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Insulin Receptor Substrate-1 as a Signaling Molecule for Focal Adhesion Kinase pp125FAK and pp60

1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês

10.1074/jbc.273.48.32244

ISSN

1083-351X

Autores

Patricia Lebrun, Isabelle Mothe‐Satney, Laurent Delahaye, Emmanuel Van Obberghen, Véronique Baron,

Tópico(s)

Cellular Mechanics and Interactions

Resumo

Insulin receptor substrate-1 (IRS-1) is a major substrate of insulin and insulin-like growth factor-I receptors, which upon phosphorylation on tyrosine docks several signaling molecules. Recently, IRS-1 was found to interact with αvβ3 integrins upon insulin stimulation. Integrins are transmembrane proteins that play an important role in adhesion between cells and between cells and extracellular matrix. One of the major proteins implicated in integrin signaling is pp125FAK, a cytosolic tyrosine kinase, which upon integrin engagement becomes tyrosine-phosphorylated and subsequently binds to c-Src. Here, we established a mammalian two-hybrid system to show that pp125FAK binds to IRS-1. This association depends largely on the C terminus of pp125FAK but not on pp125FAK tyrosine kinase activity. Furthermore, we observed co-immunoprecipitation of pp125FAK with IRS-1 in 293 cells, suggesting a possible biological function of this association. When IRS-1 was expressed in 293 cells together with pp125FAK or Src, we found extensive IRS-1 tyrosine phosphorylation. In pp125FAK-expressing cells, this was concomitant with increased association of IRS-1 with Src homology 2-containing proteins such as growth factor receptor-bound protein 2, phosphatidylinositol (PI) 3-kinase p85α subunit, and Src homology 2-containing protein-tyrosine phosphatase-2. In addition, pp125FAK-induced association of IRS-1 with PI 3-kinase resulted in increased PI 3-kinase activity. In contrast, no change in mitogen-activated protein kinase activity was observed, indicating that pp125FAK-induced association between IRS-1 and growth factor receptor-bound protein 2 does not affect the mitogen-activated protein kinase pathway. Moreover, we found that engagement of integrins induced IRS-1 tyrosine phosphorylation. Considering our results together, we suggest that integrins and insulin/insulin-like growth factor-I receptor signaling pathways converge at an early point in the signaling cascade, which is the IRS-1 protein. Insulin receptor substrate-1 (IRS-1) is a major substrate of insulin and insulin-like growth factor-I receptors, which upon phosphorylation on tyrosine docks several signaling molecules. Recently, IRS-1 was found to interact with αvβ3 integrins upon insulin stimulation. Integrins are transmembrane proteins that play an important role in adhesion between cells and between cells and extracellular matrix. One of the major proteins implicated in integrin signaling is pp125FAK, a cytosolic tyrosine kinase, which upon integrin engagement becomes tyrosine-phosphorylated and subsequently binds to c-Src. Here, we established a mammalian two-hybrid system to show that pp125FAK binds to IRS-1. This association depends largely on the C terminus of pp125FAK but not on pp125FAK tyrosine kinase activity. Furthermore, we observed co-immunoprecipitation of pp125FAK with IRS-1 in 293 cells, suggesting a possible biological function of this association. When IRS-1 was expressed in 293 cells together with pp125FAK or Src, we found extensive IRS-1 tyrosine phosphorylation. In pp125FAK-expressing cells, this was concomitant with increased association of IRS-1 with Src homology 2-containing proteins such as growth factor receptor-bound protein 2, phosphatidylinositol (PI) 3-kinase p85α subunit, and Src homology 2-containing protein-tyrosine phosphatase-2. In addition, pp125FAK-induced association of IRS-1 with PI 3-kinase resulted in increased PI 3-kinase activity. In contrast, no change in mitogen-activated protein kinase activity