p125Fak Focal Adhesion Kinase Is a Substrate for the Insulin and Insulin-like Growth Factor-I Tyrosine Kinase Receptors*

Véronique BaronDagger , Véronique Calléja, Patricia Ferrari, Françoise Alengrin, and Emmanuel Van Obberghen

From INSERM, U145, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cédex 2, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The focal adhesion kinase p125Fak is a widely expressed cytosolic tyrosine kinase, which is involved in integrin signaling and in signal transduction of a number of growth factors. In contrast to tyrosine kinase receptors such as the platelet-derived growth factor and the hepatocyte growth factor receptors, which induce p125Fak phosphorylation, insulin has been shown to promote its dephosphorylation. In this study, we compared p125Fak phosphorylation in insulin-stimulated cells maintained in suspension or in an adhesion state. We found that, in nonattached cells, insulin promotes p125Fak phosphorylation, whereas dephosphorylation occurred in attached cells. This was observed in Rat-1 fibroblasts overexpressing the insulin receptor, as well as in Hep G2 hepatocytes and in 3T3-L1 adipocytes expressing more natural levels of insulin receptors. Insulin-induced p125Fak phosphorylation correlated with an increase in paxillin phosphorylation, indicating that p125Fak kinase activity may be stimulated by insulin. Mixing of purified insulin or insulin-like growth factor-I (IGF-I) receptors with p125Fak resulted in an increase in p125Fak phosphorylation. Using a kinase-deficient p125Fak mutant, we found that this protein is a direct substrate of the insulin and IGF-I receptor tyrosine kinases. This view is supported by two additional findings. (i) A peptide corresponding to p125Fak sequence comprising amino acids 568-582, which contains tyrosines 576 and 577 of the kinase domain regulatory loop, is phosphorylated by the insulin receptor; and (ii) p125Fak phosphorylation by the insulin receptor is prevented by addition of this peptide. Finally, we observed that p125Fak phosphorylation by the receptor results in its activation. Our results show that the nature of the cross-talk between the insulin/IGF-I receptors and p125Fak is dependent on the cell architecture, and hence the interaction of the insulin/IGF-I signaling system with the integrin system will vary accordingly.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The focal adhesion kinase p125Fak is a cytosolic tyrosine kinase initially isolated from Src-transformed cells (1, 2). It is localized at focal adhesion plaques of cultured cells and binds to a number of proteins involved in the organization of the cytoskeleton and to signaling molecules, resulting in the formation of multicomponents complexes (reviewed in Refs. 3-6). Tyrosine phosphorylation of p125Fak occurs rapidly in response to integrin clustering or binding to the extracellular matrix, and this correlates with increased kinase activity (2, 7-9).

For most cell types, cooperation of adhesion-mediated and growth factor-mediated signaling pathways is required for appropriate growth control, and it is now widely accepted that p125Fak may be a point of convergence in the actions of integrins and growth factors (10, 11). Indeed, p125Fak is not only activated by integrins, but also by mitogenic neuropeptides (12), thrombin (13), sphingosine (14), the bioactive lipid lysophosphatidic acid (15-17), and by ligands for tyrosine kinase receptors such as platelet-derived growth factor and hepatocyte growth factor (18-20).

In contrast to other tyrosine kinase receptors, which induce tyrosine phosphorylation of p125Fak, it has been reported that in fibroblasts insulin promotes a decrease in p125Fak phosphorylation (21-23).

The insulin receptor is a heterotetrameric oligomer consisting of two extracellular 135-kDa alpha -subunits and two 95-kDa transmembrane beta -subunits containing a tyrosine kinase (24, 25). Insulin binding to the alpha -subunit stimulates autophosphorylation of the beta -subunit cytoplasmic domain on multiple tyrosine residues. This activates the receptor kinase, leading to phosphorylation of several substrates including IRS-1,1 IRS-2, Shc, and Gab-1 (26-31). IRS-1/2 and Gab-1 carry multiple potential tyrosine phosphorylation sites, which upon phosphorylation become docking sites for SH2 domain-containing proteins. These include the p85 regulatory subunit of phosphatidylinositol 3-kinase, Grb2, and the phosphotyrosine phosphatase SHP-2 (32-34).

IRS-1 is also found to be associated with Csk, the C-terminal Src kinase that is an inhibitor of the Src kinase family. It has been shown recently that the insulin-induced complex of IRS-1 and Csk could be involved in dephosphorylation of p125Fak observed after insulin treatment of fibroblasts (35).

