From the Molecular Cardiology Unit, Department of
Medicine, ** Departments of Cell Biology and Molecular Physiology & Biophysics, and the § Graduate Program in Cardiovascular
Sciences, Baylor College of Medicine, Houston, Texas 77030
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transforming growth factor (TGF
) signal transduction is mediated by two receptor Ser/Thr
kinases acting in series, type II TGF
receptor (T
R-II)
phosphorylating type I TGF
receptor (T
R-I). Because the failure
of interaction cloning, thus far, to identify bona fide T
R-I
substrates might reasonably have been due to the use of inactive
T
R-I as bait, we sought to identify molecules that interact
specifically with active T
R-I, employing the triple mutation
L193A,P194A,T204D in a yeast two-hybrid system. The Leu-Pro
substitutions prevent interaction with FK506-binding protein 12 (FKBP12), whose putative function in TGF
signaling we have
previously disproved; the charge substitution at
Thr204 constitutively activates T
R-I. Unlike
previous screens using wild-type T
R-I, where FKBP12 predominated,
none of the resulting colonies encoded FKBP12. A novel protein was
identified, T
R-I-associated protein-1 (TRAP-1), that interacts in
yeast specifically with mutationally activated T
R-I, but not
wild-type T
R-I, T
R-II, or irrelevant proteins. In mammalian
cells, TRAP-1 was co-precipitated only by mutationally activated
T
R-I and ligand-activated T
R-I, but not wild-type T
R-I in the
absence of TGF
. The partial TRAP-1 protein that specifically binds
these mutationally and ligand-activated forms of T
R-I can inhibit
signaling by the native receptor after stimulation with TGF
or by
the constitutively activated receptor mutation, as measured by a
TGF
-dependent reporter gene. Thus, TRAP-1 can
distinguish activated forms of the receptor from wild-type receptor in
the absence of TGF
and may potentially have a functional role in
TGF
signaling.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The signal transduction events coupling receptors for the
TGF1 superfamily to
TGF
-dependent responses remain incompletely understood. TGF
signaling requires two transmembrane receptors acting in series,
T
R-II phosphorylating T
R-I, each characterized by a cytoplasmic
serine/threonine kinase domain (1), but the subsequent signaling
pathways are less clear-cut (2). Several candidates have been
identified by interaction cloning in yeast "two-hybrid" systems,
which interact with the cytoplasmic domain of T
R-I. These include
both the immunophilin-binding protein, FKBP12 (3), a target for the
macrolides FK506 and rapamycin, and the
subunit of Ras
farnesyltransferase (FNTA) (4, 5). T
R-II-interacting protein 1, a
Trp-Asp domain protein, was isolated analogously (6). FKBP12 associates
with inactive ALK5 and is released from receptor complexes upon ligand
binding (3). Although FKBP12 might inhibit TGF
signaling, at least
at threshold concentrations of ligand (7, 8), mutational analysis by
ourselves (9) and others (8, 10) has proven that FKBP12 recognition is dispensable for signal generation by T
R-I; FNTA likewise is
unnecessary for TGF
signaling (11). However, genetic screening in
Drosophila identified the transcription factor,
mothers against decapentaplegic (MAD), as acting downstream from the TGF
homologue, decapentaplegic (12). In vertebrates, multiple MAD-related proteins exist (Smads) that
are thought to mediate signaling by TGF
family members via phosphorylation-dependent nuclear translocation (13-17).
TGF
responses required Smad4 in concert with Smad2 (14) or Smad3
(14, 15). Because Smad proteins are substrates for type I receptors
(16, 17), these transcription factors may provide a direct link between type I receptors and the nucleus. Using genetic complementation in
yeast, a novel member of the mitogen-activated protein kinase family,
the TGF
-activated kinase, TAK1, was identified as an alternative
mediator of TGF
signaling, which may be necessary for at least a
subset of TGF
effects (18). Thus, functional pathways appear to
exist distinct from direct phosphorylation of Smad proteins.
