGR to
GHGR (aa 132-135) on the processing of lefty. 293 cells
were transfected with the lefty A mutants, and
culture media were analyzed by Western blotting (Fig. 3B).
The mutation of RGKR to GGKG (aa 74-77)
prevented the processing of the 42-kDa lefty precursor to the 34-kDa
form, whereas the mutation of RHGR to GHGR (aa 132-135) abolished the processing of the 34-kDa polypeptide to the 28-kDa form.
Moreover, the mutation of RGKR to GGKG (aa
74-77) did not inhibit the formation of the 28-kDa polypeptide,
suggesting that the two proteolytic cleavages are independent.
Therefore, the cleavage at Arg135 does not require cleavage
at Lys77 or the formation of the 34-kDa polypeptide. These
results also rule out the possibility that these point mutations
prevented the proteolytic cleavage in another site.
Biological Activity of Lefty Polypeptides--
Members of the
TGF-
Since lefty did not seem to directly signal through the Smad pathway,
we analyzed the phosphorylation and subsequent activation of MAPK after
lefty treatment. We first used the conditioned culture media from
control and lefty A-transfected 293 cells and analyzed their
effects on both the MAPK and JNK kinase pathways in P19 cells. The
activity of the MAPK and JNK kinase pathways in P19 cells was
visualized in vivo using the PathDetectTM
luciferase reporting system. The culture media from control and lefty A-transfected 293 cells were collected and used for
the treatment of P19 cells. The culture media from lefty
A-transfected 293 cells induced ~4-fold activation of the MAPK
pathway in P19 cells compared with treatment with the culture media
from control cells (Fig. 5). We analyzed
whether the activation of the MAPK pathway is specifically due to the
lefty polypeptides or other factors present in the culture media of
transfected cells. The lefty proteins present in the culture media were
"blocked" by increasing the concentration of anti-lefty antibody
A353, and their activity was analyzed by the treatment of P19 cells.
The anti-lefty antibody inhibited the MAPK activity of the culture media in a concentration-dependent fashion (Fig. 5). These
results show that lefty factors rather than other factors present in
the culture media of transfected 293 cells induced the activation of
the MAPK pathway in the pluripotent P19 cells. In addition, the results
also provide evidence that human lefty factors have biological activity
in mouse cells, unlike other cytokines with a species-specific
activity. In contrast to these results, we did not detect any effect of
the culture media of lefty-transfected cells on the JNK
pathway (data not shown).
To further validate the results obtained from the reporter assays and
to determine the time kinetics of the response, we examined the kinase
activity of Erk1/2 after treatment with lefty. To reduce the basal MAPK
activity, P19 cells were starved in culture for 2 days in the absence
of serum. These cells were incubated with lefty proteins (42, 34, and
28 kDa) purified from the culture media of lefty
A-transfected cells. Erk1/2 in the treated P19 cells was
immunoprecipitated with Erk1/2-specific antibody, and the kinase
activity of the immunocomplexes was tested on dephosphorylated MBP. The
phosphorylation of MBP was then assessed by Western blotting. The
results show that lefty proteins induced MAPK activation in a
time-dependent manner. Lefty proteins induced MAPK
activation as early as 5 min, and the activity continued to
progressively increase over a period of 60 min (Fig.
6A). This response to lefty could be blocked by antibody A353 (data not shown). To determine whether the induction of MAPK was due to the 42-kDa or cleaved forms of
lefty, the 42-kDa protein was affinity-purified and separated from the
34- and 28-kDa proteins using the A44 affinity column, and the
experiments were repeated using the 42-kDa purified protein and the
34/28-kDa protein mixture. The 34/28-kDa protein mixture induced MAPK
activity; but compared with the effect of the 42/34/28-kDa protein
mixture, the effect was delayed. MAPK activation was visualized after
30 min of incubation of the cells with lefty proteins, and the response
became pronounced after 60 min of incubation (Fig. 6B). In
contrast, purified 42-kDa lefty vigorously activated MAPK after 5 and
15 min, and this response quickly diminished to undetectable levels 30 and 60 min after incubation (Fig. 6C). These findings show
that the 42-kDa protein led to early MAPK activation, whereas the
34/28-kDa protein mixture induced a delayed response. These results
suggest that the activation of the MAPK pathway in P19 cells is related
to the endoproteolytic processing of lefty.
To further identify the bioactive cleaved form(s) of lefty, we analyzed
the biological activity of the different lefty polypeptides. 293 cells
were transfected with control wild-type lefty A, its mutant
forms (GGKG (aa 74-77) and GHGR
(aa 132-135)), and lefty B. Forty-eight hours after
transfection, cultured media were collected and used for the treatment
of P19 cells after checking the lefty protein expression. The effect of
the conditioned media from 293 cells on the MAPK pathway in P19 cells
was quantified by measuring luciferase activity (Fig.
7A). The conditioned media of
293 cells transfected with wild-type lefty A or lefty
B used as a control induced an ~2.5-fold increase in the
activation of the MAPK pathway in P19 cells. Moreover, the conditioned
media from 293 cells transfected with the GGKG
(aa 74-77) lefty A mutant induced MAPK pathway activation in P19 cells in a fashion similar to that of the conditioned media from
293 cells transfected with control wild-type lefty A.
