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

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.

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 (1)(2)(3)(4)(5)(6)(7). Protein phosphorylation on tyrosine is an imme-diate 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) pp125 FAK , 1 which upon integrin engagement becomes phosphorylated on tyrosine and activated (8 -10). pp125 FAK contains a central kinase domain flanked by large N-and C-terminal regions, but it lacks canonical interaction motifs such as pleckstrin homology (PH), phosphotyrosine binding (PTB), and SH2 and SH3 (Src homology) domains. The pp125 FAK sequence, which allows targeting of the kinase to the focal adhesions, is located in the C terminus (11). The potential physiological substrates of pp125 FAK are the cytoskeletal proteins paxillin (12)(13)(14) and tensin (15) and p130 CAS (Crk-associated substrate) (16,17). The major autophosphorylation site of pp125 FAK is tyrosine 397, which upon phosphorylation becomes a binding site for the cytosolic tyrosine kinase c-Src (18,19). Association of c-Src leads to pp125 FAK tyrosine phosphorylation, creating binding sites for SH2-containing proteins such as growth factor receptor-bound protein 2 (GRB2) and possibly for the p85␣ regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (20 -22). Evidence for a role of pp125 FAK in cell migration has been provided by the use of pp125 FAK knockout mice. Indeed, cells from pp125 FAK -deficient mice have reduced mobility and enhanced focal adhesion contact formation (23). In addition to integrins, a number of growth factors and neuropeptides, including platelet-derived growth factor, bombesin, endothelin, and lysophosphatidic acid (24 -26) have been reported to induce tyrosine phosphorylation and activation of pp125 FAK . Together, these data have led to the suggestion that pp125 FAK is a key protein linking integrin and growth factor signaling pathways (27). 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 (28 -30). Experiments performed in our laboratory have shown that insulin stimulation induces phosphorylation or dephosphorylation of pp125 FAK , depending on the adhesion state of the cells (31). Moreover, these studies suggest that pp125 FAK is a direct substrate of insulin and insulin-like growth factor-I (IGF-I) receptors. Recently, it was found that ␣ v ␤ 3 integrin associates with activated insulin receptor and platelet-derived growth factor-␤ receptor and potentiates the biological responses induced by platelet-derived growth factor (32). In addition, Vuori and Ruoslahti (33) have reported that insulin receptor substrate-1 (IRS-1) co-immunoprecipitates with ␣ v ␤ 3 integrin following insulin stimulation. IRS-1 is a docking protein implicated in the insulin and IGF-I signaling pathways (34). 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 (35)(36)(37)(38)(39)(40)(41).
The aim of our study was to investigate the potential crosstalk between the insulin receptor (IR)/IRS-1 signaling pathway and the integrin/pp125 FAK signaling circuitry. We first looked at a putative interaction between pp125 FAK 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 pp125 FAK and IRS-1 in intact cells.

Materials
The reporter vector pE1bLUC was kindly provided by Richard A. Maurer (Oregon Health Sciences University, Portland, OR). pp125 FAK cDNA, inserted at the EcoRI site into the pBluescript (KSϪ), 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. 125 I and [␥-32 P]ATP were from ICN Pharmaceuticals, Inc. (Orsay, France); 125 I-protein A was labeled using the chloramine-T method as described previously (42). Triton X-100, Nonidet P-40, leupeptin, benzamidine, pepstatin, and L-␣-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 pp125 FAK 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 pp125 FAK sequence comprising amino acids 392-406. Antibodies to phosphotyrosine and to the p85␣ subunit of PI 3-kinase used for Western blotting were from Upstate Biotechnology, Inc. Insulin was kindly provided by Novo-Nordisk (Copenhagen, Denmark).

Cell Culture and Transfection
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 g/ml Geneticin. Transfection was performed by the calcium phosphate precipitation method of Chen and Okayama (43) (5 g 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.

NHIR Cell Adherence on Extracellular Matrix
NHIR cells are NIH 3T3 cells stably transfected with the insulin receptor. They are maintained in DMEM containing 10% FCS and 0.5 g/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 Ϫ8 M) and lysed for immunoprecipitation.

