From the Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
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ABSTRACT |
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Two structural elements, the L45 loop on the
kinase domain of the transforming growth factor- The Smad family of proteins play a central role in signal
transduction by the transforming growth factor- The maintenance of specificity in this system requires that each member
of the type I receptor family be able to discriminate among different
groups of Smad proteins. The specific interaction between Smad proteins
and type I receptors is determined by two structural elements, namely,
the L3 loop in the carboxyl-terminal domain (or MH2 domain) of Smad
proteins and the L45 loop in the kinase domain of the receptors (7, 8).
Both elements consist of a short amino acid sequence that is highly
conserved among Smad proteins or receptors of similar specificity but
differs on a few critical residues between functionally distinct Smad proteins or receptors. Thus, the L45 loop sequence of the
T An important question left open by previous studies is about the Smad
specificity of a third group of type I receptors. This group includes
ALK1, ALK2, and Drosophila Saxophone. Saxophone is essential
for dorsal closure of the Drosophila embryo and is believed
to mediate Dpp signals along with Thickveins (9-12). ALK1 (also known
as TSR-I) is highly expressed in vascular endothelial cells (13).
Inactivating mutations of ALK1 in humans cause hereditary hemorrhagic
telangiectasia (14). Hereditary hemorrhagic telangiectasia is an
autosomal dominant disorder characterized by epithelial vascular
dysplasia with a high propensity to hemorrhage in the nasal and
gastrointestinal mucosa. ALK2 (also known as ActR-I or Tsk7L) is a
broadly expressed receptor that can bind BMPs, activin, and, under
certain conditions, TGF- In this study, we have investigated the Smad specificity of this third
group of type I receptors. We were intrigued by the fact that the L45
sequence of these receptors is very different from that of the T R1B/L17, COS-1, and HepG2 cells were maintained as described
previously (8, 25). Mouse embryonic P19 cells were maintained in
Dulbecco's modified Eagle's medium containing 10% fetal bovine serum
and 2 mM glucose. Mutagenesis of Smad proteins and
receptors was performed by polymerase chain reaction using appropriate
oligonucleotides, and verified by DNA sequencing. T Activation of the 3TP-luciferase reporter (20) and Mix2 A3-luciferase
reporter (26) by receptors were analyzed in R1B/L17 cells as described
previously (8, 25). To measure the activity of a XVent2-luciferase
reporter (27), P19 cells were transfected with this construct, and the
constitutively active forms of type I receptors, using Lipofectin (Life
Technologies, Inc.) according to manufacturer's instructions.
Luciferase activity was measured 40 h after transfection.
Metabolic labeling of transfected cells with
[32P]orthophosphate or [35S]Met/Cys was
performed as described previously (8, 25). Immunoprecipitates with
monoclonal anti-Flag M2 antibody (IBI, East Kodak) were visualized by
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
Subcellular localization of Smad proteins was examined in HepG2 cells
transfected with N-terminally Flag-tagged Smad proteins and the
constitutively active receptors. Immunofluorescence was carried out as
described previously (8). At least 200 cells were scored per assay. The
percentage of Flag-positive cells with predominantly or exclusively
nuclear immunofluorescence is plotted in the figures.
ALK1 and ALK2 Transduce BMP-like Signals--
To investigate the
signaling specificity of human ALK1 and ALK2, we used transcriptional
assays that discriminate between BMP-like signaling and
TGF-
We did similar experiments using two TGF- Smad Specificity of ALK1 and ALK2--
These results raised the
possibility that ALK1 and ALK2 may be able to phosphorylate and
activate Smad1 but not Smad2. To investigate this possibility, receptor
constructs were cotransfected with N-terminally Flag-tagged Smad1 or
Smad2 into R1B/L17 cells, and Smad phosphorylation was determined by
anti-Flag immunoprecipitation from the
[32P]orthophosphate-labeled transfectants. The basal
phosphorylation of Smad1 and Smad2 observed in the absence of receptor
activity in these cells (Fig.