However, the conditioned media from 293 cells transfected with the
GHGR (aa 132-135) lefty A mutant failed to
induce the activation of the MAPK pathway in P19 cells. The results
show that the MAPK activation was primarily due to the 28-kDa form of
lefty since the GHGR (aa 132-135) mutation, which
prevented the formation of the 34-kDa form, had no effect on MAPK
activation, whereas the GGKG (aa 74-77)
mutation, which blocked the formation of 28-kDa lefty, led to the loss
of the MAPK-inducing activity. Therefore, the biological activity of
the conditioned media appears to be related to the presence of the
28-kDa polypeptide. To further validate that the smallest lefty
polypeptide is the biologically active form of lefty, we used a
recombinant E. coli lefty polypeptide (aa 136-369)
corresponding to the 28-kDa form present in the culture media of
transfected cells. P19 cells were treated with the recombinant lefty
polypeptide, and the MAPK pathway activity was analyzed by the reporter
assay. The recombinant E. coli lefty polypeptide (aa
136-369) induced the activation of the MAPK pathway in a manner similar to that of the conditioned culture media and in a polypeptide concentration-dependent fashion (Fig. 7B). To
independently validate these results, the MAPK activation was assessed
by analysis of the kinase activity of Erk1/2 after treatment of P19
cells with the recombinant E. coli lefty polypeptide. The
recombinant lefty polypeptide induced the activation of the MAPK
pathway in a time-dependent fashion (Fig. 7C).
This activity was similar to that induced by the 34/28-kDa protein
mixture and could be visualized after 30-60 min of incubating the
cells with the recombinant E. coli lefty protein.
Some data suggested that lefty-1, the mouse homolog of human lefty A,
may act as an inhibitor of BMP signaling (12). Since BMP is known to
induce MAPK activation, we compared the effect of BMP and lefty alone
and together on MAPK activation. Consistent with previous results,
recombinant lefty (10 ng/ml) induced a delayed
time-dependent response (Fig.
8A). Used at the same dose, BMP induced a response within 15 min, which diminished in 30 min (Fig.
8B). Addition of BMP to lefty enhanced the MAPK activation of lefty at an early time point (5 min), but diminished the response at
later time points (Fig. 8C). These findings show that the
combination of the two cytokines produced a more balanced response in
MAPK activity compared with the effect of each individual cytokine.
There is a great interest in understanding the vertebrate body
organization that determines the location, structure, and arrangement of visceral organs. Different members of the TGF- Transfection of human embryonic kidney 293 cells and the CHO cell line
with lefty A and lefty B led to the secretion of
three polypeptides of 42, 34, and 28 kDa. The 28- and 34-kDa
polypeptides correspond to the carboxyl-terminal domain of the lefty
proteins, which indicates that lefty A and lefty B are subjected to the same endoproteolytic processing, in agreement with the 95% identity between these proteins. We did not find any immunoreactivity with endogenous proteins in the different cell lines tested, suggesting that
these cell lines do not express endogenous lefty or its possible homolog(s) or that their expression is very weak. The specificity of
the three lefty polypeptides was analyzed by Western blotting using
monoclonal anti-HA antibody and purified polyclonal anti-lefty antibody
A353. Addition of extra amino acids to the C terminus of TGF- Since the proproteins of the TGF- Our data strongly suggest that PC5A is involved in the processing of
lefty proteins in vivo. PC5A produced the in vivo
processing of the 42-kDa lefty precursor to the 34-kDa form.
Furthermore, early detection of PC5/PC6A mRNA in the neural tube
spatially matches the predominant lefty expression on the
left side of the neural tube (12, 54). Interestingly, lefty and
PC5/PC6A are coexpressed in different regions of the developing nervous
system during embryo implantation and in the female reproductive tract, suggesting PC5A as the candidate enzyme for processing of lefty in vivo (14, 5, 56). None of the PC endoproteases tested including PC5A were able to induce in vivo the formation of
the biologically active 28-kDa form. To validate that the proprotein convertase family of endoproteases is involved in the in
vivo endoproteolytic processing of lefty polypeptides, we
identified and mutated the potential consensus sequences required for
convertase cleavage. The mutations of the PC consensus sequences RGKR
(aa 74-77) and RHGR (aa 132-135) prevented the formation of the 34- and 28-kDa lefty polypeptides, respectively. Taken together, we suggest
that PC5A induces cleavage at Arg77 to release the 34-kDa
polypeptide. The 28-kDa lefty is produced by cleavage of the 42-kDa
lefty polypeptide, likely by a serine protease that may be a new
member of the PC family of endoproteases.
