Interactome of Transforming Growth Factor-β Type I Receptor (TβRI): Inhibition of TGFβ Signaling by Epac1 Paolo Conrotto, Ihor Yakymovych, Mariya Yakymovych, and Serhiy Souchelnytskyi* Ludwig Institute for Cancer Research, Uppsala University, Box 595, BMC, SE-751 24, Uppsala, Sweden Received August 22, 2006
Transforming growth factor-β (TGFβ) is a potent regulator of cell growth, differentiation, and apoptosis. Type I TGFβ receptor (TβRI) is the key receptor for initiation of intracellular signaling by TGFβ. Here we report proteomics-based identification of proteins that form a complex with TβRI. Using 2D-GE and MALDI TOF mass spectrometry, we identified 16 proteins that specifically interacted with a GST-fused TβRI Thr204Asp construct with constitutively active serine/threonine kinase. We confirmed interactions of the receptor with cAMP regulated guanine nucleotide exchange factor 1 (Epac1), R-spectrin, PIASy, and R-catenin proteins using immunoblotting. Interaction of the receptor with Epac1 required intact kinase activity of TβRI but was not affected by deletion of cAMP-binding domain of Epac1. TGFβ1induced C-terminal phosphorylation of Smad2 was inhibited in vivo and in vitro in the presence of Epac1. Epac1 inhibited also TGFβ1/TβRI-dependent transcriptional activation, as evaluated by luciferase reporter assays. We observed that expression of Epac1 counteracted TGFβ/TβRI-dependent decrease of cell adhesion and TGFβ/TβRI-induced stimulation of cell migration. Thus, we have reported novel TβRI-interacting proteins and have shown that Epac1 inhibited TGFβ-dependent regulation of cell migration and adhesion. Keywords: proteomics • TGFβ • TβRI • Epac1 • transcription • migration • adhesion
Introduction TGFβ regulates cell proliferation, migration, differentiation, and apoptosis.1,2 It binds to type II and type I (TβRI) receptors, which leads to formation of a heterotetrameric receptor complex. In this complex, type I receptor kinase is activated, and initiates signaling by phosphorylation of specific substrates. Inactivation of the kinase of TβRI abrogates the signaling, emphasizing the importance of the type I receptor for cell responsiveness to TGFβ.1-3 A number of proteins have been identified in complexes with TβRI.1-14 Searches for TβRI interacting proteins have been performed by studying selected proteins in coprecipitation assays, or by binary screens of protein arrays, e.g., using yeast two-hybrid screens.4,5,8 Identified receptor-interacting proteins provide physical connections of TβRI to various regulatory processes. TβRI has been found to form complexes with STRAP,9 PP2A BR ,10 TRAP-1,11 FKBP12,12 R-farnesyltransferase,13 and PI3 kinase .14 TβRI formed a complex with type II TGFβ receptor, Smad6, Smad7, Smad2 and Smad3, as well as via Smad proteins with a number of Smad-interacting proteins.1-4 The interaction of TβRI with FKBP12 is claimed to decrease receptor kinase activity,12 whereas interactions with Smad2 and Smad3 promote initiation of gene transcription.1,2,15 These data indicate that TβRI acts in complexes with other proteins that significantly modulate the intensity of signaling and types of * To whom correspondence should be addressed. Tel: +46 (0)18 16 04 11; Fax: +46 (0)18 16 04 20; E-mail:
[email protected]. 10.1021/pr060427q CCC: $37.00
2007 American Chemical Society
responses. Despite a number of studies,1-5 many of the TβRI effects on cells, e.g., cytoskeleton rearrangements, transport, energy and metabolism regulation, are not fully understood and still require unveiling of complete molecular mechanisms. GST-fusion proteins have been shown to be a useful tool for the detection of interacting proteins. In relation to TGFβ family signaling, GST pull-downs followed by proteomics-based analysis of interacting proteins have been performed for Smad3 and BMP type II receptor (BMPRII).6,7 These interacting proteins may provide links of TGFβ and BMP signaling to the variety of regulatory pathways. As an example, the identification of the interaction between BMPRII and c-Kit receptor introduced a novel paradigm in signaling, e.g., that serine/threonine kinase receptors may form a complex with tyrosine kinase receptors.16 Recent developments in proteomics allow large scale analysis of protein-protein interactions, with identification of 10 or more proteins in a single complex being rather routine than an exception.17,18 This breakthrough has been possible due to separation capabilities of two-dimensional gels and high sensitivity of mass spectrometry in protein identification. We describe here proteomics-based identification of 16 proteins that form a complex with the activated TβRI. We show also that one of the interacting proteins, Epac1, inhibited TGFβdependent regulation of cell adhesion and migration.
Materials and Methods Cells, Reagents, and DNA Constructs. The human breast cancer cell line MCF-7, mink lung epithelial cells Mv1Lu, and Journal of Proteome Research 2007, 6, 287-297
287
Published on Web 12/15/2006
research articles
Figure 1. Characterization of GST-TβRIca and GST constructs used in this work. (A) Proteins were expressed in bacteria, purified, and separated in SDS-PAGE. An image of a Coomassie Brilliant Blue-stained gel is shown. Arrows indicate migration positions of GST and GST-fusion with the cytoplasmic part of TβRI with the Thr204 substituted with aspartic acid (GST-TβRIca). GST-TβRIca was observed as a double band, and some GST was also detected in GST-TβRIca preparations. Arrowheads indicate migration positions of molecular mass markers. (B) Schematic presentation of full-length TβRI, GST-TβRIca, and GST alone. TM, transmembrane domain; GS, glycine-serine rich region which is involved in activation of TβRI kinase; KD, kinase domain.
the human embryonic kidney cells 293T were obtained from ATCC and cultured in 10% fetal bovine serum (Biowest, Nuaille, France), 100 U/mL penicillin, and 100 µg/mL streptomycin. The R4.2 clone of Mv1Lu cells, which is deficient in TβRI, was obtained from Dr. Massague´. Monoclonal anti-HA antibody was purchased from Roche (Basel, Switzerland), anti-Flag M5 antibody was from SigmaAldrich, and antibodies against CREB and phospho-CREB (pSer133) were obtained from Cell Signaling Technology (MA). A rabbit polyclonal (RK2) EGFR antiserum was a gift of Dr. Schlessinger; antisera against phospho-Smad2 (pS2) and TβRI (VPN) were described earlier.19,20 Antibodies against Epac1, PIASy, R-catenin, R-spectrin and c-Kit were obtained from Santa Cruz (CA). The cAMP analogue 8-pCPT-2-O-Me-cAMP was purchased from BioLog-life science institute (Bremen, Germany); human TGFβ1 was from PeproTech EC (London, UK). The constitutive active (ca) GST-TβRI consists of the complete cytoplasmic portion (amino acid residues 148-503) of constitutively active TβRI Thr204Asp mutant fused in frame with GST and inserted into the pGEX4T-1 vector (GE Healthcare-Amersham Pharmacia, Uppsala, Sweden).21 Wild-type (WT) TβRI, TβRIca and kinase dead (KD) TβRI Lys232Arg mutant were subcloned in pCDNA3 vector. Epac1∆ construct has a deletion of the cAMP-binding domain (base pairs 17773453). Full-length Epac1 and Epac1∆ constructs were obtained from Dr. Mochizuki, and GST-Epac constructs were obtained from Dr. Kuhlmann. Metabolic Labeling. For metabolic labeling, cells were grown overnight in cysteine- and metionine-free MCDB 104 medium with 0.1% FBS, in the presence of 1 µCi/mL Redivue Pro-Mix, which contained [35S]methionine/[35S]cysteine (GE HealthcareAmersham Biosciences, Uppsala, Sweden). After labeling, cells 288
Journal of Proteome Research • Vol. 6, No. 1, 2007
Conrotto et al.
