Src-family Tyrosine Kinases in Activation of ERK-1 and p85/p110-phosphatidylinositol 3-Kinase by G/CCKBReceptors*

We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc·Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.

Gastrin was initially characterized as the major hormonal regulator of gastric acid secretion. This polypeptide also functions as a growth factor on the gastrointestinal tract in vivo (1). Its trophic effects have also been observed on several cancer cell lines derived from colon stomach and pancreas (2)(3)(4). Gastrin effects have been shown to be mediated by the gastrin/ CCK B (G/CCK B ) 1 receptor, which belongs to the family of G protein-coupled receptors (5,6).
Extracellular-regulated kinase-1 and -2 (ERK-1 and ERK-2) are two members of the mitogen-activated protein kinase family known to play an important role in cell proliferation. They have been shown to be activated by a variety of receptors including receptor tyrosine kinases and G protein-coupled receptors (GPCRs). Their activation by growth factor receptors with intrinsic tyrosine kinase activity has been well documented. The best understood mechanism involves the recruitment of the Grb2⅐Sos or Shc⅐Grb2⅐Sos complexes to tyrosinephosphorylated receptors. These complexes subsequently activate the following cascade: Ras/Raf/ERK kinases/ERKs (7).
Src-family tyrosine kinases have been proposed to serve as intermediates between GPCR and ERK activation. However, their involvement in this transduction pathway seems to be highly receptor-and cell type-specific. In some cells, lysophosphatidic acid and angiotensin receptors can activate ERKs via a mechanism that involves the tyrosine phosphorylation of Shc proteins by Src-family kinases (8 -11). In other cell types, neither Src nor Shc are required for ERK activation by these ligands (12). The mechanism leading to ERK activation by ␣ 1 -B, ␣ 2 -A adrenergic receptors, muscarinic acetylcholine, or bradykinin receptors also involves Src and Shc (9,13,14), whereas thrombin or formylmethionylleucylphenylalanine induce the ERK cascade independently of these two proteins (15,16). We and others have recently shown that gastrin modulates ERK-1/2 activity; however, the contribution of Src-family kinases and Shc proteins has never been studied (17)(18)(19).
Phosphatidylinositol 3-kinases (PI 3-kinases) are lipid kinases that phosphorylate phosphatidylinositols (PtdIns) at the D-3 position of the inositol ring. Several PI 3-kinase isoforms have been described in the literature. The first cloned PI 3-kinase was a heterodimer composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit, which has been implicated in the regulation of mitogenesis and cell transformation (20 -22). This enzyme has been found to be associated and activated by growth factor receptors (23,24). In addition, inhibition of PI 3-kinase activity by specific inhibitors or antibodies results in blockage of growth factor-induced cell proliferation (25,26). Activation of the p85/p110-PI 3-kinase by tyrosine kinase receptors such as platelet-derived growth factor or colony-stimulating factor-1 receptors involves the binding of the SH2 domain (for Src-Homology 2) of p85 to specific phosphotyrosine-containing sequences of the receptors (27,28). In contrast, the interaction of p85 with a tyrosine-phosphorylated intermediate protein, the insulin receptor substrate-1 (IRS-1), represents a mechanism for p85/p110-PI 3-kinase activation by insulin or insulin growth factor-1 (29,30). Quite recently, novel forms of PI 3-kinases have been characterized. They do not interact with p85 and are directly activated by GPCRs and ␤␥ subunits of G proteins (31)(32)(33).
Several laboratories including ours have reported that some GPCRs such as the G/CCK B are capable of activating the p85/ p110-PI 3-kinase (34 -36). However, very little is known about the mechanism of activation.
In this study, we have analyzed the contribution of Srcfamily tyrosine kinases to the activation of ERK-1 or p85/ p110-PI 3-kinase pathways by gastrin. We report here that Shc tyrosine phosphorylation by Src-family tyrosine kinases and Shc⅐Grb2 complex formation are early events in ERKs activation via the GPCR, G/CCK B . Similarly, Src-family tyrosine kinases, by phosphorylating IRS-1, play an essential role in the gastrin-mediated p85/p110-PI 3-kinase-signaling cascade.

EXPERIMENTAL PROCEDURES
Cell Culture-Chinese hamster ovary (CHO) cells, stably transfected with an expression plasmid encoding the human G/CCK B receptor and designated as control cells, were grown in ␣-minimum essential medium supplemented with 10% fetal calf serum and 200 g/ml G418 at 37°C in a 95% air, 5% CO 2 atmosphere.