was observed, indicating that pp125FAK-induced association between IRS-1 and growth factor receptor-bound protein 2 does not affect the mitogen-activated protein kinase pathway. Moreover, we found that engagement of integrins induced IRS-1 tyrosine phosphorylation. Considering our results together, we suggest that integrins and insulin/insulin-like growth factor-I receptor signaling pathways converge at an early point in the signaling cascade, which is the IRS-1 protein. focal adhesion kinase insulin receptor substrate-1 insulin-like growth factor-I growth factor receptor-bound protein 2 phosphatidylinositol SH2-containing protein-tyrosine phosphatase-2 mitogen-activated protein pleckstrin homology phosphotyrosine binding Src homology 2 and 3, respectively Crk-associated substrate insulin receptor IR β-subunit polyacrylamide gel electrophoresis DNA binding domain activation domain son of sevenless Dulbecco's modified Eagle's medium fetal calf serum amino acids. Integrins are transmembrane proteins expressed in most tissues. They are involved in key biological functions including cell migration and adhesion, embryogenic development, prevention of programmed cell death, wound repair, and angiogenesis (1Frisch S.M. Francis H. J. 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Protein phosphorylation on tyrosine is an immediate event after integrin engagement following cell interaction with the extracellular matrix. One of the major phosphorylated proteins is the cytosolic tyrosine kinase (focal adhesion kinase) pp125FAK,1 which upon integrin engagement becomes phosphorylated on tyrosine and activated (8Hanks S.K. Calalb M.B. Harper M.C. Patel S.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8487-8491Crossref PubMed Scopus (723) Google Scholar, 9Kornberg L. Earp H.S. Parsons J.T. Schaller M. Juliano R.L. J. Biol. Chem. 1992; 267: 23439-23442Abstract Full Text PDF PubMed Google Scholar, 10Lipfert L. Haimovich B. Schaller M.D. Cobb B.S. Parsons J.T. Brugge J.S. J. 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Chem. 1995; 270: 17437-17441Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar) and tensin (15Bockholt S.M. Burridge K. J. Biol. Chem. 1993; 268: 14565-14567Abstract Full Text PDF PubMed Google Scholar) and p130CAS (Crk-associated substrate) (16Schlaepfer D.D. Broome M.A. Hunter T. Mol. Cell. Biol. 1997; 17: 1702-1713Crossref PubMed Scopus (398) Google Scholar, 17Tachibana K. Urano T. Fujita H. Ohashi Y. Kamiguchi K. Iwata S. Hirai H. Morimoto C. J. Biol. Chem. 1997; 272: 29083-29090Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). The major autophosphorylation site of pp125FAK is tyrosine 397, which upon phosphorylation becomes a binding site for the cytosolic tyrosine kinase c-Src (18Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 19Eide B.L. Turck C.W. Escobedo J.A. Mol. Cell. Biol. 1995; 15: 2819-2827Crossref PubMed Scopus (162) Google Scholar). Association of c-Src leads to pp125FAK tyrosine phosphorylation, creating binding sites for SH2-containing proteins such as growth factor receptor-bound protein 2 (GRB2) and possibly for the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (20Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (473) Google Scholar, 21Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 22Schlaepfer D.D. Hanks S.K. Hunter T. Van der Geer P. Nature. 1994; 372: 786-791Crossref PubMed Scopus (1426) Google Scholar). Evidence for a role of pp125FAK in cell migration has been provided by the use of pp125FAK knockout mice. Indeed, cells from pp125FAK-deficient mice have reduced mobility and enhanced focal adhesion contact formation (23Ilic D. Furuta Y. Kanazawa S. Takeda N. Sobue K. Nakatsuji N. Nomura S. Fujimoto J. Okada M. Yamamoto T. Nature. 