were lysed in a lysis buffer (1% Triton X-100, 50 mM Tris, pH 8.0, 150 mM NaCl, and Complete Protease Inhibitor Cocktail (Roche AB, Stockholm, Sweden)) and centrifuged at 13 000 rpm for 15 min, and extracts were used for GST pull-down. GST Purification and Pull-Down. GST alone and GSTTβRIca were expressed in E.coli BL21. After growing in LB medium, bacteria where chilled on ice for 10 min, centrifuged and re-suspended in STE buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 100 µg/mL lysozyme, 5 mM dithiothreitol (DTT), 1% trazylol). Cells were then sonicated and incubated on ice in a STE buffer with 1% Triton X-100. The extract was filtered through a 0.45 µm filter and was incubated for 2 h at 4 °C with Glutathione-Sepharose beads (GE Healthcare-Amersham Biosciences, Uppsala, Sweden). Beads were then washed 2 times with 1% Triton X-100 in STE solution, 2 times with 0.5 M NaCl, 10 mM Tris-HCl, pH 8.0, once with TBS and were used for pull-down experiments. The purity of prepared proteins was evaluated by SDS-PAGE. For pull-down assay, a lysis buffer (1% Triton X-100, 50 mM Tris, pH 8.0, 150 mM NaCl, and Complete Protease Inhibitor Cocktail (Roche AB, Stockholm, Sweden)) with or without cell extract was incubated for 2 h at 4 °C with equal amount of GST or GST-TβRIca bound to Glutathione-Sepharose beads. Beads were then washed 5 times with a lysis buffer and 2 times with 20 mM Tris-HCl, pH 7.4. Before two-dimensional (2-D) gel electrophoresis, the proteins bound to GST and GST-TβRIca on beads were re-suspended in a 2-D-sample buffer (8 M urea, 4% CHAPS, 10 mM DDT, IPG buffer pH 3-10). 2-D Gel Electrophoresis. Samples were subjected to isoelectric focusing (IEF) using IPGDryStrips with immobilized pH gradient, pH range 3-10, 18 cm, linear (GE HealthcareAmersham Biosciences, Uppsala, Sweden). Samples were applied by in-gel rehydration technique. IEF was performed in an IPGphor (GE Healthcare-Amersham Biosciences, Uppsala, Sweden) according to the following protocol: rehydration, 10 h; 50 V, 3 h; 1000 V, 1 h; 8000 V, 10 h or to 50 000 Vh. After IEF, strips were equilibrated in 50 mM Tris-HCl, pH 8.8, 6 M urea, 2.0% SDS, 30% glycerol with 10 mM DTT for 10 min, and then for 10 min in the same buffer without DTT but with 20 mM iodoacetamide. Equilibrated strips were placed on the top of 10% polyacrylamide gels and fixed with 0.5% agarose in a concentrating buffer (62.5 mM Tris-HCl, pH 6.8, 0.1% SDS). SDS-PAGE was performed in a Dalt-Six (GE HealthcareAmersham Biosciences, Uppsala, Sweden), following the manufacturer’s recommendations (constant power 50 W, run for 6-8 h). Gels were fixed in 10% acetic acid and 20% methanol in water for 10-12 h. Gels were subjected to silver staining, dried, and exposed in a Fuji FLA-3000 phosphorimager (Fuji, Tokyo, Japan). In total, 20 gels with samples from five different experiments were prepared and subjected to analysis. Gel Analysis. Images of silver-stained gels and 35S-labeled proteins in 2-D gels were analyzed using Image Master 2D Platinum software (GE Healthcare-Amersham Biosciences, Uppsala, Sweden). Protein spots that appeared reproducibly in at least 4 out of 5 GST-TβRIca pull-down gels, which were 35 S-labeled and were not detected in gels of pull-down with GST alone, were selected for identification. Selection of spots was additionally controlled by manual inspection of gels. Protein Identification by Mass Spectrometry. Protein spots were excised from gels, de-stained, and subjected to in-gel digestion with trypsin (modified, sequence grade porcine; Promega, Falkenberg, Sweden). Tryptic peptides were concentrated and desalted on ZipTip (Millipore, USA). Peptide mass
TβRI-Interacting Proteins
research articles
Figure 2. Detection of proteins interacting with GST-TβRIca using 2-D gel electrophoresis. Representative images of silver-stained (A, B, E, F, G) and 35S-labeled proteins (C, D, H) are shown. Cellular proteins interacting with GST-TβRIca were separated in 2-D gels which were stained with silver (B, F) and detected after exposure in a phosphorimager (D; 35S-labeled). To detect proteins nonspecifically present in GST-TβRIca preparations, and which did not originate from mammalian cells, the GST-TβRIca preparation alone was subjected to 2D-GE, i.e., without incubation with extracts of mammalian cells (A, C, E). Enlarged images of inserts from silver stained 2-D gels of a control sample (E) and coprecipitated MCF-7 proteins (F) are shown. Panels B and F show proteins which were found to interact with GST-TβRIca. Annotation of protein spots is as in Table 1. Coprecipitation of proteins with GST alone was also analyzed (G, H). Isoelectrofocusing and direction of SDS-PAGE are indicated. Migration positions of molecular mass markers are indicated by arrows (B, D, E, H). Images of whole 2-D gels (A-D, G, H) or inserts (E, F) are shown. Representative gels out of 20 gels generated in 5 experiments, are shown. Journal of Proteome Research • Vol. 6, No. 1, 2007 289
research articles
Conrotto et al.
Table 1. List of Proteins Identified in a Complex with GST-TβRIca experimentalb spot no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
protein name
theoreticalb
pI
sequence coverage (%)b
no. of matching peptidesb
57.5 45.7 43.3 60.9 100.8 71.7 73.0 62.5
5.8 9.9 5.4 8.5 5.8 9.9 5.8 7.0
19 17 33 15 14 19 20 23
9 5 10 10 14 14 8 10
9.1
82.8
6.2
19
16
70
7.2
95.6
9.5
14
12
1.00
70
7.3
95.6
9.5
15
17
9.3 e - 1
0.58
70
8.8
84.7
9.2
20
16
7.5 e - 1 1.0 e + 0 1.0 e + 0 1.0 e + 0 1.0 e + 0
0.55 1.96 1.67 1.35 2.21
60 100 60 68 58
9.5 7.4 9.1 7.2 7.6
45.9 112.2 54.5 66.2 53.7
9.9 6.7 5.7 8.3 5.5
20 15 22 19 30
8 16 11 11 15
identification number gi no.a
probabilityb
Z valueb
Mr
pI
Mr
24850133 13899014 1709895 31544962 7019571 10047251 1177023 32394404
1.0 e + 0 1.3 e - 1 1.0 e + 0 9.6 e - 1 9.9 e - 1 8.6 e - 1 1.0 e + 0 1.0 e + 0
1.02 0.65 0.99 0.68 0.73 0.60 1.30 1.20
61 60 60 60 100 68 65 75
7.8 7.8 8.2 9.0 7.5 5.0 4.5 7.6
10439942
1.0 e + 0
1.57
70
27696046
1.0 e + 0
1.56
27696046
9.9 e - 1
2370078 20072160 20521019 189071 17318569 4504919
PIASy PIAS-NY cytoplasmic antiproteinase 2 Nedd4 binding protein 3 catenin alpha KIAA1588 Rho-GTPase-activating protein 25 cAMP regulated guanine nuclotide exchange factor 1 (Epac1) unnamed protein product (AAA domain-containing protein) unnamed protein product (AAA domain-containing protein) unnamed protein product (AAA domain-containin prot.) ortholog of Fugu SMC1(mytosis specific chromosome segregation protein) novel protein novel protein alpha-Spectrin cytokeratin 1 cytokeratin 8
a The “gi” number in NCBI protein database. b Probability, Z-value, sequence coverage, number of matching peptides, theoretical pI, and molecular mass were obtained by search with ProFound. Experimental values were calculated from migration positions in 2-D gels.