Expression Vector and Transfection-The dominant-negative Shc construct, consisting of the isolated SH2 domain of Shc, or the cDNA encoding Csk were cotransfected with pTK-Hyg selection vector in CHO cells expressing the human G/CCK B receptor using the Fugene TM 6 reagent. Transfected cell lines obtained after 3 weeks of hygromycin selection were characterized and compared with control cells. The pL-SH2-Shc-SN vector was a gift from Drs I. Baldari and P. G. Pelicci (Siena, Italia). The cDNA encoding Csk was from Dr. S. Roche (Montpellier, France).
Immunoprecipitations and Western Blotting Analysis-Control-, Shc-SH2-, or Csk-transfected CHO cells were serum-starved for 18 h before peptide addition. After stimulation, the cells were lysed, and the soluble fractions, containing identical levels of proteins, were immunoprecipitated as described previously (36) with the indicated antibodies. Proteins from immunoprecipitates or whole cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Membranes were blotted as described previously (36) with the indicated antibodies. Proteins were visualized by using 125 I-protein-A followed by autoradiography.
Src-like Kinase Assays-Immunoprecipitates were washed twice with lysis buffer and 3 times with TBS (25 mM Tris, pH 7.5, 150 mM NaCl, 0.2 mM vanadate). Kinase assays were carried out at 30°C for 10 min in 40 l of kinase buffer containing 20 mM Hepes, pH 7.5, 10 mM MnCl 2 , 1 mM dithiothreitol, 3.75 M ATP, 5 Ci of [␥-32 P]ATP, and 5 M acid-denatured enolase as an exogenous substrate. Samples were analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. The gels were then treated with 1 N KOH at 50°C for 1 h after electrophoresis and autoradiographed.
Immune Complex Assays for ERK-1 Activation-As described previously (18), lysates immunoprecipitated with specific anti-ERK-1 antibodies were incubated for 30 min at room temperature in reaction buffer containing myelin basic protein (0.2 mg/ml) and [␥-33 P]ATP. The reaction was stopped as described above under "Csk Kinase Assays." Phosphatidylinositol 3-Kinase Assay-As described previously (36), the immunoprecipitates were resuspended in reaction buffer. The kinase assay was started by addition of 50 M [␥-32 P]ATP and PtdIns at a final concentration of 0.2 mg/ml. After 15 min, the reaction was stopped by the addition of 4 M HCl, and the phosphoinositol lipids were extracted with chloroform/methanol (1:1). The phospholipids were separated by thin layer chromatography and analyzed by autoradiography.
[ 3 H]Thymidine Incorporation-DNA synthesis was estimated by measurement of [ 3 H]thymidine incorporation into the trichloroacetic acid-precipitable material. The [ 3 H]thymidine (0.6 Ci/ml) was added during the last 3 h of the 24-h treatment period. The cells are washed with serum-free medium to remove unincorporated [ 3 H]thymidine. DNA was precipitated with 10% trichloroacetic acid at 4°C for 15 min. Precipitates were washed twice with 95% ethanol, dissolved in 0.5 ml of 0.1 N NaOH, and analyzed in a liquid scintillation counter.

Role of Shc Proteins and Src-family Tyrosine Kinases in
Src tyrosine kinase activity has been previously shown to be highly regulated by tyrosine phosphorylation. Phosphorylation of the C-terminal noncatalytic tail maintained the enzyme in an inactive state, whereas the active conformation corresponded to the phosphorylation of the kinase domain (38). Previous studies reported the tyrosine phosphorylation of Srclike proteins in response to gastrin (39,40). However, whether this phosphorylation is inhibitory or stimulatory remains to be determined. We first examined the ability of gastrin to regulate the kinase activity of Src in CHO cells transfected with the G/CCK B receptor. After treatment of the cells with 10 nM gastrin, the ability of Src to phosphorylate the exogenous substrate, enolase, was measured in anti-Src immunoprecipitates as described under "Experimental Procedures." The kinetics of Src activation by gastrin was rapid, reached a peak value at 1 min (3.8-fold Ϯ 0.4), and decreased subsequently (Fig. 1B,  upper panel). To analyze the role of Src-family tyrosine kinases in the intracellular mechanisms activated by gastrin, we used a negative regulatory protein of Src, Csk, which inactivates Src-family kinases by phosphorylation (38). Overexpression of Csk in CHO cells (Fig. 1C) completely blocked gastrin-induced Src kinase activity (Fig. 1B, upper panel). Half of the lysate used for the kinase assay was immunoprecipitated with an anti-Src antibody and immunoblotted with the same antibody to verify that similar amounts of Src were recovered in controland Csk-transfected cells (Fig. 1B, lower panels).