To identify the biologically active form of lefty, we tested the
activity of various lefty polypeptides. The members of the TGF- In contrast to these inhibitory activities, lefty proteins directly
induce MAPK activation. This activity is inducible by both the 42-kDa
and cleaved forms of the protein. However, the 42-kDa protein induces a
quick response, whereas the effect induced by the cleaved forms is
delayed, taking as long as 30 min to become detectable. This latter
response appears to be the effect of the 28-kDa lefty protein since the
mutation that prevents secretion of the 28- kDa (but not the 34-kDa)
form leads to the loss of this MAPK-inducing activity. Moreover, this
effect is reproducible by the recombinant E. coli lefty
polypeptide (aa 136-369) corresponding to the 28-kDa native lefty
protein. These results show that, in contrast to the TGF- The kinetics of MAPK activation by the 42-kDa protein or the
42/34/28-kDa protein mixture of lefty was similar to that observed with
TGF- In summary, our results indicate that the lefty protein is
endoproteolytically processed to release two cleaved forms. The lefty
protein is expressed as a precursor of 42 kDa that is cleaved at
Arg77 and Arg135 to release polypeptides of 34 and 28 kDa, respectively. Lefty fails to induce activation of the Smad
pathway, but leads to MAPK activation. The 42- and 28-kDa lefty
proteins induce MAPK activation, but the 34-kDa protein does not
exhibit this activity. Among the PC endoproteases known, PC5A is the
only enzyme that is able to induce the processing of the lefty protein
to the 34-kDa form, suggesting processing as a means for regulating the
function of lefty in vivo. The data support a molecular
model of processing as a mechanism for regulation of lefty signaling.
(TGF-
) superfamily, are involved in
the formation of embryonic lateral patterning. Members of the TGF-
superfamily require processing for their activation, suggesting
cleavage to be an essential step for lefty activation. Transfection of
different cell lines with lefty resulted in expression of a
42-kDa protein, which was proteolytically processed to release two
polypeptides of 34 and 28 kDa. Since members of the proprotein
convertase (PC) family cleave different TGF-
factors and are
involved in the establishment of embryonic laterality, we studied their
role in lefty processing. Cotransfection analysis showed that PC5A
processed the lefty precursor to the 34-kDa form in vivo,
whereas furin, PACE4, PC5B, and PC7 had a limited activity. None of
these PCs showed activity in the processing of the lefty polypeptide to the 28-kDa lefty form. The mutation of the consensus sequences for PC
cleavage in the lefty protein allowed the lefty cleavage sites to be
identified. Mutations of the sequence RGKR to
GGKG (amino acids 74-77) and of RHGR to GHGR
(amino acids 132-135) prevented the proteolytic processing of the
lefty precursor to the 34- and 28-kDa forms, respectively. To identify
the biologically active form of lefty, we studied the effect of lefty
treatment on pluripotent P19 cells. Lefty did not induce Smad2 or Smad5 phosphorylation, Smad2/Smad4 heterodimerization, or nuclear
translocation of Smad2 or Smad4, but activated the MAPK pathway in a
time- and dose-dependent fashion. Further analysis showed
the 28-kDa (but not the 34-kDa) polypeptide to induce MAPK activity.
Surprisingly, the 42-kDa lefty protein was also capable of
inducing MAPK activity, indicating that the lefty precursor is
biologically active. The data support a molecular model of processing
as a mechanism for regulation of lefty signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 superfamily play an
essential role in the establishment of laterality (6-8). The earliest events are the asymmetric expression of nodal and
lefty (9-12). Lefty is a novel subfamily of the TGF-
protein superfamily and is composed of lefty-1 and
lefty-2 in mouse (11, 12) and their homologs, lefty
A and lefty B, in human (13, 14). lefty-1 and lefty-2 both exhibit an asymmetric expression on the
left side of gastrulating mouse embryos. However, the major expression domains of the two genes are different: lefty-1 expression
is predominantly confined to the left side of the ventral neural tube,
whereas lefty-2 is strongly expressed in the lateral plate mesoderm on the left side (11, 12). Asymmetric expression of
lefty and nodal are also perturbed in mouse
mutants with laterality defects (15-18). Furthermore, the knockout
mutation of lefty-1 induces a variety of left/right
positional defects in visceral organs. The most common feature of
lefty-1-deficient mice is bilateral expression of
nodal and lefty-2, which results in a thoracic
left (rather than right) isomerism (19). These observations support that lefty proteins encode a signal for "leftness."
superfamily as well as many other proteins are
synthesized as large inactive precursor proteins that must be
proteolytically processed to release the bioactive polypeptide (20).
One of the best studied examples is the proteolytic processing of the
TGF-
1 precursor, which is an essential step in the formation of the
biologically active TGF-
1 polypeptide. TGF-
1 is expressed as an
inactive precursor of 55 kDa, which is cleaved to produce TGF-
1 of
44 kDa and finally a polypeptide of 12.5 kDa, which is biologically
active in a homodimer form (21). However, despite its physiological
relevance, nothing is known about the endoproteolytic processing of
lefty proteins, and the characterization of their biologically active
form has not yet been reported. In addition, the identification of the
proteases involved in the processing of lefty will shed light on the
regulation of the TGF-
network and its modulation during embryonic
patterning. Proteins of the TGF-
superfamily are cleaved by members
of the proprotein convertase (PC) family of endoproteases (21-23).