The fact that insulin induces p125Fak dephosphorylation suggests the existence of an antagonistic action of insulin on the integrin signaling pathway. However, examples of cooperation between insulin and integrin signals have been reported. For instance, stimulation of DNA synthesis by insulin is potentiated by engagement of the alpha vbeta 3 integrin. Moreover, insulin promotes the association of IRS-1 with this integrin, which is indicative of a putative role for IRS-1 in the cooperation between the two signaling pathways (36). Finally, recent studies indicate that conjunction of insulin or IGF-I activation with integrin engagement plays an important role in dissemination of tumor cells (37).

In the present work, we show that the effect of insulin on p125Fak phosphorylation depends on the adhesion status of the cells. Indeed, in nonattached cells insulin stimulates p125Fak phosphorylation, whereas in attached cells it induces its dephosphorylation. In addition, we provide several lines of evidence for the view that p125Fak is a direct substrate of the insulin receptor and the IGF-I receptor tyrosine kinases, and that cross-phosphorylation of p125Fak by a tyrosine kinase receptor results in its activation. This may explain the positive cooperation occurring between integrins and insulin/IGF-I signaling pathways.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- p125Fak cDNA, inserted at the EcoRI site into pBluescript (KS-), was a generous gift from Dr. Thomas J. Parsons (University of Virginia, Charlottesville, VA).

Mouse monoclonal anti-p125Fak antibodies (clone 2A7) and anti-phosphotyrosine antibodies (clone AG10) came from Upstate Biotechnology, Inc, (Lake Placid, NY). Rabbit polyclonal antibody to p125Fak (A-17) used in the Western blot experiments was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit anti-mouse antibodies were purchased from DAKO (Glostrup, Denmark). Reagents for SDS-PAGE were from Bio-Rad. Hybond-C nitrocellulose was from Millipore, and protein G-Sepharose was from Sigma-France (St. Quentin Fallavier, France). [gamma -32P]ATP was purchased from ICN-France. Porcine insulin and recombinant human IGF-I were kindly provided by Novo-Nordisk (Copenhagen, Denmark) and Lilly, respectively.

Cell Lines-- Rat-1 embryo fibroblasts transfected with an expression plasmid encoding the human insulin receptor (RHIR) were a gift from Dr. A. Ullrich (Max-Planck-Institut für Biochemie, München, Germany). These cells and Rat-1 cells were grown in Ham's F-12 medium and Dulbeco's modified Eagle's medium (DMEM) (1/1) supplemented with 10% (v/v) fetal calf serum (FCS) and 10 mM glutamine.

NIH 3T3 cells transfected with an expression plasmid encoding the human IGF-I receptor were produced in our laboratory as described previously (38). 293 EBNA cells are human embryo kidney cells, which constitutively express the EBNA-I protein from Epstein-Barr virus, and Hep G2 is a cell line derived from human hepatocyte carcinoma (Invitrogen, San Diego, CA). These cells were grown in DMEM supplemented with 10% (v/v) FCS and 10 mM glutamine. All transfected cells were maintained in 400 µg/ml Geneticin (G418).

3T3-L1 fibroblasts were cultured in DMEM containing 10% (v/v) FCS and 10 mM glutamine and were induced to differentiate into adipocytes as described previously (39).

Cells were seeded into six-well plates or into 100 mm-diameter dishes and grown until confluence. They were starved overnight in DMEM containing 0.2% (w/v) bovine serum albumin (BSA) and 10 mM glutamine before use.

Production of Kinase-deficient p125Fak-- Site-directed mutagenesis was performed using the TransformerTM kit from CLONTECH. The two primers (Genset, Paris, France) were CCg CTC TAg AAC TAg Tgg gCC CCC Cgg gCT gC, which changes the BamHI site for ApaI in the pBKS polylinker, and ggC TgT AgC AAT CAg AAC ATg TAA AAA CTg C, which replaced Lys-454 with Arg in p125Fak. The manufacturer's instructions were followed without modifications. The mutated plasmid was amplified into XL-1Blue Escherichia coli strain, and the mutation was checked by sequence analysis. The cDNA was subcloned into pCEP4 expression vector (Invitrogen) by excision at the sites NheI and XhoI and ligation between the sites XbaI and XhoI of pCEP4.

Transfection of 293 EBNA Cells-- Cells were trypsinized and seeded at 250,000 cells/well of a six-well plate 2 days before use. Transfection was performed by the calcium phosphate precipitation method of Chen and Okayama (40). Plasmid DNA (1 µg/well) was incubated for 30 min at 25 °C with 0.25 M CaCl2 and BES. This solution was added dropwise to the cells, which were incubated overnight at 35 °C under 3% CO2. Cells were starved for 15 h before use in DMEM containing 0.2% BSA (w/v).