1995; 377: 539-544Crossref PubMed Scopus (1576) Google Scholar). In addition to integrins, a number of growth factors and neuropeptides, including platelet-derived growth factor, bombesin, endothelin, and lysophosphatidic acid (24Zachary I. Sinnett S.J. Rozengurt E. J. Biol. Chem. 1992; 267: 19031-19034Abstract Full Text PDF PubMed Google Scholar, 25Rankin S. Rozengurt E. J. Biol. Chem. 1994; 269: 704-710Abstract Full Text PDF PubMed Google Scholar, 26Seufferlein T. Rozengurt E. J. Biol. Chem. 1994; 269: 9345-9351Abstract Full Text PDF PubMed Google Scholar) have been reported to induce tyrosine phosphorylation and activation of pp125FAK. Together, these data have led to the suggestion that pp125FAK is a key protein linking integrin and growth factor signaling pathways (27Zachary I. Rozengurt E. Cell. 1992; 71: 891-894Abstract Full Text PDF PubMed Scopus (384) Google Scholar). However, the molecular basis underlying this cooperation remains ill defined. Recent studies suggest that synergistic interactions between growth factor and integrin signaling pathways are involved in regulation of cell proliferation, adhesion, and migration (28Schwartz M.A. Lechene C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6138-6141Crossref PubMed Scopus (83) Google Scholar, 29McNamee H.P. Ingber D.E. Schwartz M.A. J. Cell Biol. 1993; 121: 673-678Crossref PubMed Scopus (301) Google Scholar, 30Miyamoto, S., Teramoto, H., Gutkind, J. S., and Yamada, K. M. (1996) J. Cell Biol. 1633–1642Google Scholar). Experiments performed in our laboratory have shown that insulin stimulation induces phosphorylation or dephosphorylation of pp125FAK, depending on the adhesion state of the cells (31Baron V. Calléja V. Ferrari P. Alengrin F. Van Obberghen E. J. Biol. Chem. 1998; 273: 7162-7168Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Moreover, these studies suggest that pp125FAK is a direct substrate of insulin and insulin-like growth factor-I (IGF-I) receptors. Recently, it was found that αvβ3 integrin associates with activated insulin receptor and platelet-derived growth factor-β receptor and potentiates the biological responses induced by platelet-derived growth factor (32Schneller M. Vuori K. Ruoslahti E. EMBO J. 1997; 16: 5600-5607Crossref PubMed Scopus (423) Google Scholar). In addition, Vuori and Ruoslahti (33Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (338) Google Scholar) have reported that insulin receptor substrate-1 (IRS-1) co-immunoprecipitates with αvβ3 integrin following insulin stimulation. IRS-1 is a docking protein implicated in the insulin and IGF-I signaling pathways (34White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). Its tyrosine phosphorylation, induced upon insulin and IGF-I stimulation, creates binding sites for SH2-containing proteins such as the p85 regulatory subunit of PI 3-kinase, the adaptor GRB2, and the SH2-containing protein-tyrosine phosphatase-2 (SHP-2), resulting in activation of the PI 3-kinase and the MAP kinase pathways (35Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1265) Google Scholar, 36Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar, 37Rocchi S. Tartare-Deckert S. Mothe I. Van Obberghen E. Endocrinology. 1995; 136: 5291-5297Crossref PubMed Scopus (38) Google Scholar, 38Myers M.J. Sun X.J. Cheatham B. Jachna B.R. Glasheen E.M. Backer J.M. White M.F. Endocrinology. 1993; 132: 1421-1430Crossref PubMed Scopus (227) Google Scholar, 39Kuhne M.R. Pawson T. Lienhard G.E. Feng G.S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, 40Lavan B.E. Kuhne M.R. Garner C.W. Anderson D. Reedijk M. Pawson T. Lienhard G.E. J. Biol. Chem. 1992; 267: 11631-11636Abstract Full Text PDF PubMed Google Scholar, 41Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (601) Google Scholar). The aim of our study was to investigate the potential cross-talk between the insulin receptor (IR)/IRS-1 signaling pathway and the integrin/pp125FAK signaling circuitry. We first looked at a putative interaction between pp125FAK and IRS-1. To do this, we established a mammalian two-hybrid system, which allows for the analysis of direct protein/protein