Production of pp125 FAK Expression Vectors
To produce the pBKS/pp125 FAK -⌬C, pBKS/pp125 FAK was cleaved by NheI and ClaI. After a fill-in, religation was performed at 16°C for 4 h. Kinase-deficient pp125 FAK was produced by site-directed mutagenesis using the Transformer TM kit from CLONTECH Laboratories, Inc. (Palo Alto, CA). The two primers (Genset, Paris, France) were 5Ј-CCG CTC TAG AAC TAG TGG GCC CCC CGG GCT GC-3Ј, which changes the BamHI site to ApaI in the pBKS polylinker, and 5Ј-GGC TGT AGC AAT CAG AAC ATG TAA AAA CTG C-3Ј, which changes Lys 454 to Arg in pp125 FAK . pp125 FAK 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 pp125 FAK was made in the pBKS vector using Quick Change TM kit from Stratagene (San Diego, CA). The two primers were 5Ј-GAA ACA GAT GAC TTT GCA GAG-3Ј and 5Ј-CTC TGC AAA GTC ATC TGT TTC-3Ј (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 the SpeI/BglII sites and ligated into pCEP at the NheI/BamHI sites.
Transfection of 293 Cells-Cells were cultured in DMEM supplemented with 5% (v/v) FCS and 500 g/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.
Luciferase Assay-36 h after transfection, cells were solubilized in 100 l of Reporter lysis buffer from Promega (Madison, WI). 10 l of cell lysate was used to measure the luciferase activity with 50 l of luciferin substrate purchased from Promega. Substrate degradation was followed by production of photons, and this chemiluminescent reaction was measured using a luminometer.

PI 3-Kinase Assay
To measure PI 3-kinase activity, 293 cells were transfected with pp125 FAK , IRS-1, or both and were stimulated or not stimulated with 10 Ϫ6 M insulin for 5 min. Cells were washed with buffer A, supplemented with 2 mM NaVO 4 , and lysed with 500 l of lysis buffer, and cell extracts were subjected to immunoprecipitation with pp125 FAK (1 g 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.

Detection of Protein/Protein Interactions in Mammalian
Cells-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, pp125 FAK -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 (18,19), we found in our mammalian two-hybrid system that wild-type pp125 FAK interacted with c-Src. More precisely, interaction between pp125 FAK 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 pp125 FAK , our observations suggest that the kinase activity of pp125 FAK is functional in our mammalian system. This was further confirmed using the kinase-deficient mutant of pp125 FAK (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 pp125 FAK . The pp125 FAK mutant deleted of the C-terminal domain (⌬ aa 965-1065) interacted with c-Src to the same extent as wild-type pp125 FAK . 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 pp125 FAK and constitutively active c-Src. This interaction is subject to modulation by changes in the structure and kinase activity of pp125 FAK . Taken together, our results confirm the validity of this mammalian system. pp125 FAK Interacts with IRS-1-The cDNAs of pp125 FAK or the ␤IR were subcloned into pM. The cDNAs of IRS-1 or the p85␣ subunit of PI 3-kinase (p85␣) were subcloned into pVP16. 293 cells were transfected with pE1bLUC, pM/␤IR, and pVP16/ IRS-1; pM/pp125 FAK and pVP16/p85␣; or pM/pp125 FAK and pVP16/IRS-1. Interestingly, as shown in Fig. 3, we found that pp125 FAK 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 ␤IR 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 (45). Indeed, IRS-1 contains a PTB (aa 144 -318), which binds to phosphotyrosine 960 of ␤IR (46,47). In addition, and as described previously (20,21), pp125 FAK interacts directly with p85␣, since we found a 20-fold increase in luciferase activity when pp125 FAK and p85␣ are coexpressed. The interactions between pp125 FAK and p85␣, and between IRS-1 and ␤IR, indicate that these proteins are functional and correctly expressed in 293 cells.
Characterization of pp125 FAK /IRS-1 Interaction-Next, we searched for the domains of pp125 FAK and IRS-1 potentially involved in the interaction between the two molecules. Neither pp125 FAK nor IRS-1 contains SH2 or SH3 domains (35,48). However, pp125 FAK has various putative tyrosine phosphorylation sites, which can become binding sites for SH2 domaincontaining proteins (20 -22, 37-41) and for the PTB domain of IRS-1. To characterize the interaction between pp125 FAK and IRS-1, several pp125 FAK mutants were tested (see Fig. 2). 293 cells were co-transfected with the reporter vector pE1bLUC, pVP16/IRS-1, and pp125 FAK , or its mutants subcloned into pM. The interaction was measured by luminometric assay and calculated as a function of the mock condition (Fig. 4A). Similarly to the results shown in Fig. 3, interaction between pp125 FAK and IRS-1 induced a 45-fold increase in luciferase activity compared with mock. Interestingly, pp125 FAK -⌬C interaction with IRS-1 was decreased about 5-fold compared with the interaction between wild-type pp125 FAK and IRS-1 and presented only a 9-fold increased activity compared with mock. This could indicate that the pp125 FAK C-terminal region is directly involved in this interaction. However, since deletion of the C-terminal domain did not completely abolish the association, additional domain(s) of the protein might be involved. We have also determined that the N-terminal domain of pp125 FAK (aa 1-386) is not implicated in the interaction with IRS-1 (data not shown). Next, we tested the interaction of IRS-1 with the kinase-deficient mutant of pp125 FAK . pp125 FAK -KD interacted with IRS-1 similarly to wild-type pp125 FAK , indicating that pp125 FAK kinase activity is not necessary for the association of pp125 FAK with IRS-1. In conclusion, the C-terminal domain of pp125 FAK is important for the interaction with IRS-1, and this association is independent of pp125 FAK tyrosine kinase activity.