These endoproteases are Ca2+-dependent serine
proteases with a consensus cleavage site of RXXR (21-25).
Furin was the first convertase to be extensively characterized and is
required for the proper processing of several TGF-
proproteins.
Furin-deficient LoVo cells fail to cleave TGF-
1, whereas cells
transfected with furin regain the ability to properly process TGF-
1
(21). Furthermore, PCs were recently shown to be involved in the
establishment of embryonic patterning. Furin is required for ventral
closure, axis rotation, formation of the yolk sac vasculature, and
proper left-sided expression of lefty-2 and pitx2
(26). Mouse embryos lacking PACE4 develop an ambiguous situs combined
with left pulmonary isomerism and/or craniofacial malformations
including cyclopia (27). Despite the clear implication of the
proprotein convertases in the regulation of TGF-
1, nothing is really
known about the processing of the lefty cytokines by these proteases.
Here, we analyzed lefty cleavage and studied the in vivo
implication of the proprotein convertase family of endoproteases in
its processing. We also identified the cleavage sites of the
protein and carried out assays to identify the bioactive forms of lefty polypeptides.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was obtained from Sigma. Recombinant
Escherichia coli lefty A, expressed and refolded from Ser136 to Phe369, was obtained from Regeneron
Pharmaceuticals (Tarrytown, NY).
-lefty A vector was filled in
with T4 DNA polymerase and cloned into PmeI-digested
pAdCMV5. Restriction mapping and flanking DNA sequencing confirmed the
orientation of the resulting lefty A expression plasmids.
All convertases were cloned into the mammalian expression vector
pcDNA3. Construction of the cDNAs has been previously described
(28-30).
-minimal essential medium supplemented with 7.5% bovine calf serum, 2.5% fetal bovine serum, and 1%
antibiotic/antimycotic mixture. Twenty-four hours after transfection,
cells were incubated for 20 h with the conditioned media of 293 cells stably transfected with wild-type lefty A or
lefty B cDNA or the GGKG and GHGR mutant lefty
A cDNAs. Cells were then washed with phosphate-buffered saline, and the luciferase activity was quantified using the Promega luciferase assay system following the manufacturer's instructions. To
ensure equal treatment of cells with the culture media, the media of
these cultures were also examined by Western blotting. The blot was
stained with the polyclonal antibody to lefty A. The density of the
42-kDa protein measured by scanning laser densitometry was the same in
each lane.
-glycerophosphate, 5 mM EGTA, 1 mM sodium
orthovanadate, and 1 mM dithiothreitol). To 10 µl of the
immunocomplexes were added 20 µg of dephosphorylated MBP, 10 µl of
assay dilution buffer, and 10 µl of Mg/ATP mixture (500 µM unlabeled ATP and 75 mM
MgCl2), and the mixture was incubated for 20 min at
30 °C with constant shaking. Ten microliters of the reaction mixture
was added to an equal volume of 2× reducing sample buffer; the mixture
was subjected to SDS-polyacrylamide gel electrophoresis; and the
proteins were transferred to nitrocellulose membranes. The
phosphorylation of MBP was assessed by immunoblotting with 1 µg/ml
anti-phospho-MBP.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily (11-14).
Cytokines of the TGF-
superfamily require proper endoproteolytic
cleavage for their activity. However, nothing is known about the
processing and activation of lefty polypeptides. We cloned the coding
regions of human lefty A and lefty B by PCR using
Marathon-Ready cDNA from a human pancreas library. To analyze the
expression, lefty A was tagged at the carboxyl-terminal
domain with HA sequence. Human embryonic kidney 293 cells were
transfected with pcDNA3-lefty A-HA, and the expression
of lefty was analyzed in the culture media collected from the
transfected cells by Western blotting using monoclonal anti-HA antibody
(Fig. 1A). lefty A transfection induced the expression of three specific forms with respective molecular masses of 42, 34, and 28 kDa. In addition, the
location of the HA tag indicates that these fragments correspond to the
carboxyl-terminal domain of the lefty A protein. These results strongly
support the insight that, as a member of the TGF-
superfamily, lefty
A is a secreted protein.
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Fig. 1.
Identification of lefty forms and
characterization of anti-lefty antibody. A and
B, human embryonic kidney 293 cells were transfected with
pcDNA3 empty vector (Control) or with the coding
sequence for lefty A with an HA tag in the carboxyl-terminal
domain (pcDNA3-lefty A-HA). Secreted lefty forms were
analyzed in the culture media by Western blotting using anti-HA
antibody (A) and anti-lefty antibody A353 (B).
C, CHO cells were transfected with pcDNA3 empty vector
(Control) or with the coding sequence for lefty
A. The blot was probed with polyclonal anti-lefty antibody A353.
D, lefty B processing was also analyzed by transfection of
293 cells without (Control) and with lefty B and
Western blotting of the culture media using anti-lefty antibody
A353.
1-antitrypsin Portland
(
1-PDX), a genetically engineered serine protease
inhibitor derived from the trypsin inhibitor
1-antitrypsin.
