COMMUNICATION
A Novel Protein Distinguishes between Quiescent and Activated Forms of the Type I Transforming Growth Factor beta  Receptor*

Min-Ji CharngDagger §, Dou ZhangDagger §, Païvi KinnunenDagger par , and Michael D. SchneiderDagger §**Dagger Dagger

From the Molecular Cardiology Unit, Dagger  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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Transforming growth factor beta  (TGFbeta ) signal transduction is mediated by two receptor Ser/Thr kinases acting in series, type II TGFbeta receptor (Tbeta R-II) phosphorylating type I TGFbeta receptor (Tbeta R-I). Because the failure of interaction cloning, thus far, to identify bona fide Tbeta R-I substrates might reasonably have been due to the use of inactive Tbeta R-I as bait, we sought to identify molecules that interact specifically with active Tbeta 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 TGFbeta signaling we have previously disproved; the charge substitution at Thr204 constitutively activates Tbeta R-I. Unlike previous screens using wild-type Tbeta R-I, where FKBP12 predominated, none of the resulting colonies encoded FKBP12. A novel protein was identified, Tbeta R-I-associated protein-1 (TRAP-1), that interacts in yeast specifically with mutationally activated Tbeta R-I, but not wild-type Tbeta R-I, Tbeta R-II, or irrelevant proteins. In mammalian cells, TRAP-1 was co-precipitated only by mutationally activated Tbeta R-I and ligand-activated Tbeta R-I, but not wild-type Tbeta R-I in the absence of TGFbeta . The partial TRAP-1 protein that specifically binds these mutationally and ligand-activated forms of Tbeta R-I can inhibit signaling by the native receptor after stimulation with TGFbeta or by the constitutively activated receptor mutation, as measured by a TGFbeta -dependent reporter gene. Thus, TRAP-1 can distinguish activated forms of the receptor from wild-type receptor in the absence of TGFbeta and may potentially have a functional role in TGFbeta signaling.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The signal transduction events coupling receptors for the TGFbeta 1 superfamily to TGFbeta -dependent responses remain incompletely understood. TGFbeta signaling requires two transmembrane receptors acting in series, Tbeta R-II phosphorylating Tbeta 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 Tbeta R-I. These include both the immunophilin-binding protein, FKBP12 (3), a target for the macrolides FK506 and rapamycin, and the alpha  subunit of Ras farnesyltransferase (FNTA) (4, 5). Tbeta 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 TGFbeta 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 Tbeta R-I; FNTA likewise is unnecessary for TGFbeta signaling (11). However, genetic screening in Drosophila identified the transcription factor, mothers against decapentaplegic (MAD), as acting downstream from the TGFbeta homologue, decapentaplegic (12). In vertebrates, multiple MAD-related proteins exist (Smads) that are thought to mediate signaling by TGFbeta family members via phosphorylation-dependent nuclear translocation (13-17). TGFbeta 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 TGFbeta -activated kinase, TAK1, was identified as an alternative mediator of TGFbeta signaling, which may be necessary for at least a subset of TGFbeta 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 TGFbeta 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 Tbeta R-I, employing Tbeta R-IL193A,P194A,T204D as the bait. The charged amino acid substitution at Thr204 confers constitutive activity in the absence of ligand and Tbeta 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 Tbeta R-I and Tbeta R-I that is activated in the presence of TGFbeta .

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Interaction Cloning and Two-hybrid Assays in Yeast-- Constructions containing the cytoplasmic domains of Tbeta R-I and Tbeta R-II or point mutations of Tbeta R-I in the yeast expression plasmid pAS2-1 were previously described (9). The cytoplasmic domain of Tbeta 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 beta -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.

To obtain a more complete TRAP-1 cDNA, the 3' partial sequence was used to screen a human heart cDNA library in lambda gt10 (CLONTECH); 50,000 plaques were plated per 150-mm dish. Duplicate filters were hybridized with 32P-labeled TRAP-1 cDNA (Rediprime DNA labeling kit, Amersham Pharmacia Biotech) in 7% SDS, 0.5 M NaH2PO4, and 1 mM EDTA at 55 °C overnight. Filters were washed twice in 2 × SSC and 0.05% SDS at room temperature for 15 min and then in 0.1 × SSC and 0.1% SDS twice at 55 °C for 15 min. Filters were subjected to autoradiography. Purification of positive clones was done as described (20). Inserts were subcloned into pBluescript SK (Strategene) and sequenced. The GenBankTM accession number for TRAP-1 is AF022795.