Concerning IRS-1, we envisioned that its PTB domain could interact with phosphotyrosines on pp125 FAK . Moreover, IRS-1 also contains a PH domain, which is implicated in the association between protein and phospholipids and is also thought to be involved in protein/protein interactions (49). To evaluate the possible role of IRS-1 PH and PTB domains, we subcloned IRS-1 deleted of its PTB domain (⌬PTB, ⌬ aa 144 -316) or its PH domain (⌬PH, ⌬ aa 1-144) into pVP16, whereas wild-type pp125 FAK was subcloned into pM. 293 cells were co-transfected with pE1bLUC, pp125 FAK , IRS-1 wild type, ⌬PH, or ⌬PTB. Results are presented in Fig. 4B. When pp125 FAK was coexpressed with ⌬PH-IRS-1 or with ⌬PTB-IRS-1, the resulting interaction was comparable with that seen with wild-type IRS-1, indicating that neither the IRS-1 PH domain nor the IRS-1 PTB domain is involved in the interaction. To ensure the viability of our IRS-1 mutants, we verified that in this system ⌬PH-IRS-1 interacts with ␤IR, whereas ⌬PTB-IRS-1/␤IR does not (data not shown). In summary, neither the PH domain nor the PTB domain of IRS-1 participates in the interaction be- FIG. 2. Interaction of wild-type and mutant forms of pp125 FAK with c-Src using the mammalian two-hybrid system. 293 cells were co-transfected with 500 ng of pVP16/constitutively active c-Src, pp125 FAK wild type (WT) or mutants subcloned into pM vector (500 ng), and 100 ng of the reporter vector encoding luciferase. Cell lysates were prepared and used for a luciferase assay. Results are presented as -fold induction compared with the mock condition. The ⌬C mutant corresponds to pp125 FAK deleted of its last 100 amino acids, and KD is a kinase-deficient mutant of pp125 FAK (K454R). Results are representative of four independent experiments each performed in triplicate. tween pp125 FAK and IRS-1.
Co-immunoprecipitation of pp125 FAK with IRS-1 in Intact Cells-To find whether pp125 FAK and IRS-1 interact in intact cells, wild-type and mutant forms of pp125 FAK were overexpressed in 293 cells. After immunoprecipitation of endogenous IRS-1, the proteins were separated by SDS-PAGE and analyzed by Western blotting using antibodies to IRS-1 (Fig. 5A) or to pp125 FAK (Fig. 5B). Fig. 5A shows that IRS-1 immunoprecipitation was comparable in all conditions. As shown in Fig.  5B, we did not detect co-immunoprecipitation of pp125 FAK with IRS-1 when pp125 FAK was not overexpressed in 293 cells (mock). This is due to the very low level of pp125 FAK expression in these cells (data not shown). However, Fig. 5B shows that pp125 FAK co-immunoprecipitated with IRS-1 when cells were transfected with the wild-type pp125 FAK (WT). The C-terminal mutant of pp125 FAK (⌬C) weakly co-immunoprecipitated with IRS-1, a result that further supports the idea that the pp125 FAK C terminus is involved in the association with IRS-1. Moreover, in accordance with results presented in Fig. 4A, kinase-deficient pp125 FAK (KD) co-immunoprecipitated with IRS-1 to the same extent as pp125 FAK wild type. Fig. 5B (lower  part) shows the tyrosine phosphorylation level of pp125 FAK co-immunoprecipitating with IRS-1. Wild type and the C-terminal mutant are phosphorylated on tyrosine, while as expected kinase-deficient pp125 FAK is not. We have verified in total cell lysates that the expression level of pp125 FAK and its mutants was the same. In summary, in intact cells and in the mammalian two-hybrid system, the interaction between pp125 FAK and IRS-1 is independent of pp125 FAK kinase activity but requires the pp125 FAK C terminus.