2A, left lanes) is
caused by MAP kinases acting on phosphorylation sites unrelated to the
receptor-mediated phosphorylation sites
(37).3 ALK1(QD) and ALK2(QD)
increased the phosphorylation of Smad1 but not that of Smad2, which is
similar to the effect of BMPR-IB(QD) and opposite to the effect of
T
To confirm that ALK1(QD) or ALK2(QD) specifically activated Smad1, we
determined the ability of these constructs to induce nuclear
accumulation of Smad proteins. Consistent with the above results,
expression of ALK1(QD) or ALK2(QD) induced nuclear accumulation of
Flag-Smad1 but not Flag-Smad2, which again is similar to the effect of
BMPR-IB(QD) and opposite the effect of T
We repeated these assays using a Smad1 construct [Smad1(AAVA)] that
contains three Ser to Ala mutations in the C-terminal sequence SSVS
(38). These serines are the sites phosphorylated by BMPR-I, and their
mutation to alanine prevents receptor-mediated phosphorylation and
activation of Smad1 by BMP (38). The Smad1(AAVA) mutant failed to
accumulate in the nucleus when cotransfected with ALK1(QD) or ALK2(QD)
vectors (data not shown). Therefore, ALK1 and ALK2 can mimic the
ability of BMP type I receptors to specifically phosphorylate and
activate Smad1, leading to BMP-like transcriptional responses.
Determinants of Specificity in ALK1 and ALK2--
The sequence of
the L45 loop in the kinase domain of T Role of the
We searched Smad MH2 domains for other regions containing
subtype-specific residues, that is, residues that would be different between Smad1 and -2 but conserved between Smad1, -5, -8, and Mad or
between Smad2 and -3. The region immediately preceding the C-terminal
phosphorylation sites contains four such residues (7). However,
introduction of these mutations into Smad2, along with the L3 loop
mutations, did not improve the ability of ALK1(QD) or ALK2(QD) to
induce nuclear accumulation of the resulting construct (Fig.
5A, Smad2(LC1) construct). Another region with
subtype-specific residues is a sequence corresponding to
To verify that both
From these results, we conclude that two groups of Smad1-activating
receptors coexist in vertebrates and possibly Drosophila, the BMPR-I group and the ALK1 group. A similar conclusion was recently
reached by others using a different BMP reporter assay (41).
Furthermore, the present work shows that the recognition and activation
of Smad1 by ALK1 and ALK2 is specified by the L45 loop on the receptors
and the L3 loop together with the
The BMPR-I group and the ALK1 group recognize Smad1 through related but
different mechanisms which involve different L45 sequences on the
receptor kinase domain, and a differential use of two surface structures on the Smad1 MH2 domain. Smad1 recognition and activation by
these two mechanisms might be qualitatively different in ways that
cannot be discerned by the present assays. In a given cellular context,
such differences might make one receptor more suitable than the other
as an activator of the Smad1 pathway, or both receptors could activate
Smad1 in complementary ways or with different response outcomes.
(TGF-
) family
type I receptors and the L3 loop on the MH2 domain of Smad proteins,
determine the specificity of the interactions between these receptors
and Smad proteins. The L45 sequence of the TGF-
type I receptor
(T
R-I) specifies Smad2 interaction, whereas the related L45 sequence of the bone morphogenetic protein (BMP) type I receptor (BMPR-I) specifies Smad1 interactions. Here we report that members of a third
receptor group, which includes ALK1 and ALK2 from vertebrates and
Saxophone from Drosophila, specifically phosphorylate and activate Smad1 even though the L45 sequence of this group is very divergent from that of BMPR-I. We investigated the structural elements
that determine the specific recognition of Smad1 by ALK1 and ALK2. In
addition to the receptor L45 loop and the Smad1 L3 loop, the
specificity of this recognition requires the
-helix 1 of Smad1. The
-helix 1 is a conserved structural element located in the vicinity
of the L3 loop on the surface of the Smad MH2 domain. Thus, Smad1
recognizes two distinct groups of receptors, the BMPR-I group and the
ALK1 group, through different L45 sequences on the receptor kinase
domain and a differential use of two surface structures on the Smad1
MH2 domain.