To determine the contribution of Src-family kinases to gastrin-induced ERKs stimulation, we measured ERK-1 activity in CHO cells overexpressing Csk. Blockage of gastrin-induced Src kinase activity by Csk also resulted in a significant but partial inhibition (52% Ϯ 11%) of ERK-1 activation by gastrin (Fig.  1D).
Role of Src-family Tyrosine Kinases in Shc⅐Grb2 Complex Formation Induced by Gastrin- Fig. 2A depicts the effects of Csk overexpression on Shc⅐Grb2 complex activation induced by gastrin. In control cells, the maximal effects of gastrin on tyrosine phosphorylation of Shc ( Fig. 2A, left) and the association between Shc and Grb2 ( Fig. 2A, right) were, respectively, 2.5-fold Ϯ 0.2 and 2.8-fold Ϯ 0.3 of the basal level. In Csk-transfected cells, tyrosine phosphorylation of Shc ( Fig. 2A, left) and the association between Shc and Grb2 ( Fig. 2A, right) in response to gastrin were reduced by, respectively, 49% Ϯ 4% and 56% Ϯ 5% compared with control cells, indicating the involvement of Src-family tyrosine kinases for maximal gastrin effect on Shc⅐Grb2 complex formation. Reprobing of the blots with the same antibodies used for immunoprecipitation revealed equal amounts of protein in precipitates from controland Csk-transfected cells ( Fig. 2A, lower panels).
We then examined whether Src-like tyrosine kinase activity was detected in association with Shc proteins following gastrin stimulation. Tyrosine kinase activity in Shc immunoprecipitates was measured by using enolase as an exogenous substrate. A 4.65 Ϯ 0.35-fold increase in the kinase activity was detected in anti-Shc precipitates after gastrin stimulation and was abolished in CHO cells expressing high levels of Csk proteins (Fig. 2B, upper panel). Half of the lysate used for the kinase assay was immunoprecipitated with an anti-Shc antibody and immunoblotted with the same antibody to verify that similar amounts of Shc were recovered in control-and Csktransfected cells (Fig. 2B, lower panel).
Role of Src-family Tyrosine Kinases in p85/p110-PI 3-Kinase Activation Induced by Gastrin-The mechanisms leading to the activation of the p85/p110-PI 3-kinase by GPCRs are still poorly understood. To determine whether activation of G/CCK B receptors expressed in CHO cells stimulates the p85/p110-PI 3-kinase, PI 3-kinase activity was measured in anti-p85 immunoprecipitates following gastrin treatment. We observed a rapid increase in PI 3-kinase activity in response to gastrin. The activation reached a maximum at 1 min (3.6-fold Ϯ 0.3) A, lower panels, the blots were stripped and reprobed with the same antibody used for immunoprecipitation to ensure equal loading of the immunoprecipitated proteins. Representative autoradiograms from three independent experiments are shown. B, control-or Csk-transfected cells were incubated for varying lengths of time with 10 nM gastrin. B, upper panel, the Src-related kinase activity toward the exogenous substrate enolase was determined in an immune complex kinase assay using anti-Src antibodies. A representative autoradiogram from five separate experiments is shown. B, lower panel, half of the lysate used for the kinase assay was immunoprecipitated with an anti-Src antibody and immunoblotted with the same antibody to verify that similar amounts of Src were recovered in control-and Csk-transfected cells. C, left, whole cell lysates from control-or Csk-transfected cells were resolved by SDS-polyacrylamide gel electrophoresis and immunoblotted (IB) with an anti-Csk antibody. C, right, lysates from control-and Csk-transfected cells were immunoprecipitated (IP) with anti-Csk antibody. Csk activity was assessed by phosphorylation of poly(Glu-Tyr) as described under "Experimental Procedures." Data are presented as net 33 P cpm incorporated into poly(Glu-Tyr) and represent the mean ϮS.E. from three independent experiments. D, control cells-, Shc-SH2-, or Csk-transfected cells were incubated with 10 nM gastrin for 3 min. ERK-1 activity was measured as described under "Experimental Procedures." The data represent the mean ϮS.E. from five independent experiments. and decreased thereafter (Fig. 3, left panel). Gastrin-induced PI 3-kinase activation in anti-p85 immunoprecipitates was completely blocked in Csk-transfected cells (Fig. 3, right panel), suggesting the involvement of Src-like tyrosine kinases in the mechanism of PI 3-kinase activation by gastrin. We did not detect any increase in the tyrosine phosphorylation of the p85 subunit of PI 3-kinase in gastrin-treated cells (data not shown).