Expression of pp125 FAK and IRS-1 Induces IRS-1 Tyrosine Phosphorylation-Since pp125 FAK is a tyrosine kinase, we next examined whether the interaction between pp125 FAK and IRS-1 could have consequences on IRS-1 phosphorylation. In brief, 293 cells were cotransfected with IRS-1 and with plasmids encoding wild-type or mutant forms of pp125 FAK . Insulin stimulation was used as a positive control for IRS-1 phosphorylation. Cell lysates were subjected to immunoprecipitation with antibody to IRS-1; proteins were separated on a 7.5% polyacrylamide gel and transferred to a membrane, and phosphotyrosine-containing proteins were revealed with antibody to phosphotyrosine (Fig. 6A). When IRS-1 is expressed alone, its basal tyrosine phosphorylation is not detectable in 293 cells. However, IRS-1 tyrosine phosphorylation was seen when cells were stimulated with insulin. Interestingly, the expression of wild-type pp125 FAK in 293 cells induced pronounced tyrosine phosphorylation of IRS-1. In contrast, IRS-1 phosphorylation was low in cells expressing pp125 FAK -⌬C, which is in accordance with the observation that pp125 FAK -⌬C interacts with IRS-1 less efficiently than wild-type pp125 FAK (Fig. 6B, lower  part). When 293 cells were transfected with pp125 FAK -KD, no tyrosine phosphorylation of IRS-1 was observed, indicating that the tyrosine kinase activity of pp125 FAK is responsible directly or indirectly for IRS-1 phosphorylation. We also checked in these cells whether wild-type and pp125 FAK -⌬C were indeed phosphorylated, whereas the kinase-deficient pp125 FAK was not. Fig. 6B (upper part) shows that the level of IRS-1 immunoprecipitation is similar in all conditions. Moreover, the amount of pp125 FAK mutants co-immunoprecipitating with IRS-1 (Fig. 6B, lower part) was similar to the wild-type, except with pp125 FAK -⌬C, and thus could not explain the reduced tyrosine phosphorylation of IRS-1 observed with the kinase-deficient mutant. Finally, comparable expression levels of pp125 FAK (wild type and mutants) were also found in total cell lysates (Fig. 6C). In conclusion, pp125 FAK expression in 293 cells leads to an important increase in IRS-1 tyrosine phosphorylation. This phosphorylation requires an intact pp125 FAK C-terminal domain and is dependent on pp125 FAK tyrosine kinase activity. However, we cannot exclude the possibility that another associated tyrosine kinase participates in IRS-1 phosphorylation.
Expression of Src and IRS-1 Induces IRS-1 Tyrosine Phosphorylation-We next examined whether Src could be implicated in IRS-1 tyrosine phosphorylation. To this end, 293 cells were co-transfected with IRS-1 and with wild-type or mutant forms of pp125 FAK or Src. After immunoprecipitation of cell lysates with antibody to IRS-1, phosphoproteins were separated and revealed with antibody to phosphotyrosine. As described previously in this paper, expression of pp125 FAK -WT in 293 cells induced robust IRS-1 tyrosine phosphorylation (Fig.  7). In cells expressing IRS-1 and pp125 FAK -Y397F, which has lost the ability to bind Src, IRS-1 phosphorylation was strongly

FIG. 5. Co-immunoprecipitation of pp125 FAK with IRS-1 in intact cells.
293 cells were transfected with 5 g of the following constructs: pCEP only (mock), pCEP/WT (pp125 FAK wild type), pCEP/⌬C (pp125 FAK deleted of its C terminus), or pCEP/KD (kinase-deficient pp125 FAK , K454R). Cell lysates were subjected to immunoprecipitation, followed by SDS-PAGE, and analyzed by immunoblotting with antibodies to IRS-1 (1:1000) (A) or to pp125 FAK (1:200) (B, upper part). Results for IRS-1 immunoblotting were quantified by densitometry using NIH Image, and results represent the average of two independent experiments. Next, the membrane corresponding to pp125 FAK was stripped and blotted with antibodies to phosphotyrosine (1 g/ml) (B, lower part). The autoradiograms show a representative experiment out of four performed in duplicate. reduced but was still observed. This result could indicate that, without participation of Src, pp125 FAK is able to phosphorylate IRS-1, albeit to a limited extent. However, since pp125 FAK -Y397F expression led to a pronounced decrease in IRS-1 phosphorylation compared with the wild-type, we investigated whether Src by itself could be responsible for IRS-1 tyrosine phosphorylation. In 293 cells expressing IRS-1 and a constitutive active form of Src (Src-CA), extensive IRS-1 tyrosine phosphorylation was found. In contrast, expression of kinase-dead Src (Src-KD) did not lead to such phosphorylation. Immunoprecipitation of IRS-1 was the same in all conditions (data not shown). These results indicate that constitutively active Src is able to lead to IRS-1 tyrosine phosphorylation and that this process requires Src's kinase activity. Moreover, in cells expressing IRS-1 and the two kinases, pp125 FAK and Src, there was an additional effect on IRS-1 phosphorylation.