interactions. In addition, we searched for the occurrence of an interaction between pp125FAK and IRS-1 in intact cells. The reporter vector pE1bLUC was kindly provided by Richard A. Maurer (Oregon Health Sciences University, Portland, OR). pp125FAK cDNA, inserted at the EcoRI site into the pBluescript (KS−), was a generous gift from Thomas J. Parsons (University of Virginia, Charlottesville, VA). The constitutive active and the kinase-dead Src cDNAs, subcloned into pSGT vector, were kindly provided by Sara Courtneidge (EMBL, Heidelberg, Germany). Culture media and Geneticin were from Life Technologies, Inc. Bovine fibronectin, vitronectin, and poly-l-lysine were from Sigma. 125I and [γ-32P]ATP were from ICN Pharmaceuticals, Inc. (Orsay, France); 125I-protein A was labeled using the chloramine-T method as described previously (42Freychet P. Roth J. Neville D.J. Biochem. Biophys. Res. Commun. 1971; 43: 400-408Crossref PubMed Scopus (400) Google Scholar). Triton X-100, Nonidet P-40, leupeptin, benzamidine, pepstatin, andl-α-phosphatidylinositol were from Sigma. Aprotinin was from Bayer (Bayer AG, Germany), and phenylmethylsulfonyl fluoride was from Serva (Heidelberg, Germany). Protein A and protein G-Sepharose were from Amersham Pharmacia Biotech Inc. (Uppsala, Sweden). Enzymes for molecular biology were purchased from New England Biolabs (Beverly, MA). Antiserum to IRS-1 was prepared in our laboratory and was raised against a synthetic peptide corresponding to the C-terminal sequence comprising amino acids 1223–1235 of rat IRS-1. Immunoblotting of p42 MAP kinase was performed with rabbit antibodies purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and antibodies against phosphotyrosine/phosphothreonine p42/p44 MAP kinases were from New England Biolabs. Immunoprecipitation and immunoblotting of pp125FAK were performed, respectively, with a mouse monoclonal antibody from Upstate Biotechnology, Inc. (Lake Placid, NY) and with an antibody prepared in our laboratory by immunizing rabbits with a synthetic peptide derived from the pp125FAK sequence comprising amino acids 392–406. Antibodies to phosphotyrosine and to the p85α subunit of PI 3-kinase used for Western blotting were from Upstate Biotechnology, Inc. Insulin was kindly provided by Novo-Nordisk (Copenhagen, Denmark). 293 EBNA cells are human embryo kidney cells that constitutively express the EBNA-1 protein from the Epstein-Barr virus (Invitrogen, San Diego, CA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% (v/v) fetal calf serum (FCS) and 500 μg/ml Geneticin. Transfection was performed by the calcium phosphate precipitation method of Chen and Okayama (43Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4799) Google Scholar) (5 μg of DNA per 100-mm diameter dish). 18 h after transfection, cells were starved in DMEM supplemented with 0.2% (w/v) bovine serum albumin for 20 h before use. 150-mm culture dishes were incubated with poly-l-lysine (10 μg/ml) or fibronectin (10 μg/ml) together with vitronectin (3 μg/ml), in PBS at 4 °C overnight. The dishes were rinsed twice with phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 6.5 mmNa2PO4, 1.5 mmKH2PO4, pH 7.4) and dried 1 h at 37 °C before use. NHIR cells are NIH 3T3 cells stably transfected with the insulin receptor. They are maintained in DMEM containing 10% FCS and 0.5 μg/ml Geneticin. Confluent cells were detached with trypsin and plated on the extracellular matrix protein-coated dishes in DMEM, 10% FCS. 24 h after the plating, NHIR cells were starved in 0.2% bovine serum albumin medium for 4 h. Cells were then stimulated or not stimulated with insulin (10−8m) and lysed for immunoprecipitation. To produce the pBKS/pp125FAK-ΔC, pBKS/pp125FAK was cleaved by NheI and ClaI. After a fill-in, religation was performed at 16 °C for 4 h. Kinase-deficient pp125FAK was produced by site-directed mutagenesis using the TransformerTM kit fromCLONTECH Laboratories, Inc. (Palo Alto, CA). The