p125Fak Phosphorylation in Intact Cells-- Insulin was added to the cells at a final concentration of 0.1 µM. After the indicated time periods, insulin was removed by two washes in 50 mM Hepes, pH 7.5, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 2 mM sodium orthovanadate, 100 mM NaF (stop buffer). Cells were solubilized for 30 min on ice in the above buffer containing 1% (v/v) Triton X-100 and protease inhibitors (20 µM leupeptin, 100 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). Lysates were then clarified by centrifugation at 13,000 × g for 20 min at 4 °C before being added for 4 h at 4 °C to antibodies to p125Fak (1 µg/500 µg of protein) preadsorbed on protein G-Sepharose. Pellets were washed three times in 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, and were resuspended in Laemmli sample buffer containing 3% (w/v) SDS and 5% (v/v) 2-mercaptoethanol. Proteins were analyzed by SDS-PAGE using a 7.5% resolving gel before Western blot analysis.

In some experiments, we performed immunoprecipitation using antibodies to p125Fak and antibodies to paxillin mixed on protein G-Sepharose (1 and 1.5 µg/500 µg of protein, respectively).

p125Fak Phosphorylation in Detached Cells-- Cells were washed twice with phosphate-buffered saline (PBS) containing 1 mM EDTA, and detached gently from the dishes using a rubber policeman. They were washed once in PBS. Cells were maintained in suspension in PBS for 30 min at 37 °C before the addition of insulin (0.1 µM) for the indicated time periods. Cells were dipped into ice, centrifuged at 3,000 × g for 3 min at 4 °C, and washed twice with stop buffer. Cell solubilization and immunoprecipitation of lysates were performed as described above using antibodies to p125Fak mixed or not with antibodies to paxillin.

p125Fak Phosphorylation by Insulin and IGF-I Receptors in Vitro-- We used 293 EBNA cells transiently transfected with the plasmids encoding wild-type or kinase-deficient p125Fak (p125Fak/K454R).

Cells were washed twice and solubilized in lysis buffer for 30 min on ice. Cleared lysates were incubated for 4 h at 4 °C with antibodies to p125Fak (1 µg/200 µg of protein) preadsorbed on protein G-Sepharose. Pellets were washed three times in 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100, and resuspended into this buffer.

Insulin or IGF-I receptors, partially purified by chromatography on wheat germ agglutinin, were stimulated by insulin or IGF-I (0.1 µM) for 30 and 15 min, respectively. Where indicated, insulin and IGF-I receptors were purified by immunoprecipitation using specific antibodies. The receptors were mixed with pellets containing the immunoprecipitated p125Fak. Phosphorylation mix was added to each sample (50 mM MgCl2, 60 µM [gamma -32P]ATP (2.5 mCi/mmol)) for 15 min at 22 °C. In some experiments, the reaction was stopped by addition of 4-fold concentrated Laemmli sample buffer. In other experiments, the reaction was stopped by addition of ice-cold buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100), and pellets were washed twice in this buffer before SDS-PAGE analysis.

Western Blot Analysis-- Samples were submitted to one-dimensional SDS-PAGE and were then transferred to polyvinylidene difluoride membrane following the method of Towbin et al. (41). The membrane was blocked with saline buffer (10 mM Tris, pH 7.4, 140 mM NaCl) containing 5% (w/v) BSA for 4 h at 22 °C. Antibodies to phosphotyrosine (1 µg/ml) were added in blocking buffer for 15 h at 4 °C. Several washes were performed using the saline buffer containing 0.5% (v/v) Tween 20. Rabbit anti-mouse antibodies (1 µg/ml) were added for 1 h at 22 °C in blocking buffer, followed by several washes. The membrane was then incubated with 125I-protein A (250,000 cpm/ml) for 1 h at 22 °C. After several washes, the membrane was autoradiographed.

Phosphorylation of Fak Peptide (Amino Acids 568-582)-- The peptide corresponding to p125Fak amino acids 568-582 (SRYMEDSTYYKASKG) was synthesized by Eurogentec (Belgium). The insulin receptor kinase activity toward this peptide was measured as follows. Partially purified insulin receptors were incubated without or with insulin (0.1 µM) for 60 min at 22 °C in a final volume of 40 µl containing 50 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% (v/v) Triton X-100. Increasing peptide concentrations were added to the receptors. Phosphorylation was initiated by addition of 15 µM [gamma -32P]ATP (2.5 mCi/mmol), 8 mM MgCl2, 4 mM MnCl2. The reaction was stopped by spotting a constant volume of each sample onto phosphocellulose papers, which were dipped into 1% (v/v) orthophosphoric acid. Papers were washed extensively, and 32P incorporation into the peptide was quantified by Cerenkov counting. The reaction was found to be linear for at least 2 h.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

We wished to investigate whether the effect of insulin on p125Fak phosphorylation depends on cellular adhesion. To test this, we compared attached RHIR (Rat-1 fibroblasts overexpressing the insulin receptor) with RHIR in suspension, a condition that causes disassembly of focal adhesion plaques and p125Fak dephosphorylation. Tyrosine phosphorylation of p125Fak was revealed by immunoprecipitation using antibodies to p125Fak, followed by immunoblotting with antibodies to phosphotyrosine.