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 (MATalpha ) was transformed with wild-type Tbeta R-I, point mutations of Tbeta R-I, or Tbeta 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 beta -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-Delta 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 Tbeta R-I-HA-pCMV5, 8 µg of pFlag-Delta TRAP-1-CMV2, 8 µg of Tbeta R-II-SV-Sport, and 45 µl of LipofectAMINE (Life Technologies, Inc.). Human recombinant TGFbeta 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 beta -galactosidase reporter gene, driven by the SV40 early promoter (Amersham Pharmacia Biotech) (25), 0-600 ng of pFlag-Delta TRAP-1-CMV2, and 0-100 ng of a CMV-driven Tbeta 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 TGFbeta 1 (R & D Systems). Cells were harvested 24 h later, and the luciferase activity and beta -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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Interaction Cloning of a Novel Protein, TRAP-1, Using Activated Tbeta R-I as Bait-- A human lymphocyte cDNA library was screened using Tbeta R-IL193A,P194A,T204D as bait in the yeast two-hybrid system (2.4 × 106 colonies). Unlike previously reported screens using wild-type Tbeta 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 Tbeta R-I and T204D-Tbeta R-I (4). Two proteins were novel, designated Tbeta 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-Tbeta 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 beta -galactosidase. By both criteria, TRAP-1 and TRAP-2 interacted specifically with Tbeta R-IL193A,P194A,T204D but not with wild-type Tbeta R-I, Tbeta R-II, or other irrelevant proteins (Fig. 2A).


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 1.   Features of the presumptive full-length TRAP-1 sequence. Putative phosphorylation sites are denoted for protein kinase C (underlined), casein kinase 2 (double underlined), and protein kinase A (bold); putative myristylation sites are indicated by dotted lines.


View larger version (49K):
[in this window]
[in a new window]
 
Fig. 2.   TRAP-1 associates selectively with activating mutations of Tbeta R-I. The specificity for interaction of TRAP-1 and TRAP-2 with Tbeta R-I, mutations of Tbeta R-I, and control proteins was assessed by the yeast mating assay. Yeast strain Y190 was transformed with TRAP-1 or TRAP-2 in pAS2-1 and was mated with yeast strain Y187 carrying wild-type or mutant Tbeta R-I, Tbeta R-II, lamin C, or p53 in pACT2. In A and B, mating cultures were plated on nonselective medium (SD/-Leu/-Trp, a and d) to confirm the presence of both plasmids and on selective medium (SD/-Leu/-Trp/-His/+3-AT; b and e) to assess protein-protein interaction by two-hybrid induction of the HIS3 gene; lacZ activity was determined by a colony lift X-gal assay on filter replicas of the SD/-Leu/-Trp plates (c and f).

TRAP-1 Selectively Binds Active Forms of Tbeta R-I in Yeast Two-hybrid Assays-- Because three amino acid substitutions were incorporated in our Tbeta 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 Tbeta R-I were tested. TRAP-1 also bound the Tbeta R-IT204D kinase domain, containing the substitution that activates Tbeta R-I signaling; TRAP-1 did not interact with Tbeta R-IL193A or Tbeta R-IP194A (Fig. 2B). Thus, activation of Tbeta R-I, not disruption of FKBP12 binding, was responsible for the conditional binding of TRAP-1. In contrast, TRAP-2 only interacted with Tbeta R-IL193A,P194A,T204D but not Tbeta R-IT204D or other single mutations. For this reason, TRAP-1 was selected for more extensive analysis.

To obtain the full-length coding sequence of TRAP-1, the 3' 1399-bp partial cDNA was used to screen a human heart cDNA library, and the 5' sequence of TRAP-1 was obtained by performing 5'-RACE on poly(A)+ RNA from human heart. The full-length cDNA was 3100 bp in length, comprising 860 amino acid residues (Fig. 1). An in-frame stop codon (TAA) was 153 bp upstream from the ATG start codon and Kozak consensus sequence (26). A search of GenBankTM using BLAST revealed no homologous proteins; a search for potential functional motifs using the BLAST Enhanced Alignment Utility identified one potential phosphorylation site for protein kinase A, nine for protein kinase C, and eight for casein kinase 2, and seven potential myristylation sites. No other known structural motifs were identified. TRAP-1 mRNA was detected as two transcripts of 4.4 and 6 kilobases in all tissues examined, with lesser abundance in lung and liver (Fig. 3); the mechanism for generating the two transcripts remains to be determined.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 3.   Northern analysis of TRAP-1 mRNA. Blots containing poly(A)+ RNA from multiple human tissues were hybridized with the radiolabeled 3' 1399-bp TRAP-1 cDNA. Two transcripts of 4.4 and 6 kilobases were detected in all tissues examined.