two primers (Genset, Paris, France) were 5′-CCG CTC TAG AAC TAG TGG GCC CCC CGG GCT GC-3′, which changes the BamHI site toApaI in the pBKS polylinker, and 5′-GGC TGT AGC AAT CAG AAC ATG TAA AAA CTG C-3′, which changes Lys454 to Arg in pp125FAK. pp125FAK cDNAs were subcloned into pCEP by excision of pBKS constructs at the XbaI and XhoI sites and ligation with the NheI and XhoI sites of pCEP. The Y397F mutant of pp125FAK was made in the pBKS vector using Quick ChangeTM kit from Stratagene (San Diego, CA). The two primers were 5′-GAA ACA GAT GAC TTT GCA GAG-3′ and 5′-CTC TGC AAA GTC ATC TGT TTC-3′ (Eurogentec, Seraing, Belgium). The Y397F cDNA was then excised from pBKS using NotI and XhoI and inserted into pCEP cleaved by the same enzymes. To subclone constitutive active and kinase-dead Src cDNAs into pCEP vector, the Src cDNAs were excised of pSGT constructs at theSpeI/BglII sites and ligated into pCEP at theNheI/BamHI sites. Cells were washed with buffer A (50 mm Hepes, 150 mm NaCl, 100 mm NaF, 10 mm EDTA, 10 mm Na4P2O7, pH 7.5) supplemented with 2 mm sodium orthovanadate (NaVO4). Cells were then lysed for 15 min on ice with buffer A supplemented with 2 mm NaVO4, 1% (v/v) Nonidet P-40, 20 mm leupeptin, 4 mmbenzamidine, 2 mm pepstatin, 1 mmphenylmethylsulfonyl fluoride, and 100 units/ml aprotinin (lysis buffer). After centrifugation at 4 °C for 15 min, cell lysates were added to antibodies (pp125FAK (1 μg of Ig/sample) or IRS-1 (serum dilution 1:50)) preadsorbed on protein G-Sepharose (mouse monoclonal antibodies) or protein A-Sepharose (rabbit polyclonal antibodies). Immunoprecipitation was performed at 4 °C for 4 h for pp125FAK and 2 h for IRS-1. Pellets were then washed three times with buffer A supplemented with 2 mmNaVO4 and resuspended in Laemmli buffer (44Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205531) Google Scholar). The samples were analyzed by SDS-PAGE under reducing conditions, and proteins were transferred to an Immobilon membrane (Immobilon polyvinylidene difluoride; Millipore Corp.) in transfer buffer (25 mmTris, 192 mm glycine, 20% methanol, pH 8.3). The membrane was blocked with TBS (10 mm Tris, pH 7.4, 140 mm NaCl) containing 5% bovine serum albumin (w/v) for 1 h and incubated with antibodies to pp125FAK (1:200), IRS-1 (1:1000), p85α (1.6 μg/ml), GRB2 (1 μg/ml), SHP-2 (2 μg/ml), p42 MAP kinase (0.5 μg/ml), phospho-MAP kinase (1 μg/ml), or phosphotyrosine (1 μg/ml) for 90 min at room temperature. When necessary, an incubation with rabbit anti-mouse immunoglobulins was performed for 1 h at room temperature. After each incubation, the membrane was washed four times with TBS containing 1% (v/v) Nonidet P-40 and then twice with TBS. The membrane was finally incubated with125I-protein A (250,000 cpm/ml) or with chemiluminescent antibodies (enhanced chemiluminescence, Pierce) for 1 h at room temperature; washed five times with TBS, 1% (v/v) Nonidet P-40 and twice with TBS; and exposed to sensitive films. The mammalian two-hybrid system was purchased from CLONTECH. It contains two expression vectors, pM and pVP16. The reporter vector pE1bLUC was kindly provided by Richard A. Maurer. pM expression vector contains the DNA binding domain (DBD) of Gal4, and the other vector contains the VP16 activation domain (AD). We subcloned the cDNAs of the proteins of interest in-frame downstream of the DBD or the AD. The cDNAs were first amplified by polymerase chain reaction using the Pwo DNA polymerase (Boehringer Mannheim) with different sets of primers: IRS-1, 5′-ACG CGT CGA CGT ATG GCG AGC CCT CCG CCG G-3′ and 5′-CCCAAG CTT CTA TTG ACG GTC CTC TGG TTG-3′; ΔPTB-IRS-1, 5′-GGA ATT CAT GGC GAG CCC TCC GGA TAC CG-3′ and 5′-CCCAAG CTT CTC CCC TGC CTC TCC GAC GCC-3′ for the N-terminal extremity, and 5′-CCC AAG CTT GGT TCC TTC AGG GTG CGT G-3′ and 5′-CCC-AAG CTT CTA TTG ACG GTC CTC TGG-3′ for the C-terminal part; ΔPH-IRS-1, 5′-GCG-CGT CGA CAT AAT CGG GCA AAG GCC CAC-3′ and 5′-CCCAAG