1-PDX was originally
reported to selectively inhibit furin and PC5/PC6 activities (34).
However, it has recently been shown that
1-PDX can also
inhibit other PCs such as PACE4 (35, 36). To prove the implication of
serine proteases in the processing of lefty, CHO cells were transfected
with lefty A and with
1-PDX, and the
conditioned media were analyzed by Western blotting using antibody A353
(Fig. 2A). The serine protease
inhibitor prevented the processing of lefty A (Fig. 2A),
whereas
1-antitrypsin did not (data not shown),
indicating that the precursor of 42 kDa is processed in vivo
by a serine protease to release the polypeptides of 34 and 28 kDa.
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Fig. 2.
Endoproteolytic processing of lefty A
polypeptides in vivo by members of the convertase
family of endoproteases. A, CHO cells were transfected
with empty vector (Control) and with lefty A
alone (+) and cotransfected with lefty A and the serine
protease inhibitor 1-PDX. The culture media were
subjected to Western blotting and analyzed with anti-lefty antibody
A353. B, furin-deficient CHO cells (RPE40 cells) were
transfected with vector alone (Control) and with lefty
A, and the processing of the protein was analyzed by Western
blotting of the culture media using anti-lefty antibody A353.
C, CHO cells were transfected without (Control)
and with (+) lefty A alone and cotransfected with
lefty A and different members of the convertase family of
endoproteases (furin, PACE4, PC5A, PC5B, and PC7). Conditioned culture
media were analyzed by Western blotting using anti-lefty antibody A353.
D, the transfection efficiency of CHO cells transfected with
convertases was analyzed by immunoprecipitation of the endoproteases by
endoprotease-specific antibodies after metabolic labeling with
[35S]methionine. The immunoprecipitates were subjected to
gel electrophoresis, followed by autoradiography.
superfamily, we have analyzed the in
vivo processing of lefty proteins by members of this family of
endoproteases. Furin was the first convertase to be extensively characterized and is required for the proper processing of pro-TGF-
1 (21) and BMP (37, 38). We studied the lefty processing in furin-deficient CHO cells (RPE40 cells). RPE40 cells were transfected with lefty A, and the culture media were collected and
subjected to Western blotting (Fig. 2B). The culture media
from furin-deficient RPE40 cells showed the typical pattern of three
polypeptides of 42, 34, and 28 kDa, suggesting that furin is not
essential to the processing of lefty. Other members of the convertase
family of endoproteases were cotransfected with lefty A in
CHO cells, and their effect on lefty processing was analyzed by Western
blotting of the conditioned media using antibody A353 (Fig.
2C). Among the proteases, PC5A induced the in
vivo processing of the 42-kDa lefty precursor primarily to the
34-kDa form. Furin, PACE4, and PC5B exhibited a limited effect, whereas
PC7 did not affect lefty processing of the precursor. Although all
convertases were expressed properly (Fig. 2D), none of these
proteases enhanced the processing of the 34-kDa polypeptide to the
28-kDa form (Fig. 2C).
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Fig. 3.
Identification of the endoproteolytic
cleavage sites of lefty A. A, localization of the
consensus sequences for convertase cleavage in lefty A. Lefty A shows
two potential convertase cleavage sites: RGKR (aa 74-77) and RHGR (aa
132-135). These sites were mutated to analyze their significance in
the processing of the lefty protein. B, 293 cells were
stably transfected without (Control) and with the wild-type
or mutant GGKG (aa 74-77) and GHGR
(aa 132-135) forms of lefty A. The effect of these
mutations on lefty A processing was analyzed by Western blotting using
anti-lefty antibody A353.
family signal through the Smad and MAPK pathways (40-49). To
study the biological role of the endoproteolytic processing in lefty
signaling, we tested the ability of lefty to signal through the Smad
and MAPK pathways. Upon stimulation by TGF-
, Smad2/3 is
transphosphorylated by the activated TGF-
receptors. The
phosphorylated Smad2/3 protein heterodimerizes with Smad4, and the
complexes formed accumulate in the nucleus (40-49). BMP, on the other
hand, leads to the phosphorylation of Smad5. To test the effect of
lefty on the Smad pathway, the effect of lefty on phosphorylation of
Smad2 and Smad5 was compared with the activity of TGF-
1 and BMP-4.
TGF-
1 induced the phosphorylation of Smad2, and BMP-4 induced the
phosphorylation of Smad5. On the other hand, lefty A did not have any
effect on Smad2 or Smad5 phosphorylation (Fig.
4, A and B). We
next examined the effect of lefty on heterodimerization of Smad2 with
Smad4. Smad2 was immunoprecipitated from the cytosolic fraction of P19
cells treated with lefty and with TGF-
1. The immunoprecipitates were
subjected to Western blotting for Smad4. Although TGF-
induced
heterodimerization of Smad4 with Smad2, lefty had no effect (Fig.
4C). P19 cells were then treated with lefty and with
TGF-
1. After 1 h of treatment, the nuclear lysates of these
cells were subjected to Western blotting for Smad2 and Smad4. Although
TGF-
1 led to the nuclear accumulation of Smad proteins, lefty did
not change the amount of either of these Smad proteins in the cytosol
(data not shown) or in the nuclei of the treated cells (Fig.