In attached RHIR cells (Fig. 1, top panel), insulin induced rapid dephosphorylation of p125Fak. These data are in accordance with previous studies reporting p125Fak dephosphorylation in response to insulin in quiescent cultures of fibroblasts or CHO cells (21-23, 42). No effect could be seen in parental cells, or in cells expressing kinase-deficient insulin receptors, indicating that p125Fak dephosphorylation is due to overexpression of a functional insulin receptor (data not shown). To determine if the effect of insulin on p125Fak phosphorylation is also observed in cells that express physiological levels of endogenous insulin receptors, we performed similar experiments in 3T3-L1 adipocytes and in Hep G2 hepatocytes (Fig. 1, middle and bottom panels). As shown, insulin stimulation of Hep G2 hepatocytes and 3T3-L1 adipocytes also resulted in dephosphorylation of p125Fak.


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Fig. 1.   Insulin induces the dephosphorylation of p125Fak in attached cells. Cells were stimulated by insulin (0.1 µM) for different periods and solubilized. Lysates were submitted to immunoprecipitation using anti-p125Fak antibodies. Tyrosine-phosphorylated proteins were revealed by anti-phosphotyrosine immunoblotting. Representative experiments are shown. Top panel shows results from RHIR cells, whereas middle and bottom panels represent Hep G2 hepatocytes and 3T3-L1 adipocytes, respectively.

Fig. 2 shows experiments in which cells were gently detached, maintained in suspension for 30 min, and subsequently stimulated with insulin. In these conditions, we observed only a weak phosphorylation of p125Fak in unstimulated cells. Interestingly, insulin induced an increase in tyrosine phosphorylation of p125Fak, both in RHIR and in Hep G2 hepatocytes. Phosphorylation of p125Fak was undetectable in nonattached 3T3-L1 adipocytes, although this could be due to low levels of protein expression (data not shown).


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Fig. 2.   Insulin stimulates p125Fak tyrosine phosphorylation in nonattached RHIR cells and Hep G2 hepatocytes. Cells were gently detached from the culture dishes and maintained in suspension for 30 min before addition of insulin (0.1 µM) for the indicated periods. Cells were dipped into ice and washed twice with ice-cold buffer before solubilization. Lysates were submitted to immunoprecipitation using anti-p125Fak antibodies. Tyrosine phosphorylation of p125Fak was visualized by anti-phosphotyrosine immunoblotting. The amount of immunoprecipitated p125Fak was checked by reprobing the membranes with antibodies to p125Fak. Representative experiments are shown.

Quantification of p125Fak phosphorylation was performed to compare the time-courses (Fig. 3). Dephosphorylation of p125Fak in attached RHIR cells was maximal at 15 min and returned to initial phosphorylation level within 2 h. The decrease in p125Fak phosphorylation reached 67.5 ± 5% of control after 15 min of insulin treatment. The time dependence shown here is consistent with previously published results (21). No major difference was observed between the three cell lines (see Fig. 1). In contrast, the time course of p125Fak phosphorylation in detached cells was different in RHIR and Hep G2 cells. Indeed, stimulation was observed within 2 min and was maximal between 5 and 10 min in RHIR cells. At 5 min, we measured a 4-fold induction of p125Fak phosphorylation. Phosphorylation decreased rapidly and returned to basal levels within 30 min. In Hep G2 hepatocytes, p125Fak phosphorylation reached its maximal level after 30 min (6-fold induction), but fully persisted until at least 60 min.


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Fig. 3.   The effect of insulin on p125Fak phosphorylation depends on cell adhesion. Results were quantified using the Molecular Imager system from Bio-Rad. Values are the mean ± S.E. of several independent experiments and are expressed as percent of p125Fak phosphorylation in absence of insulin.

As a whole, our data indicate that the effect of insulin on the phosphorylation of p125Fak is regulated by integrin engagement. Interestingly, insulin has opposite effects compared with other growth factors such as bombesin, which induces p125Fak phosphorylation in attached cells but not in cells maintained in suspension (43).

It should be noted that stimulation of p125Fak phosphorylation was also observed in detached NIH 3T3 cells overexpressing IGF-I receptors, whereas dephosphorylation was observed in attached cells, indicating that insulin and IGF-I have similar effects (data not shown).