CTT CTA TTG ACG GTC CTC TGG-3′; pp125FAKand pp125FAK-KD, 5′-CGC GGA TCC GTA TGG AGC GTT CCC CGG GGG CC-3′ and 5′-CGC GGA TCC TTA GTG GGG CCT GGA CTG GC-3′; constitutively active c-Src, 5′-CCG GAA TTC CGG ATG GGG AGC AGC AAG AGC-3′ and 5′-CCG GAA TTC CGG CTA TAG GTT CTC TCC AGG-3′. The restriction sites are underlined. Inserts and vectors pM and pVP16 were digested with the corresponding enzymes, and ligation was performed using the Rapid DNA Ligation Kit from Boehringer Mannheim. pM/pp125FAK-ΔC, pM/βIR, and pVP16/p85α were obtained by excising pp125FAK-ΔC, IR β-subunit (βIR), and p85α cDNAs from pBTM116/pp125FAK-ΔC, PCDNA3/βIR, and pLex/p85α, respectively, and religated in-frame into pM vector or pVP16 vector using appropriate enzymes. All the constructions were verified by sequencing the open reading frame between the AD or the DBD and the insert (T7 sequence kit, Pharmacia, and SEQUAGEL, National Diagnostic, Atlanta, GA). Cells were cultured in DMEM supplemented with 5% (v/v) FCS and 500 μg/ml Geneticin. Transfections were performed by the calcium phosphate precipitation method as described previously (500 ng of each expression vector and 100 ng of reporter vector/17-mm diameter dish). 18 h after being transfected, cells were starved in DMEM supplemented with 0.5% FCS for 20 h. 36 h after transfection, cells were solubilized in 100 μl of Reporter lysis buffer from Promega (Madison, WI). 10 μl of cell lysate was used to measure the luciferase activity with 50 μl of luciferin substrate purchased from Promega. Substrate degradation was followed by production of photons, and this chemiluminescent reaction was measured using a luminometer. To measure PI 3-kinase activity, 293 cells were transfected with pp125FAK, IRS-1, or both and were stimulated or not stimulated with 10−6m insulin for 5 min. Cells were washed with buffer A, supplemented with 2 mmNaVO4, and lysed with 500 μl of lysis buffer, and cell extracts were subjected to immunoprecipitation with pp125FAK (1 μg of Ig/sample) or IRS-1 antibodies (serum dilution 1:50). The immunoprecipitates were washed twice with each of the following buffers: 1) phosphate-buffered saline containing 1% (v/v) Nonidet P40; 2) 100 mm Tris, 0.5 m LiCl, pH 7.4; and 3) 10 mm Tris, 100 mm NaCl, 1 mm EDTA, pH 7.4. The pellets were resuspended in 30 μl of 20 mm Hepes, pH 7.4, 0.4 mm EGTA, 0.4 mm Na2HPO4, and the kinase reaction was started by the addition of l-α-phosphatidylinositol (0.2 mg/ml), 10 mm MgCl2, and 50 μm [γ-32P]ATP (7000 Ci/mmol) and was performed for 15 min at room temperature. After 15 min, the reaction was stopped by the addition of 15 μl of 4 m HCl, and the phosphoinositides were extracted with 130 μl of chloroform/methanol (v/v). The phospholipids were analyzed by thin layer chromatography and Cerenkov counting. We have set up a two-hybrid system in mammalian embryonic kidney cells, 293 cells (Fig. 1). We used pM and pVP16 vectors (CLONTECH) and the reporter vector pE1bLUC obtained from R. Maurer. The pM vector contains the SV40 promoter followed by the sequence coding for the DBD of Gal4. The pVP16 vector is similar to pM but contains the AD of VP16 instead of the DBD of Gal4. The pM and pVP16 polylinkers are located, respectively, downstream of the DBD or the AD, and the proteins of interest are subcloned in-frame with these domains. The reporter vector pE1bLUC contains five copies of the upstream activation sequence to which the DBD of Gal4 binds upstream of the luciferase cDNA. Interaction between proteins of interest reconstitutes a functional transcription factor (DBD Gal4/AD VP16) and allows transcription of luciferase. Hence, the interaction is quantified by a luminometric measurement of luciferase activity. 