TRAP-1 Selectively Binds Ligand-activated Tbeta R-I in Mammalian Cells-- To confirm the prediction that TRAP-1 might also associate specifically with activated Tbeta 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 Tbeta R-I or the activated mutation, Tbeta R-IL193A,P194A,T204D, each incorporating the Hemophilus influenzae hemagglutinin (HA) tag. Two days following transfection, the cells were incubated with TGFbeta 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, Tbeta R-IL193A,P194A,T204D, was coprecipitated with TRAP-1 even in the absence of TGFbeta 1. However, wild-type Tbeta R-I-HA was coprecipitated only from TGFbeta -treated cells. This preferential binding to the ligand-activated receptor contrasts markedly with FKBP12 and farnesyltransferase, both of which were released from Tbeta R-I in the presence of TGFbeta 1 (5). Thus, unlike these proteins, TRAP-1 is recruited selectively to the active forms of Tbeta R-I.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4.   TRAP-1 specifically binds ligand-activated Tbeta R-I. Human 293 cells transfected with epitope-tagged Tbeta R-I and Delta TRAP-1 constructs were incubated with vehicle versus TGFbeta 1. Cells were lysed, antibody to the FLAG epitope was added to immunoprecipitate (IP) TRAP-1 and associated proteins, immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis, and TRAP-1-associated Tbeta R-I was detected with anti-HA antibody. Wt, wild type.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5.   A receptor-binding fragment of TRAP-1 inhibits TGFB signaling. HepG2 cells were transfected with p3TP-Lux, the constitutive lacZ gene, and increasing amounts of the Delta TRAP-1 expression vector. p3TP-Lux expression was triggered using the constitutively activated receptor, Tbeta R-IL193A,P194A,T204D (A) or 1 ng/ml TGFbeta 1 without exogenous receptor (B). Results in A and B are shown relative to expression in vector-transfected and vehicle-treated cultures (n = 9 and 6, respectively). * and **, p = 0.0001 and p = 0.0002, in comparison with the absence of TRAP-1.

TRAP-1 Can Inhibit TGFbeta -dependent Transcription-- To ascertain whether binding of TRAP-1 to the activated receptor might have functional consequences, HepG2 cells were transfected with the TGFbeta -responsive p3TP-Lux reporter gene, an internal lacZ control, and 0-600 ng of the partial TRAP-1 expression vector (Fig. 5). Tbeta 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 Delta 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 TGFbeta 1. Expression increased to 6.17 ± 0.46, relative to vehicle-treated cells (p = 0.0001), and this induction likewise was inhibited by Delta 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 TGFbeta -dependent gene transcription.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 TGFbeta superfamily, the signal transduction pathway is similar but distinct; TGFbeta ligands induce the heterodimerization of Tbeta R-II and Tbeta R-I, whereupon Tbeta R-II phosphorylates Tbeta 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 Tbeta R-I and constitutively activate the receptor even in the absence of Tbeta R-II (9, 28).

According to this model, Tbeta R-I, being the substrate for Tbeta R-II, should interact with downstream mediators and effectors. Therefore, it has been discouraging that interaction cloning using the Tbeta R-I cytoplasmic domain as bait has not succeeded to date in identifying specific molecules that confer the Tbeta 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 Tbeta R-I might associate preferentially with phosphorylated (active) Tbeta R-I rather than with the unphosphorylated (inactive) Tbeta R-I used as bait in previously reported cloning efforts. Here, we have used Tbeta 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 Tbeta R-I in yeast two-hybrid assays, the novel protein, TRAP-1, binds only mutationally activated or ligand-activated Tbeta 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 Tbeta R-I, which favors its involvement in the TGFbeta cascade. Similar binding specificity recently was demonstrated for Smad7, which preferentially associates with active Tbeta 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 TGFbeta 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 TGFbeta signaling in the absence of TRAP-1. Further work is required to validate the suggested function of TRAP-1 in Tbeta 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.

par Present address: Dept. of Pediatrics, University of California, San Francisco, CA.