Role of Src-family Tyrosine Kinases Upstream of IRS-1 and the PI 3-Kinase-Western blot analysis with anti-phosphotyrosine antibodies from immunoprecipitates obtained with anti-IRS-1 antibodies revealed an increased tyrosine phosphorylation of IRS-1 following gastrin stimulation (Fig. 4A). In addition, PI 3-kinase activity was detected in association with IRS-1 after treatment of the cells with 10 nM of gastrin (Fig. 4B,  left panel). The kinetics of activation, maximal at 1 min (2.6 Ϯ 0.3-fold), could be correlated to the rapid phosphorylation of IRS-1, suggesting that the tyrosine phosphorylation of IRS-1, which leads to the rapid recruitment of the p85/p110-PI 3-kinase, might be responsible for PI 3-kinase activation by gastrin. Both the tyrosine phosphorylation of IRS-1 and the PI 3-kinase activity in anti-IRS-1 precipitates observed in response to gastrin were abolished by Csk overexpression (Fig.  4A and Fig. 4B, right panel), suggesting that Src-family tyrosine kinases act upstream of the IRS-1/PI 3-kinase pathway activated by gastrin.
We then analyzed whether Src-like tyrosine kinases could associate IRS-1 in response to gastrin. For this purpose, cells were stimulated with 10 nM gastrin for various times and lysed as described under "Experimental Procedures." IRS-1 proteins were immunoprecipitated with specific antibodies, and their association with Src kinases was analyzed by Western blot with anti-Src antibodies. An increase in the amount of Src proteins coprecipitated with IRS-1 was detected in response to gastrin (Fig. 4C). In addition, we observed a 2.8 Ϯ 0.3-fold increase of Src-like kinase activity in anti-IRS-1 immunoprecipitates from cells treated with gastrin (Fig. 4D), suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src⅐IRS-1 complex formation in Csk-transfected cells (Fig. 4, C and D). The same amounts of proteins in precipitates from control-or Csk-transfected cells were confirmed by Western blots in all these experiments with appropriate antibodies (lower panels).
Role of Shc Proteins and Src-family Tyrosine Kinases in Gastrin-induced DNA Synthesis-Gastrin has been previously Control-or Csk-transfected cells were treated for the times indicated with 10 nM gastrin. Cell lysates were immunoprecipitated (IP) with an anti-p85 antibody, and precipitates were assayed for PI 3-kinase activity using PtdIns as substrate. The phospholipids were resolved on thin layer chromatography plates. PIP indicates the migration of the phosphorylated substrate, PtdIns-P. Lower panel, the autoradiograms were densitometrically analyzed, and the data were plotted as fold stimulation. Data from four separate autoradiograms are presented as means ϮS.E.

FIG. 2. Role of Src-family tyrosine kinases in Shc⅐Grb2 complex formation induced by gastrin.
CHO cells were stably transfected with the human G/CCK B receptor, alone (control), or with Csk. Control-or Csk-transfected cells were stimulated (ϩ) or not (Ϫ) with 10 nM gastrin. A, upper panels, cell lysates were immunoprecipitated (IP) with anti-Shc or anti-Grb2 antibodies and immunoblotted (IB) with, respectively, anti-phosphotyrosine (Py) or anti-Shc antibodies. A, lower panels, the blots were stripped and reprobed with the same antibody used for immunoprecipitation to ensure equal loading of the immunoprecipitated proteins. Representative autoradiograms from three independent experiments are shown. B, upper panel, Shc immunoprecipitates were assayed in vitro for Src-like tyrosine kinase activity using enolase as a substrate as described under "Experimental Procedures." A representative autoradiogram from five separate experiments is shown. B, lower panel, half of the lysate used for the kinase assay was immunoprecipitated with an anti-Shc antibody and immunoblotted with the same antibody to verify that similar amounts of Shc were recovered in control-and Csk-transfected cells.
shown to be mitogenic for CHO cells expressing the G/CCK B receptors (41). Therefore we examined the importance of Shc proteins and Src-family tyrosine kinases in mitogenic signaling induced by this peptide. Overexpression of the deficient Shc mutant or Csk proteins resulted in a significant inhibition (respectively 63% Ϯ 3 and 82% Ϯ 6) of the increase in [ 3 H]thymidine incorporation induced by gastrin (Fig. 5), indicating that Shc phosphorylation and Src-family kinase activation are two events involved in growth stimulation by this mitogen.