mL, 1 h) and/or with 8-pCPT (100 µM, 30 min). Then cells were lysed in a lysis buffer (125 mM NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 1.5% Trasylol, and 1% Triton X-100) for 20 min on ice. Cell lysates were immunoprecipitated with specific antibodies bound to protein-A Sepharose beads (Immunosorb; EC Diagnostics, Uppsala, Sweden) for 2 h at 4 °C. Immunocomplexes were washed 4 times with a lysis buffer. Proteins were then subjected to 1-D SDS-PAGE. Figure 3. Validation of GST-TβRIca interaction with r-spectrin, PIASy and r catenin. GST pull-down assays with GST-TβRIca and GST constructs were performed as described in Material and Methods, followed by separation of proteins by 1-D SDS-PAGE. R-spectrin (upper panel), PIASy (middle panel), and R-catenin (lower panel) were detected by immunoblotting with respective specific antibodies. Migration positions of proteins are indicated by arrows. Arrowheads indicate migration positions of molecular mass markers. A representative experiment out of two performed is shown.
fingerprinting was performed using matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (MALDI TOF/TOF MS; Ultraflex, Bruker Daltonics, Bremen, Germany), as described.22 To identify proteins, we performed searches in the NCBInr database using ProFound search engine (http://65.219.84.5/ service/prowl/profound.html). One miscut and partial oxidation of methionine were allowed. The peptide mass tolerance in searches was 0.1 Da or less; the significance of the identification was evaluated according to the probability value, “Z” value, and sequence coverage. Transient Transfection and Immunoprecipitation Assays. Cells were transfected using lipofectamine (Invitrogen, CA) or polyethyleneimine (Sigma-Aldrich Sweden AB, Stockholm, Sweden), according the manufacturer’s protocols. Forty-eight h after transfection cells where stimulated with TGFβ1 (5 ng/ 290
Journal of Proteome Research • Vol. 6, No. 1, 2007
Phosphorylation Assay. Forty-eight hours after transfection, 293T cells were incubated 5 h in a phosphate-free medium in the presence of 1 mCi/mL [32P]orthophosphate (GE HealthcareAmersham Biosciences, Uppsala, Sweden). For the last hour of treatment, TGFβ1 (5 ng/mL) was added. Cells were then washed, lysed, and proteins immunoprecipitated, as described for immunoprecipitation. In vitro kinase assay with purified GST-TβRIca, GSTSmad2∆MH1 with deleted MH1 domain but containing MH2 domain with two C-terminal serine residues which are phosphorylation sites for TβRI, GST-Epac1 and GST-Epac1∆ with deleted cAMP-binding domain was performed as described earlier.21 Luciferase Reporter Assay. Epac1 and TβRI constructs and empty pcDNA3 vector were transfected in MCF-7 cells in combinations; the total amount of transfected DNA was always normalized with the empty vector. CAGA(12)-luc and CRE-luc luciferase reporter constructs were transfected in all experimental conditions. Twenty-four h after transfection, cells were stimulated in a serum-free medium with TGFβ1 (5 ng/mL) for the next 24 h, and with 8-pCPT (100 µM) for the the last 30 min of incubation. Cells were lysed in a luciferase buffer (2 mM DTT, 2 mM EDTA, 10% glycerol, 1% Triton X-100, 25 mM Tris phosphate, pH 7.8), and the activity was measured as described earlier23 using the Dual Luciferase assay system
research articles
TβRI-Interacting Proteins
Figure 4. Interaction between TβRI and Epac1. (A) Interaction of GST-TβRIca and Epac1 was confirmed by immunoblotting of proteins co-precipitated with GST-TβRIca and GST constructs with antibodies specific to Epac1. Epac1 was detected in precipitates of GSTTβRIca, as indicated by an arrow. (B) Interaction of TβRI and Epac1 in vivo required intact kinase activity of TβRI. Various constructs of TβRI, i.e., wild-type (HA-TβRI), constitutively active kinase (HA-TβRIca) and kinase deficient (HA-TβRI-KD), were cotransfected with Epac1 in 293T cells. Interactions between Epac1 and TβRI constructs were analyzed by immunoprecipitation with anti-Epac antibodies and detection with anti-HA (upper panel), as well as by immunoprecipitation with anti-HA and detection with anti-Epac antibodies (second panel). Expression of Epac1 and TβRI constructs are shown in two lower panels. Migration positions of proteins are indicated by arrows. Treatment of cells with TGFβ1 is indicated at the top of the upper panel. (C) Interaction of Epac1 and TβRIca did not require the cAMP-binding domain of Epac1. Wild-type Flag-tagged Epac1 (Flag-Epac1) and Epac1 construct with deleted cAMP binding domain (Flag-Epac1∆) were co-transfected with HA-TβRIca in 293T cells. Complex between Flag-Epac1 and HA-TβRIca was detected by immunoprecipitation with anti-Flag antibodies, followed by immunoblotting with anti-HA antibodies. Expression levels of HA-TβRIca (middle panel) and Epac1 constructs (lower panel) are shown. (D) Endogenous Epac1 and TβRI form a complex. Mv1Lu cells were treated with TGFβ1 (5 ng/mL, 1 h), and endogenous TβRI was immunoprecipitated. Epac1 in immunoprecipitate was detected with specific antibodies (upper panel; lines 1, 2 and 3). As a control for TβRI precipitation from Mv1Lu cells immunoprecipitates were evaluated by immunoblotting with anti-TβRI antibody (lower panel; lines 2 and 3). TβRI-deficient R4.2 cells were used as a negative control (line 1, upper panel), and the whole cell extract (WCE) of Cos1 cells transfected with Flag-Epac1 was used as a positive control for Epac1 detection (line 4, upper panel). Arrows indicate migration positions for Epac1 and TβRI, respectively. Annotation of lanes is indicated below the lower panel. Representative experiments out of three performed (A-D) are shown.