Dagger Dagger 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: TGFbeta , transforming growth factor beta ; FKBP12, FK506-binding protein 12; FNTA, alpha  subunit of farnesyltransferase; RACE, 5'-rapid amplification of cDNA ends; SD, synthetic dropout; Smad, MAD-related protein; Tbeta R-I, type I TGFbeta receptor; Tbeta R-II, type II TGFbeta receptor; TRAP-1, Tbeta 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
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F., and Massagué, J. (1994) Nature 370, 341-347[CrossRef][Medline] [Order article via Infotrieve]
  2. Massagué, J. (1996) Cell 85, 947-950[Medline] [Order article via Infotrieve]
  3. Wang, T. W., Donahoe, P. K., and Zervos, A. S. (1994) Science 265, 674-676[Medline] [Order article via Infotrieve]
  4. Kawabata, M., Imamura, T., Miyazono, K., Engel, M. E., and Moses, H. L. (1995) J. Biol. Chem. 270, 29628-29631[Abstract/Free Full Text]
  5. Wang, T. W., Danielson, P. D., Li, B. Y., Shah, P. C., Kim, S. D., and Donahoe, P. K. (1996) Science 271, 1120-1122[Abstract]
  6. Chen, R. H., Miettinen, P. J., Maruoka, E. M., Choy, L., and Derynck, R. (1995) Nature 377, 548-552[CrossRef][Medline] [Order article via Infotrieve]
  7. Wang, T. W., Li, B. Y., Danielson, P. D., Shah, P. C., Rockwell, S., Lechleider, R. J., Martin, J., Manganaro, T., and Donahoe, P. K. (1996) Cell 86, 435-444[Medline] [Order article via Infotrieve]
  8. Chen, Y. G., Liu, F., and Massagué, J. (1997) EMBO J. 16, 3866-3876[Abstract/Free Full Text]
  9. Charng, M.-J., Kinnunen, P., Hawker, J., Brand, T., and Schneider, M. D. (1996) J. Biol. Chem. 271, 22941-22944[Abstract/Free Full Text]
  10. Okadome, T., Oeda, E., Saitoh, M., Ichijo, H., Moses, H. L., Miyazono, K., and Kawabata, M. (1996) J. Biol. Chem. 271, 21687-21690[Abstract/Free Full Text]
  11. Ventura, F., Liu, F., Doody, J., and Massague, J. (1996) J. Biol. Chem. 271, 13931-13934[Abstract/Free Full Text]
  12. Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H., and Gelbart, W. M. (1995) Genetics 139, 1347-1358[Abstract/Free Full Text]
  13. Massagué, J., Hata, A., and Liu, F. (1997) Trends Cell Biol. 7, 187-192[CrossRef]
  14. Lagna, G., Hata, A., Hemmati-Brivanlou, A., and Massagué, J. (1996) Nature 383, 832-836[CrossRef][Medline] [Order article via Infotrieve]
  15. Zhang, Y., Feng, X. H., Wu, R. Y., and Derynck, R. (1996) Nature 383, 168-172[CrossRef][Medline] [Order article via Infotrieve]
  16. Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224[Medline] [Order article via Infotrieve]
  17. Kretzschmar, M., Liu, F., Hata, A., Doody, J., and Massagué, J. (1997) Genes Dev. 11, 984-995[Abstract]
  18. Yamaguchi, K., Shirakabe, T., Shibuya, H., Irie, K., Oishi, I., Ueno, N., Taniguchi, T., Nishida, E., and Matsumoto, K. (1995) Science 270, 2008-2011[Abstract]
  19. Gietz, R. D., and Schiestl, R. H. (1991) Yeast 7, 253-263[Medline] [Order article via Infotrieve]
  20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  21. Gubler, U., and Hoffman, B. J. (1983) Gene (Amst.) 25, 263-269[Medline] [Order article via Infotrieve]
  22. Bendixen, C., Gangloff, S., and Rothstein, R. (1994) Nucleic Acids Res. 22, 1778-1779[Medline] [Order article via Infotrieve]
  23. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994) Current Protocols in Molecular Biology, John A. Wiley & Sons. Inc., New York
  24. Wrana, J. L., Attisano, L., Carcamo, J., Zentella, A., Doody, J., Laiho, M., Wang, X. F., and Massague, J. (1992) Cell 71, 1003-1014[Medline] [Order article via Infotrieve]
  25. Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., and Davis, R. J. (1996) Mol Cell Biol 16, 1247-1255[Abstract]
  26. Kozak, M. (1986) Cell 44, 283-292[Medline] [Order article via Infotrieve]
  27. Pawson, T. (1995) Nature 373, 477-478[CrossRef][Medline] [Order article via Infotrieve]
  28. Wieser, R., Wrana, J. L., and Massagué, J. (1995) EMBO J. 14, 2199-2208[Abstract]
  29. Kawabata, M., Chytil, A., and Moses, H. L. (1995) J. Biol. Chem. 270, 5625-5630[Abstract/Free Full Text]
  30. Hayashi, H., Abdollah, S., Qiu, Y. B., Cai, J. X., Xu, Y. Y., Grinnell, B. W., Richardson, M. A., Topper, J. N., Gimbrone, M. A., Wrana, J. L., and Falb, D. (1997) Cell 89, 1165-1173[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.