Because the failure of interaction cloning to identify bona fide TGF
receptor substrates might reasonably have been due, in part, to the use
of inactive receptor as bait, we sought to identify molecules that
interact specifically with active T
R-I, employing
T
R-IL193A,P194A,T204D as the bait. The charged amino
acid substitution at Thr204 confers constitutive activity
in the absence of ligand and T
R-II; disruption of the invariant
Leu-Pro motif abrogates binding to FKBP12 (9). Using this strategy we
identified a novel protein, TRAP-1, that discriminates between
quiescent T
R-I and T
R-I that is activated in the presence of
TGF
.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interaction Cloning and Two-hybrid Assays in
Yeast--
Constructions containing the cytoplasmic domains of TR-I
and T
R-II or point mutations of T
R-I in the yeast expression
plasmid pAS2-1 were previously described (9). The cytoplasmic domain of T
R-IL193A,P194A,T204D in pAS2-1 was used as bait, to
screen a human lymphocyte cDNA library in the pACT vector
(CLONTECH). The bait and library DNA were
introduced into yeast strain Y190 cells
(GAL1UAS-GAL1TATA-HIS3 and
GAL1UAS-GAL1TATA-lacZ) using lithium
acetate (19). Transformants were selected on synthetic dropout
(SD)/
Leu/
Trp/
His/+25 mM 3-amino-1,2,4-triazole (3-AT)
plates and tested for lacZ using a
-galactosidase colony
lift assay (9). Positive clones were counterselected in
SD/
Leu/+cycloheximide plates to eliminate the bait plasmid and then
were subjected to the yeast mating assay for interaction specificity.
As additional controls for the two-hybrid assays in yeast, pVA3-1,
encoding murine p53 (amino acids 72-390), and pLAM5'-1, encoding lamin
C (amino acids 66-230), were obtained from
CLONTECH. Candidate plasmids purified from yeast
were sequenced by the dideoxy method.
5'-Rapid Amplification of cDNA Ends (RACE)-- The 5' sequence of TRAP-1 was completed using the 5'-RACE reaction. Human heart poly(A)+ RNA (CLONTECH) was reversed transcribed using the TRAP-1-specific primer 5'-GCC TGT GCT GTA ATT GTG GAT GAT GT-3' and Moloney murine leukemia virus reverse transcriptase. Second strand cDNA synthesis was performed using Escherichia coli DNA polymerase I, RNase H, and E. coli DNA ligase (21). Double-stranded cDNA was blunt-ended with T4 DNA polymerase, ligated to the Marathon cDNA adaptor (5'-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3') (CLONTECH) and amplified by the polymerase chain reaction using a nested internal TRAP-1 specific primer (5'-ATG TTG ACC AGG CTG ATG GAG TTG TCA C-3') and an adaptor primer (5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3'). The polymerase chain reaction products were resolved by electrophoresis through 1% agarose. The resulting 460-bp band was excised, purified, subcloned into PCR-script (Strategene), and subjected to DNA sequencing.
Yeast Mating Assay--
Yeast strain Y187 (MAT) was
transformed with wild-type T
R-I, point mutations of T
R-I, or
T
R-II plasmids and was grown on SD/-Trp plates. Yeast strain Y190
(MATa) was transformed with TRAP-1 or TRAP-2 plasmids and grown on
SD/
Leu plates. One colony from each mating type was picked, placed in
0.5 ml of YPD medium (20 g/liter peptone, 10 g/liter yeast extract),
and incubated at 30 °C with shaking at 250 rpm for 8 h. Mating
cultures (15 µl) were spread on SD/
Leu/
Trp and
SD/
Leu/
Trp/-His +25 mM 3-AT plates and incubated at
30 °C for 5 days. Growth was scored on SD/
Leu/
Trp/
His +25
mM plates. The
-galactosidase colony lift filter assay
was performed on SD/
Leu/
Trp plates (22).
Northern Blot Analysis-- Filters containing poly(A)+ RNA from multiple human tissues (CLONTECH) were prehybridized in ExpressHyb solution (CLONTECH) at 68 °C for 30 min and hybridized with a 32P-labeled TRAP-1 cDNA probe at 68 °C for 1 h. Filters were washed in 2× SSC and 0.05% SDS at room temperature for 30 min, washed in 0.1× SSC and 0.1% SDS at 50 °C for 40 min, and subjected to autoradiography.