We were then interested to approach the mechanism of p125Fak phosphorylation seen in cells exposed to insulin or IGF-I. The simplest explanation could be that, in these cells, p125Fak is directly activated by insulin or IGF-I receptors, leading to its autophosphorylation. To test this, p125Fak was immunoprecipitated from unstimulated cells. Concomitantly, immunopurified insulin or IGF-I receptors were activated or not by their respective ligand. The receptors and p125Fak were then mixed together, and [gamma -32P]ATP was added to the samples. As shown in Fig. 4A, autophosphorylation of p125Fak in the absence of insulin receptor was detected (first lane). The unstimulated receptor did not modify p125Fak phosphorylation (second lane). By contrast, addition of insulin-activated receptor induced a 2.3-fold increase in the level of p125-Fak phosphorylation (third lane). When unstimulated IGF-I receptors were used, an increase in p125Fak phosphorylation was observed (Fig. 4B, second lane compared with first lane). This is due to the high basal kinase activity and autophosphorylation levels of the IGF-I receptor in vitro that we have described previously (38). IGF-I addition to the receptor further increased p125Fak phosphorylation (4.4-fold stimulation compared with control). We noticed that the intrinsic kinase activity of p125Fak was lower using the phosphorylation mixture suitable for the IGF-I receptor (i.e. containing 50 mM MgCl2). Therefore, further experiments using either the insulin or the IGF-I receptors were performed in these conditions.


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Fig. 4.   The insulin receptor and IGF-I receptor stimulate p125Fak phosphorylation in vitro. p125Fak was immunoprecipitated from unstimulated fibroblasts. Concomitantly, insulin or IGF-I receptors were immunoprecipitated using specific antibodies and incubated or not with insulin or IGF-I (0.1 µM). After several washes, pellets containing p125Fak were mixed with pellets containing one receptor. Phosphorylation was initiated by addition of 15 µM [gamma -32P]ATP, 4 mM MnCl2, 8 mM MgCl2 for 15 min at 22 °C when using the insulin receptor. The phosphorylation mixture consisted in 60 µM ATP and 50 mM MgCl2 when using the IGF-I receptor. The reaction was stopped by addition of Laemmli sample buffer. The samples were analyzed by SDS-PAGE and autoradiographed. Standard molecular weights are indicated on the right.

In summary, incubation of p125Fak with insulin receptor or IGF-I receptor results in an enhancement of p125Fak phosphorylation in vitro. In these experiments, the proteins were immunopurified, and therefore this process is unlikely to be due to an intermediate kinase. We can probably rule out the possibility that p125Fak coimmunoprecipitates with Src, since it has been shown that p125Fak and Src are not associated in resting cells.

To determine whether this augmentation in p125Fak phosphorylation is due to direct phosphorylation of the protein by the receptors or to activation of p125Fak, we constructed a kinase-deficient mutant of p125Fak by substitution of Lys-454 to Arg (44). We first compared the expression level and kinase activity of the mutant and wild-type proteins in transfected 293 EBNA cells. Protein expression was checked by immunoprecipitation with antibodies to p125Fak followed by p125Fak immunoblotting with antibodies to p125Fak (Fig. 5, left panel). The mock-transfected cells express low levels of endogenous p125Fak compared with both the wild-type and mutant-transfected cells. To measure p125Fak kinase activity, the proteins immunoprecipitated from transfected cells were submitted to autophosphorylation (Fig. 5, right panel). As expected, the wild-type protein was markedly phosphorylated, whereas the mutant protein did not show detectable kinase activity. Next, we looked for direct phosphorylation of the mutant p125Fak by the two tyrosine kinase receptors. The kinase-deficient p125Fak was overexpressed in 293 cells and immunoprecipitated with antibodies to p125Fak. Insulin receptors or IGF-I receptors, stimulated or not, were added to the p125Fak-containing pellets. Phosphorylation was performed using [gamma -32P]ATP. After extensive washes, the proteins were analyzed by SDS-PAGE (Fig. 6). Since this p125Fak is kinase-deficient, no phosphorylation was detected in absence of receptors, or in presence of unstimulated receptors. However, phosphorylation of the kinase-dead protein occurred upon addition of ligand-stimulated receptors. Our results indicate that p125Fak is a direct substrate for the insulin and IGF-I receptors, at least in vitro. In some experiments, partially purified receptors were used, whereas in others, receptors were immunoprecipitated using specific antibodies. No difference concerning the extent of p125Fak phosphorylation was observed between these procedures.


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Fig. 5.   Expression and kinase activity of wild-type and kinase-deficient p125Fak. 293 EBNA cells were transfected 2 days before use. Cells were solubilized, and p125Fak was immunoprecipitated using specific antibodies. The level of protein expression was visualized by immunoblotting with antibodies to p125Fak (left panel). Autophosphorylation was measured in presence of 4 µM [gamma -32P]ATP and 10 mM MnCl2 (right panel). The reaction was stopped by addition of Laemmli sample buffer for SDS-PAGE analysis.