pp125 FAK -induced IRS-1 Tyrosine Phosphorylation Results in IRS-1 Docking of p85␣, SHP-2, and GRB2-IRS-1 contains multiple sites for tyrosine phosphorylation, of which several are docking sites for SH2 domain-containing proteins such as the p85␣ subunit of PI 3-kinase, the adaptor protein GRB2, and the phosphotyrosine phosphatase SHP-2 (37)(38)(39)(40)(41). Therefore, we were interested in testing the ability of IRS-1, which has been phosphorylated upon pp125 FAK expression, to interact with these SH2 domain-containing proteins. To do this, 293 cells were transfected with an expression vector encoding pp125 FAK , IRS-1, or both. After immunoprecipitation of pp125 FAK or IRS-1, proteins were separated by SDS-PAGE and analyzed by Western blotting with antibodies to pp125 FAK , IRS-1, p85␣, SHP-2, or GRB2 (Fig. 8). In these cells, interactions between pp125 FAK and p85␣ or between pp125 FAK and GRB2 are not detectable by co-immunoprecipitation. This is probably due to the small portion of these endogenous proteins associated with pp125 FAK . In nontransfected and nonstimulated cells, a weak association of IRS-1 with SHP-2 and GRB2 was observed. Treatment of IRS-1-expressing cells with insulin increased the association of SHP-2, GRB2, and p85␣ with IRS-1. This is consistent with the notion that insulin induces IRS-1 tyrosine phosphorylation and its subsequent interaction with GRB2, SHP-2, and p85␣. Interestingly, cell transfection with pp125 FAK increased severalfold the interaction of IRS-1 with p85␣, SHP-2, and GRB2. In conclusion, IRS-1 tyrosine phosphorylation induced by pp125 FAK results in an increased association of IRS-1 with p85␣, SHP-2, and GRB2.
pp125 FAK Increases IRS-1-associated PI 3-Kinase Activity in 293 Cells-Next we approached the putative biological consequences of IRS-1 phosphorylation in the presence of pp125 FAK . Since we found that the interaction between IRS-1 and p85␣ increases in the presence of pp125 FAK , we anticipated that pp125 FAK overexpression could stimulate IRS-1-associated PI 3-kinase activity. To test this, 293 cells were overexpressed with pp125 FAK , IRS-1, or both. Insulin was used as a positive control of PI 3-kinase activation. pp125 FAK or IRS-1 was immunoprecipitated, and the associated PI 3-kinase activity was measured in the immunocomplexes. As shown in Fig. 9, no PI 3-kinase activity was associated with transfected pp125 FAK . This correlates with our previous experiments (Fig. 8) showing no detectable co-immunoprecipitation of p85␣ with pp125 FAK . Moreover, there was also no detectable PI 3-kinase activity associated with endogenous IRS-1. In contrast, in cells overexpressing IRS-1, PI 3-kinase activity associated with IRS-1 was observed in basal conditions, and a further 2-fold stimulation was obtained after insulin treatment. Transfection with both IRS-1 and pp125 FAK induced an 8-fold stimulation of PI 3-kinase activity associated with IRS-1 compared with PI 3-kinase activity from cells overexpressing IRS-1 only. Expression of p85␣ was verified in all conditions (data not shown). Taken together, our observations show that pp125 FAK -induced tyrosine phosphorylation of IRS-1 creates docking sites for p85␣, which upon binding to IRS-1 leads to activation of p110 PI 3-kinase.