Co-precipitation in Mammalian Cells--
To engineer the
mammalian expression plasmid pFlag-TRAP-1-CMV2, the partial TRAP-1
cDNA (encoding the C-terminal 387 amino acids) was excised from
pAS2-1 and inserted into pFlag-CMV-2 (IBI Kodak). Mammalian 293 cells,
plated at a density of 5 × 104 cells/cm2
in 100-mm tissue culture dishes, were cotransfected for 5 h with 8 µg of wild-type or mutant T
R-I-HA-pCMV5, 8 µg of
pFlag-
TRAP-1-CMV2, 8 µg of T
R-II-SV-Sport, and 45 µl of
LipofectAMINE (Life Technologies, Inc.). Human recombinant TGF
1 (1 nM, R & D Systems) versus the diluent was
added 48 h later for 15 min. Cells were solubilized in 500 µl of
lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH
7.4, 1 mM EDTA, 0.5% Triton X-100, 1 mM
dithiothreitol, 1 mg/ml Pefabloc, 10 µg/ml leupeptin, 10 µg/ml
pepstatin, 1 µg/ml aprotinin, 50 mM NaF, 1 mM
sodium orthovanadate, 1 µM okadaic acid). Lysates (450 µl) were incubated with 20 µl of protein A-Sepharose CL-4B (Amersham Pharmacia Biotech), supernatants were incubated with 4 µg
of anti-Flag M5 (IBI Kodak) for 1 h, and 20 µl of protein A-Sepharose CL-4B beads were added overnight at 4 °C with gentle agitation. The beads were washed four times with 1 ml of lysis buffer
and were eluted by boiling for 5 min with 5 µl of 6×
SDS-polyacrylamide gel electrophoresis sample buffer. The proteins were
analyzed by 10% SDS-polyacrylamide gel electrophoresis and were
transferred electrophoretically to nitrocellulose membranes. The
membranes were incubated for 1 h at room temperature with 1 µg/ml anti-HA-biotin (Boehringer Mannheim), washed 4 × with
Tris-buffered saline:0.05% Tween, and incubated with 0.2 units/ml
streptavidin-peroxidase (Boehringer Mannheim). Bound antibody was
detected using the enhanced chemiluminescence system (Amersham
Pharmacia Biotech).
Transcriptional Control--
HepG2 hepatocellular
carcinoma cells (ATCC) were grown in Dulbecco's modified Eagle's
medium (DMEM) containing 20 mM HEPES, pH 7.4, 6 mM NaHCO3, 10% fetal bovine serum. For
transfection, cells were seeded at 1 × 105
cells/24-mm well in 12-well tissue culture dishes. Cells were transfected 36 h after plating by a calcium phosphate method (23), using 100 ng of p3TP-Lux (24), derived from the promoter for plasminogen activator inhibitor-1, 100 ng of the pCH110
-galactosidase reporter gene, driven by the SV40 early promoter
(Amersham Pharmacia Biotech) (25), 0-600 ng of
pFlag-
TRAP-1-CMV2, and 0-100 ng of a CMV-driven T
R-I
L193A,P194A,T204D expression vector (9). Cells were fed 0.5 ml of DMEM with 10% fetal bovine serum 1 h before transfection.
Calcium phosphate-DNA precipitates were formed by slowly mixing 20 µl
of 50 mM HEPES, pH 7.1, 280 mM NaCl,
Na2HPO4·7 H20 with 20 µl of 125 mM CaCl2 containing 0.8 µg of DNA. Cells were
incubated with DNA precipitates (40 µl/well) for 5 h and were
cultured overnight in DMEM with 10% fetal bovine serum. Medium was
replaced on the following day by DMEM with 0.03% fetal bovine serum in
the absence or presence or 1 ng/ml TGF
1 (R & D Systems). Cells
were harvested 24 h later, and the luciferase activity and
-galactosidase activity were measured. For all comparisons, total
DNA and promoter content were kept constant using equivalent amounts of
vector. Luciferase activity was corrected for the internal constitutive
lacZ plasmid, and results (mean ± S.E.) are expressed
relative to expression in vehicle-treated, vector-transfected cells
(six to nine cultures for each condition, from two or three independent
experiments). Results were compared by analysis of variance and
Scheffe's test using a significance level of p < 0.01.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interaction Cloning of a Novel Protein, TRAP-1, Using Activated
TR-I as Bait--
A human lymphocyte cDNA library was screened
using T
R-IL193A,P194A,T204D as bait in the yeast
two-hybrid system (2.4 × 106 colonies). Unlike
previously reported screens using wild-type T
R-I, where FKBP12 was
the predominant protein detected, none of the resulting colonies
encoded FKBP12, as expected from the obligatory role of the Leu-Pro
dipeptide. Five clones corresponded to FNTA, which previously was shown
to interact with both T
R-I and T204D-T
R-I (4). Two proteins were
novel, designated
T
R-I-associated protein-1 and -2 (TRAP-1 and
TRAP-2; Fig. 1). To define the binding specificity of TRAP-1 and TRAP-2, we employed the yeast mating assay to
ensure co-introduction of plasmids encoding both chimeric proteins into
a single yeast host (22). Yeast Y190 cells containing GAL4-BD-TRAP-1 or
GAL4-BD-TRAP-2 plasmids were mated with yeast Y187 cells containing the
"bait" (GAL4-AD-T
R-IL193A,P194A,T204D) or other test
cDNAs, in frame with the GAL4 activator domain. In this assay
system, interaction between two hybrid proteins induces two
GAL4-dependent genes, HIS3 and lacZ,
which allows both growth on histidine-deficient plates and induction of
-galactosidase. By both criteria, TRAP-1 and TRAP-2 interacted
specifically with T
R-IL193A,P194A,T204D but not with
wild-type T
R-I, T
R-II, or other irrelevant proteins (Fig.