4D).
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Fig. 4.
Effect of lefty A on Smad2 and Smad5
phosphorylation, Smad2/Smad4 heterodimerization, and nuclear
translocation of Smad2 and Smad4. A, P19 cells
were treated for 30 min with culture medium alone (negative control)
and with culture media supplemented with TGF- 1 (5 ng/ml; positive
control) and recombinant E. coli lefty A (5 ng/ml) in
presence of [32P]orthophosphate (15 µCi/ml). Smad2 was
immunoprecipitated (IP) with anti-Smad2 antibody, and the
immunoprecipitates of the untreated (
) and treated (+) cells were
subjected to SDS-polyacrylamide gel electrophoresis, followed by
autoradiography (upper panel). The overall amount of Smad2
was assessed by Western blotting (WB) of cell lysates (10 µg of protein/lane) (lower panel). B, P19 cells
were treated for 30 min with culture medium alone (negative control)
and with culture media supplemented with BMP-4 (5 ng/ml; positive
control) and recombinant E. coli lefty A (5 ng/ml) in
presence of [32P]orthophosphate (15 µCi/ml). Smad5 was
immunoprecipitated with anti-Smad5 antibody, and the immunoprecipitates
of the untreated (
) and treated (+) cells were subjected to
SDS-polyacrylamide gel electrophoresis, followed by autoradiography
(upper panel). The overall amount of Smad5 was assessed by
Western blotting of cell lysates (10 µg of protein/lane) (lower
panel). C, P19 cells were treated for 30 min with
medium alone (negative control) and with TGF-
1 (5 ng/ml; positive
control) and with lefty A (5 ng/ml). The proteins in the cytosolic
preparations of untreated (
) and treated (+) cells were
immunoprecipitated with an antibody to Smad2, and the immunocomplexes
were subjected to Western blot analysis for Smad4 (upper
panel). The cell lysates (10 µg of protein/lane) were analyzed
by Western blotting for Smad2 and Smad4 to assess the overall amount of
these proteins (middle and lower panels).
D, P19 cells were treated for 30 min with medium alone
(negative control) and with TGF-
1 (5 ng/ml; positive control) and
with recombinant E. coli lefty A (5 ng/ml). Nuclear
fractions were prepared from the untreated (
) and treated (+) cells,
and equal amounts of proteins (10 µg of protein/lane) were subjected
to Western blot analysis for Smad2 and Smad 4. Equal loading was
established by Western blotting for histone 3. Arrows point
to Smad2 and Smad4 accumulated in the nuclei of TGF-
-treated
cells.
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Fig. 5.
Activation of the MAPK pathway by lefty
A. The culture media of 293 cells transfected with
pcDNA3 empty vector (control) or lefty A were incubated
with the indicated concentrations of anti-lefty antibody A353 to block
the activity of lefty A in the medium. The media were used to treat the
serum-starved pluripotent mouse P19 embryonic carcinoma cells, and the
activation of the MAPK pathway was analyzed in vivo using
the PathDetectTM luciferase reporting system as indicated
under "Experimental Procedures." The error bars show the
S.D. of three different experiments.
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Fig. 6.
Activation of the MAPK pathway by lefty
A. Serum-starved P19 cells were incubated, for the
durations shown, without ( ) and with (+) 10 ng/ml affinity-purified
42/34/28-kDa lefty A protein mixture (A), affinity-purified
34/28-kDa lefty A protein (B), and affinity-purified 42-kDa
lefty A protein (C). Cells incubated without lefty (
)
served to show the basal activity of MAPK after 30 min. After cytokine
treatment, Erk1/2 was immunoprecipitated, and the kinase activity of
the immunocomplexes on dephosphorylated MBP was assessed by
immunoblotting using an antibody reactive with phosphorylated MBP as
described under "Materials and Methods."
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Fig. 7.
Activation of the MAPK pathway by lefty
A. A, 293 cells were transfected with pcDNA3 empty
vector (Control), lefty A, lefty B,
and the mutant GGKG (aa 74-77) and
GHGR (aa 132-135) forms of lefty A. The
conditioned media were used for the treatment of serum-starved P19
cells, and the activation of the MAPK pathway was quantitated using the
PathDetectTM reporting system. The error bars
show the S.D. of three different experiments. B, P19 cells
were incubated without and with the indicated amounts of recombinant
E. coli lefty A. The activation of the MAPK pathway was
quantitated using the PathDetectTM reporting system. The
error bars show the S.D. of three different experiments.
C, P19 cells were incubated without ( ) and with (+)
recombinant E. coli lefty A (10 ng/ml) for the durations
shown. Cells incubated without lefty (
) served to show the basal
activity of MAPK after 30 min. After cytokine treatment, Erk1/2 was
immunoprecipitated, and the kinase activity of the immunocomplexes on
dephosphorylated MBP was assessed by immunoblotting using an antibody
reactive with phosphorylated MBP as described under
"Results."