Increased Interaction of IRS-1 with GRB2 in the Presence of pp125 FAK Does Not Lead to MAP Kinase Activation-Tyrosine kinases induce recruitment of GRB2⅐SOS complexes to the plasma membrane and allow activation of Ras, which then initiates the MAP kinase cascade (50). Thus, pp125 FAK -induced tyrosine phosphorylation of IRS-1 and subsequent association with GRB2 could lead to increased MAP kinase activity. To test this possibility, 293 cells were cotransfected with IRS-1, pp125 FAK , or both and stimulated or not stimulated with insulin or okadaic acid as controls for MAP kinase activation. A fraction from each lysate was separated on a 10% acrylamide/ bisacrylamide gel. As expected, pp125 FAK overexpressed in 293 cells was tyrosine-phosphorylated, and this was not modified FIG. 6. Overexpression of pp125 FAK and IRS-1 induces IRS-1 phosphorylation on tyrosine. 293 cells were cotransfected with 5 g of pCEP only (mock) or pCEP/pp125 FAK (wild-type and mutants) and 5 g of pCEP/IRS-1. Where indicated, cells were stimulated with insulin (10 Ϫ6 M). Immunoprecipitation of IRS-1 was carried out on cell lysates. The immunocomplexes were separated into two fractions, and proteins were separated by 7.5% SDS-PAGE and transferred to membranes. One membrane was incubated with antibody to phosphotyrosine (1 g/ml) (A), and the second one was incubated with anti-IRS-1 (1:1000) or anti-pp125 FAK (1:200) (B). C, immunodetection of pp125 FAK was also carried out using a fraction of the total cell lysate by Western blot using the anti-pp125 FAK to check expression levels of pp125 FAK mutants. A representative experiment out of four is shown performed in duplicate.
by IRS-1 overexpression. IRS-1 is phosphorylated on tyrosine when cells are stimulated with insulin or when pp125 FAK is overexpressed (Fig. 10A). Fig. 10B shows that p42 MAP kinase is present at similar levels in all conditions. Fig. 10C shows activation of p42 and p44 MAP kinases revealed using antibodies, which detect doubly phosphorylated threonine 202/tyrosine 204 of p42/p44 MAP kinases. No activated MAP kinase was seen in mock condition in the absence of induction. Okadaic acid induced the appearance of the phosphorylated and activated form of p42 and p44 MAP kinase. In cells overexpressing only pp125 FAK , no activation of MAP kinase was detected. In addition, a slight MAP kinase activation was observed in the presence of insulin in cells transfected with IRS-1. However, no activated MAP kinase was observed in 293 cells co-transfected with IRS-1 and pp125 FAK . In conclusion, despite increased formation of the complex IRS-1⅐GRB2 induced by pp125 FAK , stimulation of MAP kinase is not observed.

Engagement of Integrins Induces IRS-1 Phosphorylation-To
address the physiological relevance of our findings, we studied the effect of the engagement of integrins on IRS-1 tyrosine phosphorylation. To this end, NHIR cells were plated onto polylysine or fibronectin plus vitronectin and stimulated or not stimulated with insulin as a positive control for IRS-1 phosphorylation. Cell lysates were subjected to immunoprecipitation of IRS-1, and proteins were separated by 7.5% SDS-PAGE and transferred onto a membrane. Immunoblotting using antibody to phosphotyrosine was performed (Fig. 11). In cells plated onto polylysine, basal tyrosine phosphorylation of IRS-1 was observed. As expected, insulin stimulation induced an important increase in IRS-1 phosphorylation. When NHIR cells were adherent onto fibronectin/vitronectin, integrins were engaged and IRS-1 phosphorylation was increased compared with cells plated onto polylysine. Taken together, these results mean that engagement of integrins induces IRS-1 tyrosine phosphorylation, which can further be stimulated by insulin. DISCUSSION Previous studies have shown that the focal adhesion kinase pp125 FAK is a key player in integrin signaling pathways. Thus, upon integrin engagement pp125 FAK is activated (8 -10) and becomes implicated in interactions with signaling proteins such as PI 3-kinase and GRB2 (20 -22). In addition, neuropeptides and growth factors are also able to activate pp125 FAK (24 -26), indicating that this kinase is involved in growth factor signaling pathways. However, the molecular basis of this integrin/growth factor receptor cross-talk is unknown. In contrast, a coherent picture is starting to emerge concerning the early events mediated by the insulin and IGF-I receptors (34). A major substrate of these two tyrosine kinase receptors is IRS-1. Vuori and Ruoslahti (33) have reported that insulin stimulation leads to co-immunoprecipitation of IRS-1 with the ␣ v ␤ 3 integrin. More recently, evidence for interactions between integrins and insulin receptors and between integrins and plate- FIG. 7. Expression of Src and IRS-1 induces IRS-1 tyrosine phosphorylation. 293 cells were transfected with 1 g of pCEP/IRS-1; 0.1 g of pCEP/ pp125 FAK -WT or Y397F; and 5 ng of pCEP/Src-CA or -KD alone or together. After lysis, an immunoprecipitation of IRS-1 was realized (1:50). Proteins were separated by 7.5% SDS-PAGE and transferred to a membrane. The membrane was blotted with antiphosphotyrosine (1 g/ml). An autoradiogram is shown. FIG. 8. IRS-1 phosphorylation on tyrosine induces its interaction with p85␣, SHP-2, and GRB2. 293 cells were co-transfected with 5 g of pCEP vector only or with 5 g of pCEP/IRS-1 and/or 5 g of pCEP/pp125 FAK . Where indicated, cells were stimulated with 10 Ϫ6 M insulin. Cells were lysed, and immunoprecipitation was performed using antibodies to pp125 FAK (1 g/sample) or to IRS-1 (1:50). Proteins were separated by 10% SDS-PAGE and transferred to a membrane. Membrane portions corresponding to the molecular masses of the proteins studied were immunoblotted with anti-IRS-1 (1: 1000), anti-pp125 FAK (1:200), anti-p85␣ (1.6 g/ml), anti-SHP-2 (2 g/ml), or anti-GRB2 (1 g/ml). An autoradiogram representative of four independent experiments performed in duplicate is shown. Ϫ, buffer; ϩ, 10 Ϫ6 M insulin.