(Promega, Falkenberg, Sweden). In all reporter assays, the β-galactosidase reporter plasmid pCMV-LacZ was cotransfected for normalization of the transfection efficiency. Cell Adhesion Assay. Twenty-four hours after transfection, Mv1Lu cells were treated for 18 h with TGFβ1 (5 ng/mL), or not. Then cells were detached with a detachment salt-balanced solution containing 20 mM EDTA, and 1 × 105 cells/well were seeded in 6-well plates with or without TGFβ1 (5 ng/mL) in a complete serum-free medium. After 2 h, the medium was removed, cells were washed twice with PBS, fixed for 3 min with 11% glutaraldehyde and stained with Coomassie Brilliant Blue. For each condition, cells present in three equally sized fields were counted. Cell Migration Assay. Twenty-four hours after transfection, Mv1Lu cells were detached and resuspended in a medium containing 1% serum. 5 × 103 cells were seeded on the upper side of a porous membrane (Neuro Probe Inc., MD), whereas the lower chamber was filled with a medium containing 1% serum, with or without TGFβ1 (5 ng/mL). After 18 h of
incubation, the medium on the top of the membrane was removed, and cells on the membrane upper side were gently taken out with cotton tips. Cells on the lower side, which migrated through the membrane, were fixed for 3 min with 11% glutaraldehyde, and stained with Coomassie Brilliant Blue. For each condition, cells present in three equally sized fields were counted. Statistics. Statistical analysis of 2D gel images was performed with an integrated in the ImageMaster Patinum software using Student’s t-test. Luciferase reporter, cell migration, and adhesion assays were repeated at least 3 times, with 3 repeats for each experimental condition in every assay. Significance of observed differences was evaluated using Student’s t-test.
Results Identification of Proteins which Form a Complex with Constitutively Active TβRI. To gain novel insights into intracellular signaling initiated by TβRI, we searched for proteins Journal of Proteome Research • Vol. 6, No. 1, 2007 291
research articles
Figure 5. Epac1 interacted with TβRI but not with the EGF receptor and c-Kit. 293T cells were co-transfected with Flag-Epac1 (all conditions), and HA-TβRIca, EGF receptor (EGFR) and c-Kit (c-Kit), as indicated on the top of panels. Cell extracts were immunoprecipitated with anti-HA, anti-EGFR and anti-c-Kit antibodies (4 panels), or with anti-Flag antibody (lower panel), as indicated. Co-precipitated Flag-Epac1 was detected by immunoblotting with anti-Flag antibody (upper panel). Expression of receptors was monitored by immunoblotting with respective antireceptor and anti-HA antibodies, as indicated (3 middle panels). Expression of Flag-Epac1 was monitored by immunoblotting with anti-Flag antibody (lower panel). Migration positions of respective proteins are indicated by arrows.
which form a complex with the receptor. We used a construct of TβRI that contained intracellular part of the receptor with kinase activating mutation Thr204Asp and was fused in its N-terminus with GST (GST-TβRIca; Figure 1). GST-TβRIca and GST alone were expressed in bacteria and purified (Figure 1). Preservation of the kinase activity and specificity of GST-TβRIca in phosphorylation of Smad2 protein were described earlier.21,24 This suggests that the GST-TβRIca construct used in this work preserved features of the intact endogenous kinase of TβRI. Constitutive activation of the kinase in the GST-TβRIca construct suggests that we would detect predominantly proteins which interact with the activated receptor. Proteins which interact with nonactive TβRI or proteins which interact transiently, e.g., Smad2 and Smad3, would probably not be detected. As activated TβRI initiates intracellular signaling, we expected that interacting proteins would reflect events relevant to the initiation of signaling. To promote protein-protein interactions, purified GSTTβRIca and GST were incubated with protein extracts of MCF-7 human breast cancer cells. MCF-7 cells were metabolically labeled with [35S]methionine and [35S]cysteine prior to extraction. Complexes of cellular proteins with GST-TβRIca and GST constructs, as well as GST constructs which were not incubated with cell extracts were separated by two-dimensional gel electrophoresis (Figure 2). Gels were stained with silver to detect proteins and were exposed in a phosphorimager to detect 35S-labeled proteins which originated from MCF-7 cells. We observed in average 25 spots of 35S-labeled proteins pulled down with GST-TβRIca, while in average 5 weakly 35S-labeled 292
Journal of Proteome Research • Vol. 6, No. 1, 2007
Conrotto et al.
proteins were precipitated with GST alone (Figure 2). It has to be noted that metabolic labeling allowed us to distinguish MCF-7 proteins from GST constructs, GST break-down products and bacterial proteins co-purified with GST constructs, which were detected by silver staining but were not labeled with 35S. Proteins that interacted with GST alone were considered as nonspecific interactors when they were observed in GST-TβRIca pull-down, and therefore they were excluded from analysis. Variability in intensities of spots precipitated with GST alone and GST-TβRIca may be due to sterical interference of TβRIca with proteins that interacted with GST. We evaluated quantities of proteins in protein spots, i.e., volumes of spots, in gel-repeats for GST-TβRIca pull-down, and considered for further analysis only spots which varied in their volumes less than 30% between gel-repeats. All mammalian protein spots that were 35S-labeled, and that were found specifically and reproducibly precipitated with GST-TβRIca, were excised and proteins were subjected to identification by mass spectrometry. We identified 16 proteins in 17 protein spots (Table 1), as a novel AAA domain-containing protein was identified in 2 proteins spots (spots #10 and #11). We identified proteins involved in transcriptional regulation, e.g., PIASy and PIAS-NY (spots #1 and #2). PIASy was reported to interact with Smad4 and Smad3, which leads to enhanced sumoylation of Smad4 and changes in Smads transcriptional activity.25,26 Two of the identified proteins contain AAA domains, with AAA standing for “ATPases associated with diverse cellular activities” (spots #9, #10, and #11).27 AAA proteins have been found to regulate such processes as membrane fusion, peroxisome assembly, endosomal transport, vacuolar sorting and protein degradation. The common molecular mechanism of AAA-domain containing proteins includes the energy-dependent unfolding of proteins. However, as functions of identified proteins have not been studied, their involvement in TGFβ signaling awaits further exploration. Identification of Structural Maintenance of Chromosome (SMC)-similar protein SMC1 (spot #12) suggests that TβRI may directly affect chromosomal integrity. SMC1 protein is a component of protein complexes involved in DNA damage repair and response.28 Another TβRIca-interacting protein, cAMP regulated guanine nucleotide exchange factor 1 (Epac1; spot #8) was reported as an activator of the small G proteins Rap1 and Rap2.29 Activated Rap1 then promotes cell