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Fig. 6.   Kinase-deficient p125Fak(K454R) is an in vitro substrate of the insulin receptor and the IGF-I receptor kinases. 293 EBNA cells were transfected with the plasmid encoding p125Fak mutated at lysine 454 (K454R). After 2 days, cells were lysed and p125Fak/K454R was immunoprecipitated using antibodies to p125Fak. Insulin or IGF-I receptors, stimulated or not by their respective ligand, were added to the washed pellets containing p125Fak. Phosphorylation was initiated by addition of 60 µM [gamma -32P]ATP and 50 mM MgCl2. The reaction was stopped by addition of ice-cold buffer and the pellets were washed twice. Samples were analyzed by SDS-PAGE.

That p125Fak is a direct substrate for insulin and IGF-I receptors is further supported by the observation that a synthetic peptide corresponding to p125Fak sequence can be phosphorylated by the insulin receptor. The sequence of p125Fak contains two twin tyrosine residues, Tyr-576 and Tyr-577, which are localized in the regulatory loop of its kinase domain and are phosphorylated by the Src kinase both in vitro and in vivo (45). The peptide corresponding to p125Fak amino acids 568-582, containing tyrosines 570, 576, and 577, was synthesized and used in an in vitro kinase assay with the insulin receptor. As shown in Fig. 7, the peptide was heavily phosphorylated by the insulin-stimulated receptor. For each experiment, a Lineweaver-Burk plot was obtained using a computer program for calculation of the best fit of the points. We calculated a Km = 184 ± 4 µM in the presence of insulin. For comparison, we previously obtained a Km = 120 µM using a common substrate of the insulin receptor, i.e. a synthetic peptide corresponding to the receptor sequence 1142-1158, which also contains three tyrosine residues (46). Another peptide corresponding to p125Fak amino acids 369-406, which contains p125Fak major autophosphorylation site Tyr-397, was tested in a similar kinase assay. However, we did not detect phosphorylation of this peptide by the insulin receptor (data not shown), suggesting that phosphorylation of p125Fak peptide 568-582 by the receptor is specific.


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Fig. 7.   A peptide corresponding to p125Fak sequence amino acids 568-582 is phosphorylated by the insulin receptor. Insulin receptors were incubated without (open circle ) or with (bullet ) insulin (0.1 µM). The following peptide concentrations were added to the receptors: 20, 40, 60, 80, 160, and 320 µM. Phosphorylation was performed in the presence of [gamma -32P]ATP. Samples were analyzed using a filter paper assay.

We hypothesized that incubation of this peptide with the insulin receptor would block p125Fak phosphorylation by the receptor. Thus, immunopurified p125Fak from overexpressing cells was incubated with or without the insulin receptor, in the presence of increasing peptide concentrations. Phosphorylation was measured by Western blotting with anti-phosphotyrosine antibodies. Fig. 8 shows that p125Fak peptide (568-582) inhibits p125Fak phosphorylation by the insulin receptor. Inhibition was nearly complete (almost 80%) at 250 µM peptide. We conclude from these experiments that the peptide competes with p125Fak for phosphorylation by the insulin receptor.


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Fig. 8.   p125Fak phosphorylation by the insulin receptor is inhibited by addition of the peptide corresponding to p125Fak sequence amino acids 568-582. p125Fak was immunoprecipitated from transfected 293 cells and mixed with the insulin receptor in the absence or in the presence of increasing peptide concentrations for 90 min at 4 °C. Insulin (0.1 µM) was then added and phosphorylation was performed in the presence of 60 µM [gamma -32P]ATP, 50 mM MgCl2. The reaction was stopped by addition of ice-cold buffer, and the pellets were washed twice. Phosphorylation of p125Fak was detected by Western blotting with antibodies to phosphotyrosine. We show a representative experiment of four experiments, each run in duplicate.

Phosphorylation of p125Fak on tyrosines 576 and 577 by the Src kinase has been shown to activate p125Fak enzymatic activity (45). Therefore, we tested the possibility that p125Fak phosphorylation by the insulin receptor could result in its activation. Overexpressed wild-type p125Fak was immunoprecipitated from 293 cells before being incubated without or with receptors, activated or not. Phosphorylation was performed using nonradioactive ATP. The p125Fak-containing pellets were washed extensively to remove the receptors, and the kinase activity of the protein was measured in the presence of [gamma -32P]ATP. The intrinsic kinase activity of p125Fak (determined in the absence of the receptor) is shown in Fig. 9. Addition of unstimulated receptor did not alter p125Fak kinase activity. However, phosphorylation by the ligand-stimulated receptor resulted in an increase in p125Fak activity. We conclude therefore that p125Fak is phosphorylated by the insulin receptor, and that this phosphorylation results, in vitro, in activation of its protein kinase.