FIG. 9. Increased PI 3-kinase activity in cells transfected with pp125 FAK and IRS-1. Experiments were performed as described in Fig. 8. After immunoprecipitation of pp125 FAK or IRS-1, a PI 3-kinase assay was performed. Samples were analyzed by silica thin layer chromatography and counted (Cerenkov). The experiment shown was performed in duplicate and is representative of three independent experiments. f, buffer; o, 10 Ϫ6 M insulin. let-derived growth factor receptors has been provided (32). In addition, ␣ v ␤ 3 integrin engagement potentiates platelet-derived growth factor-and insulin-induced DNA synthesis.
Taken together, these observations suggest the existence of an interplay between integrin and insulin/IGF-I signaling pathways. To search for the molecular basis of such a crosstalk, we looked for a possible link between pp125 FAK and IRS-1. To do so, we established an experimental system that permits the analysis of direct protein/protein interactions in mammalian cells. Interestingly, we found that the tyrosine kinase pp125 FAK actually binds IRS-1. This interaction occurs through the C terminus of pp125 FAK and does not require pp125 FAK kinase activity, suggesting that tyrosine-phosphorylated sites of pp125 FAK may not be involved. This hypothesis is strengthened by the observation that the PTB domain of IRS-1 is not implicated in the interaction with pp125 FAK . We also determined that the PH domain of IRS-1 is not involved in this association. Next, we demonstrated an association between pp125 FAK and IRS-1 in intact cells. Consistent with such a direct molecular interplay, we found that expression of pp125 FAK leads to IRS-1 tyrosine phosphorylation. Our studies also show that pp125 FAK kinase activity is required for IRS-1 tyrosine phosphorylation, since a kinase-deficient pp125 FAK mutant does not induce IRS-1 phosphorylation. Expression of the pp125 FAK -Y397F mutant (mutated on the c-Src binding site) was still associated with increased IRS-1 tyrosine phosphorylation, although to a minute extent compared with that seen with wild-type pp125 FAK . This indicates that pp125 FAK is able to increase IRS-1 phosphorylation but that most of the phosphorylation obtained with pp125 FAK expression is due to c-Src. This corroborates with the fact that expression of a constitutive active Src results in strong IRS-1 tyrosine phosphorylation. This was not observed upon expression of a kinase-dead Src. Moreover, the two kinases, pp125 FAK and Src, appear to have an additional effect on this phosphorylation. So far we cannot conclude whether pp125 FAK or Src directly phosphorylate IRS-1, since our results were obtained after immunoprecipitation of expressed proteins. The most likely interpretation of our data taken as a whole is that pp125 FAK functions as a scaffold protein for both Src and IRS-1 and by doing so allows the phosphorylation of IRS-1 by Src. Whether pp125 FAK by itself phosphorylates IRS-1 is not known at present.