2A).
|
|
TRAP-1 Selectively Binds Active Forms of TR-I in Yeast
Two-hybrid Assays--
Because three amino acid substitutions were
incorporated in our T
R-I bait, to establish which amino acid(s)
conferred this interaction, the associations between TRAP-1 and TRAP-2
and each individual mutation of T
R-I were tested. TRAP-1 also bound
the T
R-IT204D kinase domain, containing the substitution
that activates T
R-I signaling; TRAP-1 did not interact with
T
R-IL193A or T
R-IP194A (Fig.
2B). Thus, activation of T
R-I, not disruption of FKBP12 binding, was responsible for the conditional binding of TRAP-1. In
contrast, TRAP-2 only interacted with
T
R-IL193A,P194A,T204D but not T
R-IT204D
or other single mutations. For this reason, TRAP-1 was selected for
more extensive analysis.
|
TRAP-1 Selectively Binds Ligand-activated TR-I in Mammalian
Cells--
To confirm the prediction that TRAP-1 might also associate
specifically with activated T
R-I in mammalian cells, the
receptor-binding portion of TRAP-1 isolated in the yeast two-hybrid
screen was epitope-tagged at its N terminus using the FLAG sequence and
was cotransfected into human 293 cells together with wild-type T
R-I or the activated mutation, T
R-IL193A,P194A,T204D, each
incorporating the Hemophilus influenzae hemagglutinin (HA) tag. Two days following transfection, the cells were incubated with
TGF
1 or the vehicle. To recover TRAP-1 itself, plus associated proteins, lysates were immunoprecipitated with antibody to the FLAG
epitope and then were blotted with antibody to the HA tag (Fig.
4). The constitutively activated
receptor, T
R-IL193A,P194A,T204D, was coprecipitated with
TRAP-1 even in the absence of TGF
1. However, wild-type T
R-I-HA
was coprecipitated only from TGF
-treated cells. This preferential
binding to the ligand-activated receptor contrasts markedly with FKBP12
and farnesyltransferase, both of which were released from T
R-I in
the presence of TGF
1 (5). Thus, unlike
these proteins, TRAP-1 is recruited selectively to the active forms of
T
R-I.
|
|
TRAP-1 Can Inhibit TGF-dependent
Transcription--
To ascertain whether binding of TRAP-1 to the
activated receptor might have functional consequences, HepG2 cells were
transfected with the TGF
-responsive p3TP-Lux reporter gene, an
internal lacZ control, and 0-600 ng of the partial TRAP-1
expression vector (Fig. 5). T
R-IL193A,P194A,T204D was
sufficient to up-regulate expression more than 30-fold (33.5 ± 3.4; p = 0.0001 versus vector-transfected
cells). Co-transfection with increasing amounts of the
TRAP-1
expression vector progressively decreased the induction of p3TP-Lux.