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Fig. 8.
Activation of the MAPK pathway by lefty A and
BMP-4. Serum-starved P19 cells were incubated, for the durations
shown, without ( ) and with (+) 10 ng/ml recombinant lefty A protein
(A), recombinant BMP-4 (B), and recombinant lefty
and BMP-4 (C). Cells incubated without lefty (
) served to
show the basal activity of MAPK after 30 min. After cytokine treatment,
Erk1/2 was immunoprecipitated, and the kinase activity of the
immunocomplexes on dephosphorylated MBP was assessed by immunoblotting
using an antibody reactive with phosphorylated MBP as described under
"Results."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily play
essential roles in the establishment of embryonic patterning (6-8).
Among these, lefty is a novel subfamily of the TGF-
factors. Both
lefty-1 and lefty-2 are expressed on the left
side during gastrulation and encode a signal for leftness (11, 12, 19). However, despite their physiological role, nothing is really known about the regulation and processing of these new factors. In addition, members of the PC family of endoproteases were shown to be involved in
the establishment of embryonic patterning (26, 27). Since these
proteases are also required for the activation of the cytokines of the
TGF-
superfamily (20-25), we studied their role in the processing
of lefty proteins and identified their cleavage sites. Furthermore, we
assessed the biological activity of lefty polypeptides to identify the
bioactive forms of lefty.
interfered with the normal dimerization of the protein product and
totally inhibited the normal proteolytic processing and glycosylation
of the precursor protein (50). However, addition of the HA tag to the C
terminus of lefty did not interfere with the processing of lefty. The
difference in the processing of TGF-
and lefty might be related to
the fact that the C terminus of lefty extends 12 amino acids beyond the
C terminus of most other TGF-
family members (13).
superfamily are cleaved by members
of the PC family, we studied their role in lefty processing. We first
showed that
1-PDX prevented lefty processing.
1-PDX was originally reported to selectively inhibit
furin and PC5/PC6 activities (34). However, it has recently been shown
that
1-PDX can also inhibit other PCs like PACE4 (35,
36). The serine protease inhibitor
1-PDX interfered with
processing of lefty and prevented the formation of the 34- and 28-kDa
lefty polypeptides, indicating that lefty A and lefty B are expressed
as a 42-kDa precursor that is proteolytically processed by a PC. We
analyzed lefty processing in furin-deficient RPE40 cells. The results
showed that furin is not required for processing of lefty; hence, we studied the potential role of other PC endoproteases. Among these proteases, furin, PACE4, PC5B, and PC7 showed a very limited or no
effect on the processing of the lefty precursor. Recently, it was
reported that PCs are involved in the establishment of embryonic
patterning (26). The knockout mutation of furin and PACE4
induces a variety of left/right positional defects and perturbs the
left-sided expression of lefty proteins. Furin is required for ventral
closure, axis rotation, formation of the yolk sac vasculature, and
proper left-sided expression of lefty-2 and pitx2 (26). Mouse embryos lacking PACE4 develop an ambiguous situs combined
with left pulmonary isomerism and/or craniofacial malformations including cyclopia (27). Our results support the insight that furin and
PACE4 act by induction of the processing of cytokines other than lefty
proteins. Indeed, furin and PACE4 promote maturation of other members
of the TGF-
family such as nodal and BMP in tissue culture cells
(51-55). Furin also accounts for TGF-
1 and endoglin maturation
(20-27, 34-39, 49). Despite the presence of several perfect
Arg-X-(Lys/Arg)-Arg consensus sequences for PC cleavage
(51-53), furin and PC4 could not cleave lefty proteins in
vivo (13). Other than this consensus sequence, additional structural features are presumably needed for the specificity of the PC
endoproteases. Likewise, PC endoproteases are modulated in
vivo by the specificity of substrate and the pattern of expression.
superfamily are pleiotropic factors that modulate diverse cellular
responses in a variety of cell types. TGF-
signals through two
distinct mechanisms, MAPK activation and the Smad-mediated signaling
pathway. The latter includes Smad2/3 phosphorylation and is followed by
heterodimerization of Smad2/3 with Smad4, nuclear accumulation of these
complexes, and subsequent gene transcriptional activity (41-49). The
former cascade consists of Ras, MAPKKs, MKK4, MEK1, the MAPKs SAPK and
Erk, and the specific AP1 proteins Fra-2 and JunD (57). Lefty fails to
induce phosphorylation of Smad2; heterodimerization of Smad2 with
Smad4; and nuclear accumulation of these heteromeric complexes, which
is essential to the transcriptional activity of TGF-
(41-49).
Likewise, lefty does not lead to the phosphorylation of Smad5, which is
involved in BMP-mediated signaling (58). Thus, the biological role of
lefty does not appear to directly depend on Smad-mediated signaling.
However, we have observed that lefty perturbs the TGF-
signaling by
inhibiting the phosphorylation of R-Smad following activation of
the TGF-
receptor as well as events that lie downstream from R-Smad
phosphorylation, including heterodimerization of R-Smad proteins with
Smad4 and nuclear translocation of the R-Smad·Smad4 complex.