293 cells were transfected with the reporter vector pE1bLUC, pp125FAK-WT, or several mutants subcloned into pM and constitutively active c-Src subcloned into pVP16. Direct interaction between the proteins was revealed by a luminometric assay, and results have been calculated as a function of the mock condition (Fig. 2). The mock condition is the highest basal luciferase activity given by either the construct in pM or the construct in pVP16 alone. As shown in Fig. 2, and in accordance with previous reports of co-immunoprecipitations (18Schaller M.D. Hildebrand J.D. Shannon J.D. Fox J.W. Vines R.R. Parsons J.T. Mol. Cell. Biol. 1994; 14: 1680-1688Crossref PubMed Scopus (1113) Google Scholar, 19Eide B.L. Turck C.W. Escobedo J.A. Mol. Cell. Biol. 1995; 15: 2819-2827Crossref PubMed Scopus (162) Google Scholar), we found in our mammalian two-hybrid system that wild-type pp125FAKinteracted with c-Src. More precisely, interaction between pp125FAK and c-Src induced a 6-fold increase in luciferase activity compared with the mock condition. Since it is known that c-Src binds to the autophosphorylated tyrosine 397 of pp125FAK, our observations suggest that the kinase activity of pp125FAK is functional in our mammalian system. This was further confirmed using the kinase-deficient mutant of pp125FAK (obtained by mutation of lysine 454 in the ATP binding site), which did not associate with c-Src. Next we looked at the possible participation of another domain of pp125FAK. The pp125FAK mutant deleted of the C-terminal domain (Δ aa 965–1065) interacted with c-Src to the same extent as wild-type pp125FAK. This is expected, since the c-Src binding site is not included in the deleted part. In summary, our mammalian two-hybrid system is able to detect the interaction between pp125FAKand constitutively active c-Src. This interaction is subject to modulation by changes in the structure and kinase activity of pp125FAK. Taken together, our results confirm the validity of this mammalian system. The cDNAs of pp125FAK or the βIR were subcloned into pM. The cDNAs of IRS-1 or the p85α subunit of PI 3-kinase (p85α) were subcloned into pVP16. 293 cells were transfected with pE1bLUC, pM/βIR, and pVP16/IRS-1; pM/pp125FAK and pVP16/p85α; or pM/pp125FAK and pVP16/IRS-1. Interestingly, as shown in Fig. 3, we found that pp125FAK and IRS-1 interact strongly in this system, since their coexpression led to a 48-fold increase in luciferase activity compared with the mock condition. This is the first evidence demonstrating a direct molecular interplay between these two molecules. Moreover, coexpression of βIR and IRS-1 led to a 15-fold increase in luciferase activity, indicating that the two proteins interact in our mammalian system. Such interaction has been previously demonstrated using the classical yeast two-hybrid system (45Rocchi S. Tartare-Deckert S. Sawka-Verhelle D. Gamha A. Van Obberghen E. Endocrinology. 1996; 137: 4944-4952Crossref PubMed Scopus (0) Google Scholar). Indeed, IRS-1 contains a PTB (aa 144–318), which binds to phosphotyrosine 960 of βIR (46Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M.F. Miyazaki M. Lee J. Shoelson S.E. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 47Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (320) Google Scholar). In addition, and as described previously (20Chen H.C. Guan J.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10148-10152Crossref PubMed Scopus (473) Google Scholar, 21Chen H.-C. Appeddu P.A. Isoda H. Guan J.-L. J. Biol. Chem. 1996; 271: 26329-26334Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar), pp125FAK interacts directly with p85α, since we found a 20-fold increase in luciferase activity when pp125FAK and p85α are coexpressed. The interactions between pp125FAKand p85α, and between IRS-1 and βIR, indicate that these proteins are functional and correctly expressed in 293 cells. Next, we searched for the domains of pp125FAK and IRS-1 potentially involved in the interaction between the two molecules. Neither pp125FAK nor IRS-1 contains SH2 or SH3 domains (35Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (126

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