adhesion and secretion.30,31 TGFβ is a potent regulator of cytoskeleton rearrangements.1-3 Thus, identification of R-catenin (spot #5), cytokeratin 1 (CK1; spot #16), cytokeratin 8 (CK8; spot #17), R-spectrin (spot #15) in a complex with TβRI may provide additional insight into the molecular mechanisms of TGFβ action. Three novel proteins without assigned functions have also been identified (spots #6, #13, and #14). Thus, we identified TβRI-interacting proteins involved in such diverse activities as chromatin maintenance, cytoskeleton rearrangements, and cell adhesion. To validate observed interactions by an alternative technique, we performed immunoblotting of GST-TβRIca- and GSTprecipitated proteins with antibodies specific to the identified proteins. Antibodies to some of the proteins, but not to all, were commercially available. With available antibodies, we confirmed the interactions between the receptor and R-spectrin, PIASy, R-catenin and Epac1. Notably, proteins of expected molecular masses were observed in complexes with the receptor construct, but not in GST precipitates (Figure 3; Figure 4A). Thus, the identities of GST-TβRIca-interacting proteins were
TβRI-Interacting Proteins
research articles
Figure 6. Coexpression of Epac1 and TβRI inhibited activation of Smad2, enhanced TGFβ1-dependent phosphorylation of Epac1, but did not affect phosphorylation of CREB. (A) 293T cells were cotransfected with HA-TβRI and Flag-Epac1. Phosphorylation of endogenous Smad2 was monitored by immunoblotting of the whole cell extract (WCE) with antibodies which recognize phosphorylated C-terminal serine residues (anti-pSmad2; upper panel). Expression of Smad2 proteins was monitored by immunoblotting of the whole cell extracts with antibodies to Smad2 (anti-Smad2; second panel from the top). (B) Epac1 inhibited TβRI-induced phosphorylation of Smad2 in vitro. In vitro kinase assay was performed with purified GST-TβRIca, GST-Smad2∆MH1, GST-Epac1 and GST-Epac1∆. Incorporation of 32P-label in Smad2 construct and autophosphorylation of TβRI construct are shown in upper panel, as a 32P-image of the gel with separated in vitro kinase assay mixture (32P). Migration positions of Smad2 and TβRI constructs are indicated by arrows. Lower panel shows the same gel stained with Coomassie Brilliant Blue (CBB), to indicate loading of various constructs. Migration positions of the constructs are indicated by arrows. (C) TGFβ1/TβRI stimulated phosphorylation of Epac1. Flag-Epac1 and wild-type HA-TβRI were transfected in 293T cells, and cells were treated with TGFβ1 (5 ng/mL, 1 h), as indicated. Cells were metabolically labeled with [32P]orthophosphate, and Flag-Epac1 and HA-TβRI were precipitated with respective antibodies. 32P-labeled proteins migrating at positions of Flag-Epac1 (upper panel) and HA-TβRI (second panel from the top), are indicated by arrows. Expression of Flag-Epac1 and HATβRIca are shown in two lower panels. (D) Phosphorylation of endogenous CREB protein was monitored by immunoblotting with antibody which recognizes phosphorylated Ser133 in CREB (anti-pCREB; top panel), and expression of CREB was also monitored (antiCREB; second panel from the top). Migration positions of respective proteins are shown by arrowheads. Cells were treated with TGFβ1 (5 ng/mL for 1 h), and 8-pCPT (100 mM, 30 min), as indicated. Expression of HA-TβRIca and Flag-Epac1 are shown in the two lower panels. Representative experiments out of three performed (A-D) are shown.
established by mass spectrometry, and were confirmed by immunoblotting for selected proteins. Interaction of Epac1 with TβRI Required Intact Kinase Activity of the Receptor. Epac1 (spot #8, Table 1) is a potent regulator of cell adhesion, and could therefore contribute to TGFβ-dependent effects on cell adhesion and migration. Thus we studied further mechanism and functional importance of the interaction between TβRIca and Epac1. First we explored whether the kinase activity of TβRI is essential for the interaction with Epac1. Epac1 was co-transfected with wild-type, constitutively active and kinase-deficient TβRI constructs. Detection of TβRI receptors in Epac1 immunoprecipiates showed that the Epac1-receptor complex was formed only upon activation of the receptor (Figure 4B, upper panel). TGFβdependent activation of the wild-type TβRI or constitutive activation of the kinase were required to stimulate interaction with Epac1. The similar dependency of the interaction was confirmed also with reverse order of immunoprecipitation-
immunoblotting, e.g., with immunoprecipiation of the receptor followed by immunoblotting for Epac1. Notably, Epac1 was detected in complexes with TβRI with activated kinase, and not when the kinase was inactive (Figure 4B, second from the top panel). To explore whether cAMP binding of Epac1 is important for the interaction with TβRI, we performed a co-precipitation assay of TβRI with wild-type Epac1 and Epac1 with deleted cAMP-binding domain. We observed that TβRIca was coprecipitated with both Epac1 constructs (Figure 4C). This suggests that the cAMP-binding domain is not essential for Epac1 interaction with TβRI. We investigated also whether endogenous Epac1 and TβRI form a complex in Mv1Lu cells (Figure 4D). We found that Epac-specific antibodies detected a protein which was coprecipitated with TβRI upon treatment of Mv1Lu cells with TGFβ1. Epac-specific antibodies were not efficient in immunoprecipitation of Epac1, preventing us from confirmation by Journal of Proteome Research • Vol. 6, No. 1, 2007 293
research articles
Conrotto et al.
Figure 7. Epac1 inhibited TGFβ1/TβRI-dependent transcriptional activation. (A, B, C) Transcriptional activation assays were performed with CAGA(12)-luc reporter upon transfection of TβRI, Epac1 and siRNA to Epac1 in Mv1Lu cells (A, B) and R4.2 TβRI-deficient mutant of Mv1Lu cells (C), as indicated. Epac1 was transfected at different levels, with quantity of Epac1-expression vector indicated in panels A and C (ng; ng/well). Cells were treated with TGFβ1 (5 ng/mL, 24 h), as indicated. Efficiency of siRNA to Epac1 is shown in the insert in panel B. Lines indicate siRNA which were transfected: 1, control siRNA, 2-4, siRNA to Epac1 at 0.05 µM (lane 2), 0.1 µM (lane 3) ,and 0.2 µM (lane 4). In the luciferase reporter assay, siRNA to Epac1 was used at the concentration 0.2 µM. Epac1 was detected by immunoblotting (WB) of the whole cells extract (WCE). Migration positions of Epac1 and actin used as a loading control are indicated by arrows. (D) Activation of TGFβ signaling had marginal effect on 8-CPT- and Epac1-dependent stimulation of CRE-luc reporter. Mv1Lu cells were transfected with HA-TβRI and Flag-Epac1, and were treated with TGFβ1 (5 ng/mL, 24 h) and 8-pCPT (100 mM, last 30 min of incubation), as indicated. Transfection efficiency in all luciferase reporter assays was normalized to co-transfected β-galactosidase. Representative experiments out of 4 (A, C, D) and 3 (B), are shown. *, p < 0.01, Mv1Lu and R4.2 cells treated with TGFβ1 and tansfected as indicated (A, C). #, p < 0.01, Mv1Lu cells, transfected with siRNA to Epac1 or not, as indicated, and treated with TGFβ1 (B).