INTRODUCTION
Top
Abstract
Introduction
References
(TGF-
)1 family (1-3).
Smad proteins are directly phosphorylated by type I TGF-
family
receptors, an event that induces their accumulation in the nucleus
where they activate transcription of specific genes. Smad proteins act
as signal transducers for different members of the TGF-
family,
including TGF-
itself, the activins, and the bone morphogenetic
proteins (BMPs). The type I receptors for TGF-
and activin, which
are known as T
R-I and ActR-IB, respectively, signal via Smad2 and
its close homolog Smad3. The BMP type I receptors BMPR-IA and BMPR-IB
signal via Smad1 and possibly its close homologs, Smad5 and Smad8. Upon
receptor-mediated phosphorylation, and on their way to the nucleus, all
these Smad proteins associate with Smad4, a member of a separate
subclass that is required for the formation of transcriptional
complexes. The Drosophila orthologs of BMP (Dpp), BMPR-I
(Thickveins), Smad1 (Mad), and Smad4 (Medea) are functionally linked in
a similar fashion (4-6).
R-I/ActR-IB group (which also includes the orphan receptors ALK7,
XTrR-I and Atr-I) is compatible with the L3 loop sequence common to
Smad2 and -3, allowing functional interactions between these receptors and Smad proteins. A similar relationship exists between the L45 loop
of the BMPR-I group (which also includes Thickveins) and the L3 loop of
Smad1, -5, -8, and Mad (see Figs. 1A and 4A for L45 and L3 sequences).
in vitro (1-3, 13, 15-19).
However, ALK2 does not mediate activin responses like those mediated by
ActR-IB (20). In Xenopus, the activity of ALK2 (21) is akin
to that of BMPR-I (22, 23), because both receptors signal ventral
mesoderm induction, and defects in ventral mesoderm formation caused by
a dominant-negative ALK2 construct can be rescued by overexpression of
Smad8 (24).
R-I
group or the BMPR-I group (see Fig. 1A). Nonetheless, we
observed that ALK1 and ALK2, like BMPR-I, recognize and activate Smad1.
This was paradoxical because T
R-I, whose L45 sequence is much closer
to that of BMPR-I, does not recognize Smad1. Insights from the crystal
structure of the Smad MH2 domain allowed us to address this paradox.
EXPERIMENTAL PROCEDURES
R-I(LA) contains
a replacement of the L45 sequence ADNKDNGTW with the sequence
SDMTSRHSS. Smad2(H1-1) contains the mutations A323S, V325I, and M327N,
and Smad2(HL1) contains the mutations A323S, V325I, M327N, R427H, and
T430D. Other constructs have been reported previously (7, 8).
RESULTS AND DISCUSSION
/activin-like signaling. Since the natural ligands for ALK1 and
ALK2 have not been conclusively defined, we generated ALK1 and ALK2
mutant constructs [ALK1(QD) and ALK2(QD)] containing a Gln to Asp
mutation in the penultimate residue of the regulatory domain (GS
domain), which activates type I receptors in a ligand-independent
manner (28-30). The corresponding mutant forms of the BMP type I
receptors, BMPR-IA(QD) and BMPR-IB(QD), activate luciferase expression
when cotransfected with the XVent2-Luc reporter construct into mouse
P19 cells (Fig. 1B).
XVent2-Luc contains a BMP-responsive region from the Xenopus
Vent.2 gene driving expression of luciferase (27) and can be
activated via Smad1 but not Smad2 (8). ALK1(QD) and ALK2(QD) were also
able to activate XVent2-Luc, whereas the constitutively active TGF-
receptor construct, T
R-I(TD), had no effect (Fig.
1B).
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Fig. 1.
A, the L45 loop sequences of the type I
receptors. The residues that are distinct among different groups are
boxed, and the variant residues within a group are
italicized. B, XVent2-luciferase reporter was
transfected into P19 cells with or without the active forms of
receptors as indicated. 24 h after transfection, cells were
treated with or without 1 nM BMP2 for 20 h, and
subsequently, luciferase activity was determined. C, R1B/L17
cells were transfected with 3TP-luciferase reporter and the indicated
receptors. Luciferase activity was measured 48 h later.