Several IRS-1 tyrosine residues become docking sites for SH2 domain-containing molecules upon phosphorylation (38 -41). We show here that expression of pp125 FAK promotes the interaction of IRS-1 with p85␣, SHP-2, and GRB2. Furthermore, pp125 FAK -induced association of IRS-1 with p85␣ results in increased PI 3-kinase activity. Data have been accumulating showing that p85␣ interacts directly with pp125 FAK and that upon integrin engagement PI 3-kinase activity associated with pp125 FAK is increased (20,21). We propose that two pathways may lead to activation of PI 3-kinase by pp125 FAK , the first one consisting of a direct interaction between pp125 FAK and PI 3-kinase, the second one using IRS-1 as a docking molecule. Whether both of these pathways occur in intact cells and what the precise contribution of each one is in a physiological context remain open questions. It has been demonstrated that pp125 FAK is an antiapoptotic factor (51,52). Moreover, pp125 FAK cleavage by caspases is one of the earliest events occurring during apoptosis (53,54). It was previously assumed that the antiapoptotic effect of pp125 FAK was mediated by PI 3-kinase (55). The involvement of PI 3-kinase in preventing FIG. 10. Increased IRS-1⅐GRB2 complex formation in the presence of pp125 FAK does not lead to MAP kinase activation. 293 cells were transfected with vector only, with pCEP/ pp125 FAK or pCEP/IRS-1, or with both. After incubation with buffer, 2 ϫ 10 Ϫ6 M okadaic acid, or 10 Ϫ6 M insulin, cells were lysed. Proteins were separated on a 10% polyacrylamide gel and transferred to a membrane that was blotted with antibodies to phosphotyrosine (1 g/ml) (A) or to phospho-MAP kinase (1 g/ml) (C). After stripping, the blot was incubated with antibody to p42 MAP kinase (0.5 g/ml) (B) to check the amount of MAP kinase in all conditions (enhanced chemiluminescence). This experiment is representative of three performed in duplicate. OKA, 2 ϫ 10 Ϫ6 M okadaic acid; INS, 10 Ϫ6 M insulin.
FIG. 11. Engagement of integrins induces IRS-1 tyrosine phosphorylation. NHIR cells were plated on poly-Llysine 10 g/ml or fibronectin (10 g/ml) plus vitronectin (3 g/ml) for 24 h. Then after 4 h of starvation, cells were stimulated or not stimulated with insulin (10 Ϫ8 M). Immunoprecipitation of IRS-1 was performed on cell lysates, and proteins were separated by SDS-PAGE. Phosphotyrosine proteins transferred onto a membrane were revealed with antiphosphotyrosine (1 g/ml). An autoradiogram is shown. apoptosis has been reported by several groups (56 -61). In addition, a series of experiments has shown that IRS-1 expression partially prevents apoptosis induced by interleukin-3 deprivation in 32D cells (62). This is consistent with other studies describing IRS-1 as an antiapoptotic factor (63). As a whole, these observations and our present work favor the idea that pp125 FAK , IRS-1, and PI 3-kinase could participate in common signaling pathways, leading for instance to antiapoptotic effects. Although expression of pp125 FAK increases the association of GRB2 with IRS-1, it does not lead to MAP kinase activation. This is in accordance with observations suggesting that pp125 FAK is not implicated in MAP kinase activation (64). A previous report has shown that GRB2 located at focal adhesions interacts with the cytoskeletal protein dynamin (65). This GRB2 pool may not be capable of interacting with SOS and thus would not be implicated in activation of the MAP kinase cascade. Moreover, the GRB2-dynamin complex is able to bind to tyrosine-phosphorylated IRS-1 (66). We can envision that in our system and in the presence of pp125 FAK , IRS-1 associates with this pool of GRB2-dynamin, which is not actively involved in MAP kinase activation.
To address the question of the physiological significance of our results, we investigated whether engagement of integrins could induce IRS-1 tyrosine phosphorylation. Indeed, we find that upon integrin binding to fibronectin and vitronectin, IRS-1 tyrosine phosphorylation is increased. Accordingly, we also found that in attached cells tyrosine phosphorylation of IRS-1 is constantly observed, whereas it disappears in suspended cells (data not shown). Thus cell adhesion clearly controls the level of IRS-1 tyrosine phosphorylation.
Our results are reminiscent of other findings showing the occurrence of converging effects induced by integrin and growth factor signaling pathways (30,34). Moreover, our recent studies suggest that integrin engagement modulates the effect of insulin on pp125 FAK phosphorylation (31). These observations strongly favor the idea that integrin and insulin or growth factor receptor signaling pathways cross-react.
From our results, we propose the following two hypotheses schematized in Fig. 12: 1) IRS-1 participates in signaling mediated by the pp125 FAK /pp60 src module, which is part of the integrin cascade functioning independently of the insulin or IGF-I circuitry (A); 2) IRS-1 forms a point of convergence between integrin and insulin/IGF-I signaling pathways (B). Previous work from J. Pessin's laboratory (67) and our own (31) demonstrating that insulin modulates pp125 FAK phosphorylation leads us to believe that the latter hypothesis is the more likely one. For both scenarios, the most pressing questions relate to the biological consequences of this interplay and to its participation in physiological processes and, more importantly, in disease states associated with altered cell growth and apoptosis.