reversed order of precipitation-detection, i.e., precipitation of Epac1 and detection of TβRI (data not shown). However, we did not detect Epac1-specific band in immunoprecipitate from R4.2 cells which are clone of Mv1Lu cells and which do not express TβRI on the cell surface. This confirmed a complex formation between endogenous TβRI and Epac1. The specificity of anti-Epac antibodies was confirmed with ectopically expressed Flag-Epac1 and endogenous Epac1 (Figure 4; data not shown). To explore whether the observed interaction of Epac1 is specific for TβRI, we performed a co-precipitation assay with TβRI, c-Kit and EGF receptors (Figure 5). We observed coprecipitation of Epac1 with only TβRI, while no Epac1 was detected in immunoprecipitates of c-Kit and EGF receptors. Thus, we found that Epac1 interacted specifically with TβRI receptor and that the kinase activity of TβRI is required for the interaction. Epac1 Inhibited TGFβ-Dependent Phosphorylation of Smad2. TβRI initiates intracellular signaling by phosphorylation of its substrates. Smad2 and Smad3 are phosphorylated at two C-terminal serine residues by TβRI, and are considered as main substrates of TβRI.32,33 To monitor phosphorylation of the C-terminal serine residues, we used antibodies specific to these phosphorylated residues.19 We observed that transfection of Epac1 in the Mv1Lu cells inhibited TGFβ/TβRI-dependent phosphorylation of Smad2 (Figure 6A). Epac1 did not block 294
Journal of Proteome Research • Vol. 6, No. 1, 2007
Smad2 phosphorylation completely. As Epac1 interacts with activated TβRI only, its inhibitory effect on Smad2 phosphorylation may be observed only if there is a certain level of receptor activation. Thus, Epac1 would decrease Smad2 phosphorylation due to enhanced activity of TβRI, but would not completely inhibit it. To confirm inhibitory effect of Epac1 on Smad2 phosphorylation by TβRI, we performed in vitro kinase assay with purified GST-TβRIca, GST-Smad2∆MH1, GST-Epac1, and GSTEpac1∆ (Figure 6B). As expected, GST-TβRIca efficiently phosphorylated Smad2 construct. Addition to the reaction mixture of Epac1 constructs strongly inhibited TβRIca-induced incorporation of 32P-label into Smad2. This in vitro reconstitution of the inhibitory effect of Epac1 on Smad2 phosphorylation by TβRI confirmed data obtained in vivo (Figure 6A, B). It also indicated that the cAMP-binding domain of Epac1 is not essential for Epac1 role as an inhibitor of substrate phosphorylation by TβRI. To explore whether Epac1 phosphorylation is affected by TGFβ and especially by TβRI, we performed metabolic labeling of 293T cells with [32P]orthophosphate, which was followed by immunoprecipitation of transfected Flag-Epac1. We found that treatment of cells with TGFβ1 enhanced Epac1 phosphorylation, which was further increased upon cotransfection of wildtype TβRI (Figure 6C, top panel). As a control, we monitored phosphorylation of transfected TβRI, and observed phospho-
TβRI-Interacting Proteins
research articles
Figure 8. TGFβ1/TβRI-dependent inhibition of cell adhesion and stimulation of cell migration are inhibited by Epac1. (A) Mv1Lu cells were transfected with HA-TβRI and Flag-Epac1, and treated with TGFβ1 (5 ng/mL, 12 h), as indicated. Migration assay was performed with porous membrane chambers, and migration rate was evaluated by counting the number of cells which migrated through the membrane. *, p < 0.02, cells transfected with Flag-Epac1 and HA-TβRI, as compared to cells transfected with HA-TβRI. (B) Expression of Flag-Epac1 and HA-TβRI was monitored by immunoprecipitation and immunoblotting of cell extracts with respective antibodies, as indicated. (+) indicates which antibodies were used for immunoprecipitation, (-) indicates that nonspecific antibodies were used. Arrows indicate migration positions of HA-TβRI and Flag-Epac1. (C) Mv1Lu cells were transfected with HA-TβRI and Flag-Epac1, and treated with TGFβ1 (5 ng/mL, 12 h), as indicated. Cells which adhere to the substrate were stained and counted. *, p < 0.02, cells transfected with Flag-Epac1 and HA-TβRI, as compared to cells transfected with Flag-Epac1. (D) Images of adherent cells are shown, with indication of HA-TβRI (TβRI) and Flag-Epac1 (Epac1) transfections and treatment with TGFβ1. Representative experiments out of three performed, are shown.
rylation of the receptor upon treatment of cells with TGFβ1, as expected (Figure 6C, second from the top panel). Multiple phosphorylation of TβRI resulted in a broader band, as compared to the total TβRI (Figure 6C). Thus, Epac1 was phosphorylated in a TGFβ/TβRI-dependent manner, though the level of 32P-incorporation was low. Phosphorylation of CREB protein at Ser133 is the triggering signal for transcriptional activation of CREB.34 We found that CREB phosphorylation was not affected upon treatment of cells with TGFβ and/or expression of TβRI (Figure 6D, upper panel). Significant enhancement of CREB phosphorylation was observed only upon transfection of Epac1 and treatment of cells with cAMP analogue 8-pCPT. This suggested that TGFβ may have no effect on CREB activity even upon Epac1 overexpression. Thus, our results showed that Epac1 inhibited TGFβ/TβRIdependent phosphorylation of Smad2 in vivo and in vitro, and that TGFβ promoted phosphorylation of Epac1, but did not affect CREB phosphorylation. Epac1 Inhibited TGFβ / TGFβ-Dependent Transcriptional Activation. Receptor-dependent phosphorylation of Smad2/3 proteins initiates a number of Smad2/3-protein interactions and translocation of the Smad complexes into nucleus where they participate in regulation of transcription.1-3,32,33 Thus, we explored whether Epac1 affects TGFβ- and TβRI-dependent
transcriptional activity. We used a luciferase reporter construct that contains CAGA elements which are sites of direct DNA binding for activated Smad3 (CAGA(12)-luc).35 We observed that Epac1 strongly inhibited TGFβ/TβRI-dependent activation of CAGA(12)-luc in Mv1Lu cells, as compared to cells transfected with TβRI alone (Figure 7A, B). This effect was dependent on quantity of transfected Epac1. Moreover, transfection of siRNA to Epac1 enhanced TGFβ-dependent induction of the luciferase reporter, as compared to non-transfected Mv1Lu cells (Figure 7B). To explore further whether the inhibitory effect of Epac1 is dependent on TβRI, we used the R4.2 clone of Mv1Lu cells; R4.2 cells have a deficient TβRI and are not responsive to TGFβ.36 We observed that R4.2 cells gained sensitivity to TGFβ only upon expression of TβRI, as was expected (Figure 7C). Transfection of Epac1 alone in R4.2 cells did not affect the luciferase reporter activity, and the dose-dependent inhibitory effect of Epac1 was observed only upon its coexpression with the receptor. This suggests that Epac1 affected TGFβ signaling in a TβRI-dependent way (Figure 7C). To explore whether TβRI may affect Epac1/cAMP-dependent signaling, we studied transcriptional activation of CREBresponsive CRE-luc luciferase reporter.37 We found that TGFβ1 and 8-pCPT enhanced its transcriptional activity, and when Journal of Proteome Research • Vol. 6, No. 1, 2007 295
research articles cells were treated with both TGFβ1 and 8-pCPT, a further increase was observed. However, the increase from combined treatment was rather weak for non-transfected cells or even negligible for TβRI- and/or Epac1-transfected cells, as compared to the levels of induction by TGFβ1 or 8-pCPT. This suggests that TGFβ1/TβRI had only a marginal effect on Epac1and cAMP/CREB-dependent transcription (Figure 7D). We observed that Epac1 with deleted cAMP-binding domain was as potent as the wild-type Epac1, except that the responsiveness to 8-pCPT was lost for the truncated Epac1 (data not shown). Thus, our data indicate that Epac1 inhibited TGFβ1/TβRIdependent transcription. Epac1 Inhibited TGFβ/TβRI-Dependent Regulation of Cell Migration and Adhesion. TGFβ is known to stimulate rearrangement of the cytoskeleton, which may affect cell migration and adhesion.1-3 Epac1 is also involved in regulation of the cytoskeleton.38,39 We therefore explored whether Epac1 could affect TGFβ-dependent regulation of cell migration and adhesion. We found that TGFβ1 enhanced Mv1Lu cell migration, which was strongly increased upon expression of TβRI (Figure 8A, B). Coexpression of Epac1 with TβRI significantly decreased cell migration, as compared to cells transfected with TβRI only. Transfection of Epac1 alone also decreased cell migration, as compared to control cells (Figure 8A, B). However, upon transfection of Epac1, we did not observe significant cell death and changes in cell proliferation, as evaluated by the appearance of apoptotic bodies and by number of cells, respectively (data not shown). TGFβ-dependent stimulation of cell migration suggests that the cell adhesion to substrate may also be affected. In agreement with this possibility, we observed that TGFβ1 inhibited cell adhesion, and transfection of TβRI further enhanced this inhibition (Figure 8C, D). Epac1, on the contrary, strongly promoted cell adhesion, which was decreased upon cotransfection of the receptor (Figure 8C, D). Down-regulation of Epac1 with specific siRNA inhibited adhesion of TGFβ1-treated and non-treated cells (data not shown), suggesting importance of endogenous Epac1 for cell adhesion and supporting data obtained with ectopically expressed protein. Thus, Epac1 inhibited TGFβ/TβRI-dependent effects on migration and adhesion of cells.