D, R1B/L17 cells were transfected with A3-luciferase
reporter, Xenopus FAST-1 and the indicated receptors.
Luciferase was measured 48 h later. Data are the average of three
or more assays ± SD.
/activin-responsive
reporters, 3TP-Lux and A3-Luc. 3TP-lux contains
TGF-
/activin-response elements from plasminogen activator
inhibitor-1 and collagenase (20). A3-Luc contains three copies of the
activin response element from the Xenopus Mix.2 gene (26).
Both reporters are activated by TGF-
or activin signals mediated by
Smad2 (31-33) or Smad3 (34-36).2 Both reporters
were activated by the TGF-
receptor mutant T
R-I(TD) and the
activin receptor mutant ActR-IB(TD) in transfected R1B/L17 mink lung
cells (Fig. 1, C and D). ALK1(QD) and ALK2(QD),
like BMPR-IB(QD), failed to activate 3TP-lux and A3-Luc (Fig. 1,
C and D). Thus, the activation pattern of these
three reporter constructs suggests that the signaling specificity of
ALK1 and ALK2 is similar to that of the BMP type I receptors BMPR-IA
and IB.
R-I(TD) (Fig. 2A). These results were consistent with
ALK1 and ALK2 acting as specific activators of Smad1 but not Smad2.
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Fig. 2.
A, phosphorylation of Smad proteins was
examined in R1B/L17 cells by transient transfection with N-terminally
Flag-tagged Smad constructs and the active forms of receptors. 40 h later, cells were metabolically labeled with
[32P]orthophosphate. Anti-Flag immunoprecipitates were
analyzed by SDS-polyacrylamide gel electrophoresis and autography.
Protein expression was verified in parallel by anti-Flag
immunoprecipitation from [35S]Met/Cys-labeled cells.
B, nuclear translocation of Smad proteins was determined in
HepG2 cells. Flag-tagged Smad constructs and the indicated receptors
were cotransfected into HepG2 cells. Immunostaining was processed
48 h later first with mouse anti-Flag and second with fluorescein
isothiocyanate-conjugated goat anti-mouse antibodies. About 200-300
fluorescence-positive cells were scored per condition. The percentage
of Flag-positive cells with predominantly or exclusively nuclear
immunofluorescence is plotted.
R-I(TD) (Fig. 2B). Under these conditions, nuclear localization of
Flag-Smad proteins was observed in only a fraction of the cells. The
incompleteness of this response may be because of limitations in the
ability of the receptors to quantitatively activate the overexpressed Smad proteins. In untransfected cells, TGF-
induces a quantitative translocation of endogenous Smad2 and -3 into the
nucleus.3
R-I determines TGF-
signaling activity (39). Furthermore, the L45 loop of T
R-I and
BMPR-I specifies the choice of Smad proteins by these receptors in the
cell (8). Five of nine residues in this sequence are
conserved between T
R-I and BMPR-I (see Fig. 1A). Thus,
the three nonconserved residues are responsible for the different Smad
specificity of these two receptors. Mutant T
R-I and BMPR-I
constructs with these residues swapped show a switch in their ability
to recognize and activate Smad1 and Smad2 (8). The L45 loop is
conserved between ALK1 and ALK2 but is very divergent between these
receptors and T
R-I or BMPR-I (see Fig. 1A). Because the
Smad specificity of ALK1 and ALK2 is similar to that of BMPR-I
receptors, we investigated whether this specificity is determined by
the L45 sequence of ALK1 and ALK2 despite their lack of similarity to
the BMPR-I L45 sequence. To this end, we generated a mutant T
R-I
[T
R-I(LA)] containing the L45 sequence SDMTSRHSS, which
corresponds to the ALK2 L45 sequence. The signaling specificity of this
construct was compared with those of the wild type T
R-I and the
previously described mutant T
R-I(LB) containing the L45 sequence of
BMPR-I (8). T
R-I(LA) was similar to T
R-I(LB) and BMPR-I in its
ability to induce nuclear accumulation preferentially of Smad1 (Fig.