Lefty represses TGF-
-induced expression of reporter genes
for the p21, cdc25, and connective tissue growth factor promoters and of a reporter gene driven by the
SBE. Thus, lefty provides a repressed state of
TGF-
-responsive genes and participates in negative regulation of
TGF-
signaling by inhibition of phosphorylation of R-Smad proteins
(59). Moreover, lefty also inhibits BMP-induced phosphorylation of
Smad5 as well as BMP-mediated gene transcription, suggesting that lefty
is an inhibitor of BMP actions (59). Interestingly, injection of lefty
into mouse blastocysts leads to neurogenesis, a function attributable to BMP inhibitors such as chordin, noggin, and follistatin (12, 60-63). Antivin, a lefty-related gene product, acts as a specific competitive inhibitor for activin during embryo development in zebrafish, and the lefty-related factor Xatv acts as a feedback inhibitor of nodal signaling in induction of mesoderm and left/right axis development in Xenopus (60, 64, 65). These findings imply that lefty is an inhibitory member of the TGF-
family.
precursor,
the precursor of the lefty protein is biologically active. Moreover,
the data show that the precursor induces a response that is distinctly
different, both temporally and in terms of its magnitude, from that
induced by its cleaved forms. These findings establish that processing
of lefty leads to formation of two different forms: a 28-kDa form that
induces MAPK activation and a 34-kDa protein that is inactive. Since
PC5A processes lefty to its 34-kDa form, which is incapable of inducing MAPK activity, the processing by this enzyme could provide a mechanism for regulating lefty function by producing a bias toward production of
its inactive form. The results reported here also suggest that lefty
proteins, like other members of the TGF-
superfamily, are expressed
as a precursor that is processed for activation. The best studied
example is TGF-
1, which is expressed as an inactive precursor of 55 kDa. The TGF-
1 precursor is cleaved to produce pro-TGF-
1 of 44 kDa and finally a polypeptide of 12.5 kDa, which becomes biologically
active in a homodimer form. However, in contradistinction to the
TGF-
precursor, the 42-kDa lefty protein is biologically active and
competent to drive the activation of MAPK within minutes in treated cells.
and BMP. Both TGF-
1 and TGF-
2 lead to rapid,
dose-dependent activation of Erk1. This activation takes
place within 5-10 min after addition of the growth factor to
exponentially proliferating cultures of intestinal epithelial cells
(66). Similarly, the BMP-2-induced Erk activation shows a latent but
sustained activity during osteoblastic differentiation (58). The effect
of recombinant E. coli lefty and BMP-4 on P19 cells was
delayed and became apparent after only 15-30 min of treatment of P19
cells. The immediate effect of lefty on MAPK activation was enhanced by
BMP, although this effect later became less pronounced or appeared to
be inhibited by BMP. A similar inhibitory effect of BMP on the activity
of both platelet-derived growth factor- and epidermal growth
factor-stimulated MAPKs has been demonstrated in mesangial cells (66,
67). MAPK is a key regulator of multiple cellular processes, including
cell growth, apoptosis, and differentiation, in a wide range of
cell types (68-71). For example, macrophage apoptosis, chondrocyte
cell growth, and inhibition of DNA synthesis in intestinal epithelial cells induced by TGF-
were all found to be mediated by MAPK
activation (70, 72). Because of the ability to induce MAPK activation, lefty is a suitable candidate for modulation of growth, apoptosis, and/or differentiation of target cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank E. Plets for excellent technical
assistance and the core facility at the North Shore-Long Island
Jewish Research Institute for technical assistance in
oligonucleotide synthesis and DNA sequencing. We thank Michele Barcia
for technical assistance and Dr. Gary Thomas for providing
1-PDX and
1-antitrypsin. CHO and RPE40
cells were a kind gift of Dr. Thomas Moehring.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant CA46866 from the National Institutes of Health and by a grant from Lexon Inc. (to S. T.).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.
§ These authors contributed equally to this work.
Recipient of a fellowship from the Fonds voor Wetenschappelijk
Onderzoek Vlaanderen.
To whom correspondence and reprint requests should be
addressed: Dept. of Pathology, Biomedical Research Center, 350 Community Dr., Manhasset, NY 11030. Tel.: 516-484-0813; Fax:
516-484-2831; E-mail: tabibzadeh@bioscience.org.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M006933200
2 The sequences of the primers used for PCR and site-directed mutagenesis are available upon request.
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ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
PC, proprotein convertase;
MBP, myelin
basic protein;
Erk, extracellular signal-regulated kinase;
MAPK, mitogen-activated protein kinase;
HA, hemagglutinin;
PCR, polymerase
chain reaction;
CHO, Chinese hamster ovary;
JNK, c-Jun N-terminal
kinase;
MOPS, 4-morpholinepropanesulfonic acid;
1-PDX,
1-antitrypsin Portland;
aa, amino
acids;
MAPKK, mitogen-activated protein kinase kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase;
SAPK, stress-activated protein kinase;
BMP, bone
morphogenetic protein;
R-Smad, receptor regulated Smad.
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