Discussion Multiplicity of TGFβ regulatory effects on cells has indicated that initiation of the intracellular signaling may require a variety of mechanisms.1-3 Protein-protein interaction screens have been efficient tools in unveiling components of the signaling mechanisms.1-3,17,18 We described here a proteomics-based analysis of protein complexes formed by activated TβRI (Table 1). These interacting proteins provide possible molecular links to regulation of protein degradation, chromosome maintenance, cytoskeleton rearrangements, cell migration, and adhesion. Moreover, detection of TβRI-interacting proteins which were described as “novel” may provide insights into uncovered yet regulatory processes. We do not exclude that some of the interactions may be not direct, although conditions of GST pull-down assay favor direct interactions. We also do not exclude that TβRIca may form different complexes, as it is unlikely that all identified proteins may be in one complex. Even if further studies are required to clarify all details, reported here TβRIca-interacting proteins represent directions to be pursued to understand multiple functions of TGFβ. 296
Journal of Proteome Research • Vol. 6, No. 1, 2007
Conrotto et al.
As an example of such exploration, we observed that Epac1 inhibited TGFβ signaling on the level of TβRI receptor activation. This prevented TGFβ-dependent stimulation of cell migration and inhibition of cell adhesion (Figures 7-8). Observed effects of Epac1 on TGFβ signaling may represent a cAMP-independent function of Epac1. Our data indicate that expression of Epac1 has a role in desensitization of cells to TGFβ. The molecular mechanism of the Epac1 action on TβRI may include inhibition of the receptor kinase or interference with the Smad2/3 recognition by the receptor. These possibilities however require structural studies of TβRI in a complex with Epac1 to identify interacting surfaces. Epac1 regulates cytoskeleton and affects cell adhesion and migration.38,39 Thus, the interaction between Epac1 and TβRI may serve to balance pro-adhesion and antimigration effects of Epac1 with the anti-adhesion and promigration activities of TGFβ. TGFβ-dependent enhancement of cell migration and accompanying changes of cytoskeleton are hallmarks of pro-metastatic changes in cells which are known as epithelial-to-mesenchymal transdifferentiation.1-3 Epac1 and especially its downstream target Rap1 were found to be dysregulated in cancer, e.g., constitutive activation of Rap1 was observed in leukemia.40 Our data suggest that Epac1 may inhibit prometastatic effects of TGFβ on cell migration and adhesion (Figure 8). Thus, our results confirm the efficiency of proteomics techniques in the search for novel TβRI interacting proteins. Further studies aimed to describe dynamics of TGFβ signaling are required to fully understand functional relations between various interacting proteins. Abbreviations: TGFβ, transforming growth factor-β; Epac1, cAMP regulated guanine nucleotide exchange factor 1; BMP, bone morphogenetic protein; HA, hemagglutinin; PIAS, protein inhibitors of activated Stats; 2D-GE, two-dimensional gel electrophoresis; MALDI TOF MS, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.
Acknowledgment. We thank Dr. Ulf Hellman for discussions, comments, and access to mass spectrometry, Drs. Carl-Henrik Heldin and Johan Ericsson for comments on the manuscript. We thank Drs. Mochizuki, Massague, and Schlessinger for constructs, cells and antibody. P.C. was supported by fellowship from AIRC (Associazione Italiana Ricerca sul Cancro), ICRETT fellowship from UICC, and a short-term fellowship from EMBO. This work was supported in part by grants from the Swedish Cancer Society, the Swedish Research Council, INTAS, UICC, and Hiroshima University to S.S. References (1) Feng, X. H.; Derynck, R. Specificity and versatility in TGF-beta signaling through Smads. Annu. Rev. Cell Dev. Biol. 2005, 21, 659-693. (2) Shi, Y.; Massague, J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell 2003, 113 (6), 685-700. (3) de Caestecker, M. The transforming growth factor-beta superfamily of receptors. Cytokine Growth Factor Rev. 2004, 15, 1-11. (4) Souchelnytskyi, S. Proteomics of TGF-beta signaling and its impact on breast cancer. Expert Rev. Proteomics 2005, 2 (6), 925935. (5) Colland, F.; Jacq, X.; Trouplin, V.; Mougin, C.; Groizeleau, C.; Hamburger, A.; Meil, A.; Wojcik, J.; Legrain, P.; Gauthier, J. M. Functional proteomics mapping of a human signaling pathway. Genome Res. 2004, 14, 1324-1332. (6) Hassel, S.; Eichner, A.; Yakymovych, M.; Hellman, U.; Knaus, P.; Souchelnytskyi, S. Proteins associated with type II bone morphogenetic protein receptor (BMPR-II) and identified by twodimensional gel electrophoresis and mass spectrometry. Proteomics 2004, 4, 1346-1358.