3A) and its pattern of
activation of XVent2-Luc (Fig. 3B) and 3TP-Lux (Fig.
3C). These results suggest that the ability of the BMPR-I
L45 sequence to specify an interaction with Smad1 is shared by the very
divergent L45 sequence of ALK1 and -2 but not by the closely related
L45 sequence of T
R-I.
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Fig. 3.
A, T R-I(LA) induces nuclear
translocation of Smad1 but not Smad2. N-terminally Flag-tagged Smad1 or
Smad2 were cotransfected into HepG2 cells with wild type or mutant
T
R-I, or wild type BMPR-IB, as well as their corresponding type II
receptors. Cells were treated with 1 nM TGF-
1 or 5 nM BMP2 for 1 h and then processed for anti-Flag
immunostaining. 200-300 fluorescence-positive cells were scored. The
percentage of Flag-positive cells with predominantly or exclusively
nuclear immunofluorescence is plotted. B and C,
T
R-I(LA) activates the BMP-responsive XVent-Luc reporter but not the
TGF-
-responsive 3TP-lux reporter. XVent-luciferase and
3TP-luciferase reporters were transfected into P19 or R1B/L17 cells,
respectively, with wild type or mutant T
R-I as well as T
R-II.
Cells were then incubated with 1 nM BMP2 (B) or
0.5 nM TGF-
(T) for 1 day, and luciferase
activity was determined. Data are the average of three or more
assays ± SD.
-Helix 1 in Smad Recognition of ALK1 and
ALK2--
In light of this, we wondered whether Smad1 might recognize
ALK1 and -2 and BMPR-I by different mechanisms. The ability of Smad1
and Smad2 to recognize BMPR-I and T
R-I, respectively, is determined
by the sequence of the L3 loop (7). As inferred from the crystal
structure of the Smad4 MH2 domain, the L3 loop protrudes from the
surface of this domain (40). The sequence of the L3 loop is identical
within the Smad2/3 and Smad1/5/8 subgroups, but differs at two critical
positions between these two subgroups (Fig.
4, A and B).
Swapping these two residues between Smad1 and Smad2 switches their
ability to interact with specific receptors (7). Thus, a Smad2 mutant
containing the two Smad1-specific residues in the L3 loop [Smad2(L1)
construct] is preferentially activated by BMPR-I (7) (Fig.
5A). Using this approach, we determined that ALK1(QD) and ALK2(QD) are relatively weak inducers of
nuclear accumulation of Smad2(L1) even though they induce nuclear accumulation of Smad1 (Fig. 5A). These results indicate that
the L3 sequence alone is not sufficient to specify Smad1 recognition by
ALK1 or ALK2.
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Fig. 4.
A, -helix 1 and L3 sequences of
receptor-regulated Smad proteins. Structural elements
(arrows,
-strands; rounded boxes,
-helices)
correspond to the Smad4 MH2 domain (40). Subtype-specific residues are
boxed. The corresponding sequences of Smad4 is also shown.
Numbering of the last residue in each sequence corresponds to the Smad
species not in the parentheses. B, a close-up view of the
Smad4 MH2 structure showing the L3 loop (yellow) with
subtype-specific residues (red) and the
-helix 1 (purple) with subtype-specific residues (green).
The insert shows a frontal view of the location of the L3 loop and
helix 1 of each MH2 monomer in the crystallographic trimer.
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Fig. 5.
A, activation of mutant Smad proteins as
measured by nuclear accumulation in HepG2 cells. Flag-tagged Smad
constructs were cotransfected with the active forms of receptors as
indicated, and cells were analyzed for Flag immunostaining. 200-300
fluorescence-positive cells were scored per condition. B,
phosphorylation of Smad proteins was examined in HepG2 cells by
transient transfection with N-terminally Flag-tagged wild type and
mutant Smad2 constructs and the active forms of receptors. 40 h
later, cells were metabolically labeled with
[32P]orthophosphate. Anti-Flag immunoprecipitates were
analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography.