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Fig. 9.   p125Fak phosphorylation by the insulin receptor stimulates its kinase. Wild-type p125Fak was immunoprecipitated from transfected 293 cells and mixed with the insulin receptor. Phosphorylation was performed in the presence of 60 µM nonradioactive ATP and 50 mM MgCl2. Pellets were washed six times to remove the receptor. p125Fak kinase activity was then measured in presence of 4 µM [gamma -32P]ATP and 10 mM MnCl2. The reaction was stopped by addition of Laemmli sample buffer before SDS-PAGE analysis.

To determine if, in intact cells, insulin-induced phosphorylation of p125Fak results in the activation of its kinase activity, we measured in parallel the phosphorylation of p125Fak and paxillin, which is thought to be a physiological substrate of p125Fak kinase. In these experiments, dual immunoprecipitation was performed using antibodies to p125Fak mixed with antibodies to paxillin. In Table I, we show that phosphorylation of paxillin varied similarly to that of p125Fak. Indeed, insulin induced dephosphorylation of both p125Fak and paxillin in attached Hep G2 hepatocytes, whereas in suspended Hep G2 cells p125Fak and paxillin phosphorylation was enhanced. We conclude, therefore, that in intact hepatocytes insulin may induce both p125Fak dephosphorylation and deactivation, or p125Fak phosphorylation and activation, depending on integrin engagement.

                              
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Table I
Paxillin phosphorylation correlates with p125Fak phosphorylation in intact cells
Hep G2 hepatocytes were left attached or were detached before insulin stimulation for various times and solubilization. Immunoprecipitation was performed using antibodies to p125Fak and antibodies to paxillin mixed on protein G-Sepharose. Tyrosine-phosphorylated proteins were revealed by immunoblotting using antibodies to phosphotyrosine. Two independent experiments were quantified by the Molecular Imager System.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The cytosolic tyrosine kinase p125Fak is thought to coordinate integrins and growth factors signaling pathways. Thus, tyrosine phosphorylation of p125Fak is modulated both by cell adhesion and by growth factor treatment.

Cross-talk between integrin and insulin signalings is suggested by the finding that insulin stimulation of DNA synthesis is modulated by engagement of alpha vbeta 3 integrin. Moreover, in this system, insulin promotes association of IRS-1 with the integrin (36). Finally, tyrosine-phosphorylated insulin receptors rapidly associate integrins at focal adhesion contacts (47).

In this paper, we present evidence that cell adhesion regulates the effect of insulin on p125Fak phosphorylation. This was observed in Rat-1 fibroblasts expressing high levels of insulin receptors, but also in Hep G2 hepatocytes and 3T3-L1 adipocytes, which are insulin-sensitive cells. Moreover, we show that insulin regulates the phosphorylation of both p125Fak and paxillin. Since paxillin is thought to be a physiological substrate of p125Fak tyrosine kinase, our results would indicate that p125Fak is activated by insulin. Paxillin may also be phosphorylated by c-Src, which becomes activated by p125Fak upon association to the autophosphorylated form of p125Fak. In this case, insulin-induced phosphorylation of paxillin could reflect the activation of the p125Fak-dependent signaling pathway.

Our present data, together with previously published ones, point to a complex interaction between signaling pathways of p125Fak and the insulin/IGF-I receptors. A recent study has shown that IRS-I phosphorylation by the activated insulin receptor induces its association with Csk (35). This protein is an inhibitor of c-Src, and could therefore abolish the input provided by Src. Another report indicates that SHP-2 is required for p125Fak dephosphorylation (48). SHP-2 is a phosphotyrosine phosphatase, which is stimulated by insulin through its association with phosphorylated IRS-1. Thus, inhibition of Src and activation of SHP-2 could cooperate to promote p125Fak dephosphorylation.