research articles
TβRI-Interacting Proteins (7) Grimsby, S.; Jaensson, H.; Dubrovska, A.; Lomnytska, M.; Hellman, U.; Souchelnytskyi, S. Proteomics-based identification of proteins interacting with Smad3: SREBP-2 forms a complex with Smad3 and inhibits its transcriptional activity. FEBS Lett. 2004, 577 (1-2), 93-100. (8) Barrios-Rodiles, M.; Brown, K. R.; Ozdamar, B.; Bose, R.; Liu, Z.; Donovan, R. S.; Shinjo, F.; Liu, Y.; Dembowy, J.; Taylor, I. W.; Luga, V.; Przulj, N.; Robinson, M.; Suzuki, H.; Hayashizaki, Y.; Jurisica, I.; Wrana, J. L. High-throughput mapping of a dynamic signaling network in mammalian cells. Science 2005, 307, 16211625. (9) Datta, P. K.; Chytil, A.; Gorska, A. E.; Moses, H. Identification of STRAP, a novel WD domain protein in transforming growth factor-beta signaling. J. Biol. Chem. 1998, 52, 34671-34674. (10) Griswold-Prenner, I.; Kamibayashi, C.; Maruoka, E. M.; Mumby, M. C.; Derynck, R. Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A. Mol. Cell. Biol. 1998, 18, 6595-6604. (11) Charng, M. J.; Zhang, D.; Kinnunen, P.; Schneider, M. D. A novel protein distinguishes between quiescent and activated forms of the type I transforming growth factor beta receptor. J. Biol. Chem. 1998, 273 (16), 9365-9368. (12) Wang, T.; Donahoe, P. K.; Zervos, A. S. Specific interaction of type I receptors of the TGF-beta family with the immunophilin FKBP12. Science 1994, 265, 674-676. (13) Kawabata, M.; Imamura, T.; Miyazono, K.; Engel, M. E.; Moses, H. L. Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha. J. Biol. Chem. 1995, 270 (50), 29628-29631. (14) Yi, J. Y.; Shin, I.; Arteaga, C. L. Type I transforming growth factor beta receptor binds to and activates phosphatidylinositol 3-kinase. J. Biol. Chem. 2005, 280 (11), 10870-10876. (15) Nakao, A.; Imamura, T.; Souchelnytskyi, S.; Kawabata, M.; Ishisaki, A.; Oeda, E.; Tamaki, K.; Hanai, J.; Heldin, C. H.; Miyazono, K.; ten Dijke, P. TGF-beta receptor-mediated signaling through Smad2, Smad3 and Smad4. EMBO J. 1997, 16 (17), 5353-5362. (16) Hassel, S.; Yakymovych, M.; Hellman, U.; Ronnstrand, L.; Knaus, P.; Souchelnytskyi, S. Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor. J. Cell. Physiol. 2006, 206, 457-467. (17) Monti, M.; Orru, S.; Pagnozzi, D.; Pucci, P. Interaction proteomics. Biosci. Rep. 2005, 25, 45-56. (18) de Hoog, C. L.; Mann, M. Proteomics. Annu. Rev. Genomics Hum. Genet. 2004, 5, 267-293. (19) Persson, U.; Izumi, H.; Souchelnytskyi, S.; Itoh, S.; Grimsby, S.; Engstrom, U.; Heldin, C. H.; Funa, K.; ten Dijke, P. The L45 loop in type I receptors for TGF-beta family members is a critical determinant in specifying Smad isoform activation. FEBS Lett. 1998, 434 (1-2), 83-78. (20) Franzen, P.; ten Dijke, P.; Ichijo, H.; Yamashita, H.; Schulz, P.; Heldin, C. H.; Miyazono, K. Cloning of a TGF beta type I receptor that forms a heteromeric complex with the TGF beta type II receptor. Cell 1993, 75 (4), 681-692. (21) Yakymovych, I.; Engstro¨m, U.; Grimsby, S.; Heldin, C. H.; Souchelnytskyi, S. Inhibition of Transforming Growth Factor-beta signaling by low molecular weight compounds interfering with ATP- or substrate-binding sites of the TGF-beta type I receptor kinase. Biochemistry 2002, 41 (36), 11000-11007. (22) Stasyk, T.; Dubrovska, A.; Lomnytska, M.; Yakymovych, I.; Wernstedt, C.; Heldin, C. H.; Hellman, U.; Souchelnytskyi, S. Phosphoproteome profiling of transforming growth factor (TGF)-beta signaling: abrogation of TGFbeta1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription. Mol. Biol. Cell 2005, 16 (10), 47654780. (23) Dubrovska, A.; Kanamoto, T.; Lomnytska, M.; Heldin, C. H.; Volodko, N.; Souchelnytskyi, S. TGFbeta1/Smad3 counteracts BRCA1-dependent repair of DNA damage. Oncogene 2005, 24 (14), 2289-2297.
(24) Yakymovych, I.; Heldin, C. H.; Souchelnytskyi, S. Smad2 phosphorylation by type I receptor: contribution of arginine 462 and cysteine 463 In the C terminus of Smad2 for specificity. J. Biol. Chem. 2004, 279 (34), 35781-35787. (25) Lee, P. S. W.; Chang, C.; Liu, D.; Derynck, R. Sumoylation of Smad4, the common Smad mediator of transforming growth factor-beta family signaling. J. Biol. Chem. 2003, 278, 2785327863. (26) Long, J.; Matsuura, I.; He, D.; Wang, G.; Shuai, K.; Liu, F. Repression of Smad transcriptional activity by PIASy, an inhibitor of activated STAT. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 97919796. (27) Lupas, A. N.; Martin, J. AAA proteins. Curr. Opin. Struct. Biol. 2002, 12, 746-753. (28) Musio, A.; Montagna, C.; Mariani, T.; Tilenni, M.; Focarelli, M. L.; Brait, L.; Indino, E.; Benedetti, P. A.; Chessa, L.; Albertini, A.; Ried, T.; Vezzoni, P. SMC1 involvement in fragile site expression. Hum. Mol. Genet. 2005, 14, 525-533. (29) Dremier, S.; Kopperud, R.; Doskeland, S. O.; Dumont, J. E.; Maenhaut, C. Search for new cyclic AMP-binding proteins. FEBS Lett. 2003, 546 (1), 103-107. (30) Kopperrud, R.; Krakstad, C.; Selheim, F.; Doskeland, S. O. cAMP effector mechanisms. Novel twists for an “old” signaling system. FEBS Lett. 2003, 546, 121-126. (31) Bos, J. L.; de Bruyn, K.; Enserink, J.; Kuiperij, B.; Rangarajan, S.; Rehmann, H.; Riedl, J.; de Rooij, J.; van Mansfeld, F.; Zwartkruis, F. The role of Rap1 in integrin-mediated cell adhesion. Biochem. Soc. Transactions 2003, 31, 83-86. (32) Souchelnytskyi, S.; Tamaki, K.; Engstrom, U.; Wernstedt, C.; ten Dijke, P.; Heldin, C. H. Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-beta signaling. J. Biol. Chem. 1997, 272, 28107-28115. (33) Abdollah, S.; Macias-Silva, M.; Tsukazaki, T.; Hayashi, H.; Attisano, L.; Wrana, J. L. TbetaRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J. Biol. Chem. 1997, 272 (44), 27678-27685. (34) Johannessen, M.; Delghandi, M. P.; Moens, U. What turns CREB on? Cell. Signalling 2004, 16, 1211-1227. (35) Dennler, S.; Itoh, S.; Vivien, D.; ten Dijke, P.; Huet, S.; Gauthier, J. M. Direct binding of Smad3 and Smad4 to critical TGF betainducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J. 1998, 17 (11), 30913100. (36) Boyd, F. T.; Massague, J. Transforming growth factor-beta inhibition of epithelial cell proliferation linked to the expression of a 53-kDa membrane receptor. J. Biol. Chem. 1989, 264 (4), 22722278. (37) Bhat, N. R.; Feinstein, D. L.; Shen, Q.; Bhat, A. N. p38 MAPKmediated transcriptional activation of inducible nitric-oxide synthase in glial cells. Roles of nuclear factors, nuclear factor kappa B, cAMP response element-binding protein, CCAAT/ enhancer-binding protein-beta, and activating transcription factor-2. J. Biol. Chem. 2002, 277 (33), 29584-29592. (38) Quilliam, L. A.; Rebhun, J. F.; Castro, A. F. A growing family of guanine nucleotide exchange factors is responsible for activation of Ras-family GTPases. Prog. Nucleic Acid Res. Mol. Biol. 2002, 71, 391-444. (39) Zwartkruis, F. J.; Bos, J. L. Ras and Rap1: two highly related small GTPases with distinct function. Exp. Cell Res. 1999, 253 (1), 157165. (40) Hattori, M.; Minato, N. Rap1 GTPase: functions, regulation, and malignancy. J. Biochem. 2003, 134, 479-484.
PR060427Q
Journal of Proteome Research • Vol. 6, No. 1, 2007 297