Protein expression was verified in parallel by anti-Flag western
immunoblotting. B, BMPR-IB(QD); A1, ALK1(QD);
A2, ALK2(QD). The number underneath the phosphorylation gel
indicates the relative band intensity quantitated by a
PhosphorImager.
-helix 1, which is a surface structure in Smad4 (40). The corresponding sequence is conserved between Smad1 and Smad2 except for two residues that are
subtype-specific (Fig. 4A). These residues in Smad4 (Glu-374 and Arg-378) are separated by one turn of the
-helix and are exposed
to solvent in the vicinity of the L3 loop (40) (Fig. 4B).
Because of their properties and location, we investigated whether these
residues play a role as determinants of the interaction of Smad1 and
ALK1 and -2. A Smad2 mutant containing these two residues from Smad1
[Smad2(H1-1)] did not respond to BMPR-IB(QD), ALK1(QD) or ALK2(QD)
in the nuclear accumulation assay (Fig. 5A). However, a
Smad2 construct containing the two
-helix 1 residues and the two L3
loop residues from Smad1 [Smad2(HL1)] underwent a similar extent of
nuclear accumulation when cotransfected with ALK1(QD), ALK2(QD) or
BMPR-IB(QD) (Fig. 5A). These results suggest a critical role
of
-helix 1 in Smad1 recognition by ALK1 and ALK2.
-helix 1 and the L3 loop are required for Smad1
recognition by ALK1 and 2, wild type and mutant Smad2 constructs were
transfected into HepG2 cells with the constitutively active forms of
receptors, and phosphorylation of Smad2 was determined. Consistent with
the pattern of nuclear accumulation, coexpression of BMPR-IB(QD)
increased the phosphorylation level of Smad2(L1) and Smad2(HL1), but
not wild type Smad2, whereas coexpression of ALK1(QD) and ALK2(QD) only
enhanced phosphorylation of Smad2(HL1) (Fig. 5B).
-helix 1 on Smad1 (Fig.
6). It is likely that Saxophone
recognizes Mad in a similar fashion because the L45 sequences of
Saxophone and the L3 sequence and
-helix 1 sequence of Mad are very
similar or identical to those of their counterparts in vertebrates. It is important to note that the structural elements identified here and
previously (7, 8), although playing a major role in dictating the
specificity of the recognition between type I receptors and Smad
proteins, may be only part of larger regions mediating the association
between these proteins. Additionally, the correct assembly of receptor
complexes and Smad protein complexes and/or the possible intervention
of "adaptor" molecules might be important for the association of
receptors and Smad proteins.
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Fig. 6.
Structural motifs in the MH2 domains of Smad1
and Smad2 that determine the specificity of interactions with
representative members of three groups of type I receptors. Smad1
recognition by ALK1 and ALK2 requires a structure, the -helix 1, which is not essential for Smad1 interaction with BMPR-I (8). Smad
proteins are represented as homotrimers based on the Smad4 MH2
structure (40). See text for additional details.
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ACKNOWLEDGEMENTS |
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We thank Yigong Shi and Nikola Pavletich for insightful discussions and for the structure image. We thank Christof Niehrs for XVent2-Luc, the Genetics Institute for generously providing BMP, and Roger S. Lo for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA34610 (to J. M.) and a Cancer Center grant to the Memorial Sloan-Kettering Cancer Center.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.
Research Associate of the Howard Hughes Medical Institute.
§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Box 116, Memorial Sloan-Kettering Cancer Ctr., 1275 York Ave., New York, NY 10021. Tel.: 212-639-8975; Fax: 212-717-3298; E-mail: j-massague{at}ski.mskcc.org.
The abbreviations used are:
TGF-, transforming growth factor
; BMP, bone morphogenetic protein.
2 F. Liu and J. Massagué, unpublished work on A3-Luc.
3 M. Kretzschmar, J. Doody, and J. Massagué, unpublished work.
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REFERENCES |
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