Alternatively, we show that p125Fak can also be a substrate for the insulin/IGF-I receptor kinases. Indeed, we found that (i) kinase-deficient p125Fak is directly phosphorylated by insulin and IGF-I receptors in vitro, and (ii) a peptide corresponding to p125Fak sequence amino acids 568-582 is phosphorylated by the insulin receptor. Moreover, this peptide inhibits phosphorylation of p125Fak by the insulin receptor. p125Fak sequence amino acids 568-582 is localized in the kinase domain regulatory loop, which corresponds to the subdomain VII highly conserved among tyrosine kinases (49). It contains the kinases' consensus motif VXXXDFG, and two twin tyrosines, 576 and 577. Tyrosine 576 corresponds to tyrosine 416 of the Src kinase, which is involved in regulation of Src catalytic activity (50, 51). The two tyrosine residues present in this subdomain are also found in one group of tyrosine kinase receptors, including the insulin and IGF-I receptors, Met, and Trk. Interestingly, these twin tyrosines are involved in regulation of the receptors' enzymatic activity. Autophosphorylation on these sites is correlated with increased kinase activity, and their mutation severely impairs this kinase activity (52-61). Tyrosines 576 and 577 of p125Fak are phosphorylated by the Src kinase, both in vivo and in vitro, a phosphorylation event that is associated with activation of p125Fak kinase activity (45). Similarly, phosphorylation by the Src kinase of tyrosines 1135 and 1136 in the kinase domain regulatory loop of the IGF-I receptor correlates with receptor activation (57). Our study provides further evidence for the existence of such cross-phosphorylation resulting in activation of a protein-tyrosine kinase, since we show that p125Fak is phosphorylated and stimulated by the insulin receptor.

The tyrosine kinase receptors Met and Trk both contain a kinase regulatory loop similar to that of the insulin receptor and p125Fak. Met is a potent activator of p125Fak in carcinoma cells, and this correlates with its effects on cellular migration (19). Hence, we suggest that transactivation of p125Fak may represent a common capability of this family of tyrosine kinase receptors.

IGF-I/insulin-induced dephosphorylation of p125Fak depends on integrin engagement, since it does not occur in cells that are not in an adhesion state. It is not clear which integrin(s) mediate(s) this response, but we hypothesize that, depending on which integrins are engaged, IGF-I/insulin-induced dephosphorylation of p125Fak may or may not occur. In contrast, phosphorylation of p125Fak in response to insulin or IGF-I is independent of integrin engagement. In our study, cell adhesion was disrupted by maintaining cells in suspension. Although this might correspond to an artificial system, it is a powerful way to study a particular cell response without interference of adhesion-mediated signals. Although complete suspension is unlikely to occur in most cells, adhesion-dependent responses will vary depending on which cell type is considered, which integrins are expressed, and which panel of integrins are engaged on a particular extracellular matrix. The integrin system is highly complex, and it will be necessary to decipher all signals transmitted by each integrin to obtain a comprehensive picture of the possible cooperations with hormone and growth factors signals.

We propose that, under particular cellular circumstances, the integrins, which control IGF-I- or insulin-induced dephosphorylation of Fak, will not be expressed or activated. In this case, only phosphorylation will occur, since this signal is independent from integrin engagement. This hypothesis is supported by recent work of Leventhal et al. (62), who reported IGF-I stimulation of p125Fak phosphorylation in SH-SY5Y neuronal cells associated with IGF-I-induced morphological changes and cell motility. Although this is in contrast with our observation of p125Fak dephosphorylation in attached fibroblasts overexpressing IGF-I receptors, it supports the idea that IGF-I is also able to stimulate p125Fak phosphorylation depending on the cellular context. This apparent difference may be due to the type of cell system used, or to the specific integrin involved in the motility of SH-SY5Y neuronal cells. Another type of modified cell situation is found in transformed cells, which migrate to different locations and metastasize. Interestingly, insulin or IGF-I treatment also appears to play an active role in dissemination of certain tumor cells, in combination with alpha vbeta 5 integrin (37).

To conclude, our data provide evidence that, in intact cells, insulin and IGF-I are able to induce p125Fak phosphorylation and activation, a result that would be compatible with a situation of positive cooperation between insulin/IGF-I and integrin signaling pathways. Moreover, cell adhesion appears to control the effect of insulin and IGF-I on p125Fak-dependent signaling pathways. We propose therefore that hormone/growth factor responses should be considered in a global cellular context, which has to take into account other cooperating sytems such as adhesion-dependent signaling systems.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Thomas J. Parsons for the generous gift of p125Fak cDNA. We thank Y. LeMarchand-Brustel and J. F. Tanti for helpful discussions, and I. Mothe, P. Gual, and R. Quarck for critical reading of the manuscript. We acknowledge J. Duch for secretarial assistance.

    FOOTNOTES

* This work was supported by INSERM, by Grant 93.123 from Groupe Lipha (Lyon, France), and by the Ligue Contre le Cancer ("Axe Oncogenese").The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Fax: 33-4-93-81-54-32; E-mail: baron{at}unice.fr.

1 The abbreviations used are: IRS, insulin receptor substrate; Fak, focal adhesion kinase; IGF-I, insulin-like growth factor-I; Shc, Src homology collagen; SHP-2, SH2-containing protein-tyrosine phosphatase-2; SH, Src homology; RHIR, Rat-1 human insulin receptor; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; BSA, bovine serum albumin; PBS, phosphate-buffered saline; BES, (N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic acid).

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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