Using 600 ng of this TRAP-1 construct, only 12-fold induction was seen
(12.2 ± 1.4; p = 0.0001 versus the
absence of TRAP-1). Analogous results were seen following stimulation
of endogenous type I receptor with 1 ng/ml TGF
1. Expression
increased to 6.17 ± 0.46, relative to vehicle-treated cells
(p = 0.0001), and this induction likewise was inhibited by
TRAP-1 (2.51 ± 0.24, using 600 ng; p = 0.0001 versus the absence of TRAP-1). Thus, a partial TRAP-1
cDNA, which is sufficient to recognize the activated receptor
selectively, can function as an inhibitor of
TGF
-dependent gene transcription.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ligand-induced receptor homodimerization, causing reciprocal
cross-phosphorylation in trans, is a strategy commonly
employed by receptor tyrosine kinases for transmembrane signaling (27). Phosphorylated tyrosine residues of the receptor, in turn, recruit SH2
domain-containing molecules that are required for signaling. For
receptor serine/threonine kinases and the TGF superfamily, the
signal transduction pathway is similar but distinct; TGF
ligands
induce the heterodimerization of T
R-II and T
R-I, whereupon T
R-II phosphorylates T
R-I on serine and threonine residues of its
Gly/Ser-rich domain. This directional phosphorylation event and the
kinase activity of both receptors functioning sequentially are required
for signal generation (1). Conversely, we and others have shown that
negatively charged amino acids can serve as surrogates for the
phosphorylation of T
R-I and constitutively activate the receptor
even in the absence of T
R-II (9, 28).
According to this model, TR-I, being the substrate for T
R-II,
should interact with downstream mediators and effectors. Therefore, it
has been discouraging that interaction cloning using the T
R-I cytoplasmic domain as bait has not succeeded to date in identifying specific molecules that confer the T
R-I signal (3, 5, 7, 29). Based
on precedents with receptor tyrosine kinases, one foreseeable
explanation for this failure may be that some effector molecules acting
downstream of T
R-I might associate preferentially with
phosphorylated (active) T
R-I rather than with the unphosphorylated (inactive) T
R-I used as bait in previously reported cloning efforts. Here, we have used T
R-IL193A,P194A,T204D as bait, which
has two potentially advantageous properties for this purpose: being
autonomously active and not binding FKBP12. Beyond its ability to
distinguish activated from wild-type T
R-I in yeast two-hybrid
assays, the novel protein, TRAP-1, binds only mutationally activated or
ligand-activated T
R-I in mammalian cells not the inactive type I
receptor, reminiscent of the signaling tactics used by receptor
tyrosine kinases. This novel protein is selective in binding active
forms of T
R-I, which favors its involvement in the TGF
cascade.
Similar binding specificity recently was demonstrated for Smad7, which
preferentially associates with active T
R-I, preventing receptor
association with and phosphorylation of Smad2 (30). Thus, intracellular
antagonists as well as mediators might be identified by use of the
activated receptor as bait. Indeed, at least for Smad proteins, only
the inhibitory forms like Smad7 have been shown to bind stably to the
activated receptor. In keeping with this model, the C-terminal portion
of TRAP-1 (which is sufficient for stable interaction with the
activated receptor both in yeast and in mammalian cells) was found to
inhibit TGF
signaling, as measured by the p3TP-Lux reporter gene.
This does not preclude a more complex role for the full-length protein, which we have not yet expressed, or address the status of TGF
signaling in the absence of TRAP-1. Further work is required to validate the suggested function of TRAP-1 in T
R-I signaling via Smad, TAK1, or an alternative pathway.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge C.-H. Heldin, H. Lodish, J. Massagué, A. Marks, and K. Miyazono for reagents cited, B. Boerwinkle and F. Ervin for technical assistance, and R. Roberts for encouragement and support.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants R01 HL47567, R01 HL52555, P01 HL49953, and P50 HL42267 (to M. D. S.).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) AF022795.
¶ Present address: Dept. of Medicine, Veterans General Hospital-Taipei, and National Yang-Ming University, Taipei, Taiwan.
Present address: Dept. of Pediatrics, University of
California, San Francisco, CA.
To whom correspondence should be addressed: Molecular
Cardiology Unit, One Baylor Plaza, Rm. 506C, Baylor College of
Medicine, Houston, TX 77030. Tel.: 713-798-6683; Fax: 713-798-7437;
E-mail: michaels{at}bcm.tmc.edu.
1
The abbreviations used are: TGF, transforming
growth factor
; FKBP12, FK506-binding protein 12; FNTA,
subunit
of farnesyltransferase; RACE, 5'-rapid amplification of cDNA ends;
SD, synthetic dropout; Smad, MAD-related protein; T
R-I, type I
TGF
receptor; T
R-II, type II TGF
receptor; TRAP-1,
T
R-I-associated protein-1; 3-AT, 3-amino-1,2,4-triazole; bp, base
pair(s); DMEM, Dulbecco's modified Eagle's medium; HA,
hemagglutinin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|