DISCUSSION
The intracellular mechanisms activated by the G/CCK B receptors are not completely elucidated; particularly the early events upstream of gastrin-activated signaling pathways remain to be characterized. The important role of Src-family tyrosine kinases in the transmission of mitogenic signals induced by growth factors whose receptors possess intrinsic tyrosine kinase activity is well documented (38). In quiescent NIH-3T3 fibroblasts, kinase-inactive Src prevents platelet-derived growth factor-induced DNA synthesis. In a similar fashion, cells expressing inactive forms of Src and Fyn are unable to respond to proliferative signals. Overexpression of Src enhances the mitogenic response to epidermal growth factor treatment, whereas expression of Src inhibitory mutants decreases the response.
In the present study, we demonstrate that gastrin, acting through a GPCR, rapidly and transiently stimulates the enzymatic activity of Src. We also analyzed the downstream targets phosphorylated and activated by this tyrosine kinase following gastrin stimulation. The increase in Src-like kinase activity observed in anti-Shc immunoprecipitates from gastrin-stimulated cells suggested that Shc may be a direct substrate of Src, phosphorylated on tyrosine residues in response to gastrin. This hypothesis was supported by the inhibition of gastrininduced Shc phosphorylation and Shc⅐Grb2 complex formation in CHO cells transfected with Csk, known to inactivate Srcfamily tyrosine kinases. Our data also demonstrate that maximal activation of ERK-1 by gastrin requires Shc phosphorylation by Src-family tyrosine kinases. Indeed, we observed a FIG. 4. Involvement of Src-family tyrosine kinases in the IRS-1/PI 3-kinase pathway activated by gastrin. CHO cells were stably transfected with the human G/CCK B receptor, alone (control), or with Csk. A, control-or Csk-transfected cells were stimulated (ϩ) or not (Ϫ) with 10 nM gastrin for 1 min. A, upper panel, cellular proteins were immunoprecipitated (IP) with an antibody against IRS-1 and immunoblotted (IB) with an anti-phosphotyrosine (Py) antibody. A, lower panel, the blots were stripped and reprobed with the same antibody used for immunoprecipitation to ensure equal loading of the immunoprecipitated proteins. B, control-or Csk-transfected cells were incubated or not with 10 nM gastrin for the times indicated. Cell lysates were immunoprecipitated (IP) with an anti-IRS-1 antibody, and precipitates were assayed for PI 3-kinase activity using PtdIns as substrate. The phospholipids were resolved on thin layer chromatography plates. PIP indicates the migration of the phosphorylated substrate, PtdIns-P. B, lower panel, the autoradiograms were densitometrically analyzed, and the data were plotted as fold stimulation. Data from four separate autoradiograms are presented as means ϮS.E. C, control-or Csk-transfected cells were stimulated with 10 nM gastrin as indicated. C, upper panel, cell lysates were immunoprecipitated (IP) with antibodies against IRS-1 and immunoblotted (IB) with an anti-Src antibody. C, lower panel, the blots were stripped and reprobed with the same antibody used for immunoprecipitation to ensure equal loading of the immunoprecipitated proteins. D, control-or Csk-transfected cells were stimulated (ϩ) or not (Ϫ) with 10 nM gastrin for 1 min. D, upper panel, IRS-1 immunoprecipitates were assayed in vitro for Src-like tyrosine kinase activity using enolase as a substrate as described under "Experimental Procedures." A representative autoradiogram from five separate experiments is shown. D, lower panel, half of the lysate used for the kinase assay was immunoprecipitated with an anti-IRS-1 antibody and immunoblotted with the same antibody to verify that similar amounts of IRS-1 were recovered in control-and Csk-transfected cells. decrease of gastrin-induced ERK-1 activation in CHO cells expressing Csk or a dominant inhibitory mutant of Shc. However, the partial inhibition obtained in these transfected cells suggests the existence of an additional pathway for gastrinmediated ERK-1 stimulation independent of Shc and Src-family tyrosine kinases.
The focal adhesion kinase-related protein tyrosine kinase (PYK2) as well as Bruton's tyrosine kinase, which belongs to a family of Pleckstrin homology domain-containing tyrosine kinases, have also been proposed to link GPCRs to the activation of the ERKs cascade (9,42). However, it seems that these nonreceptor tyrosine kinases interact with Src-family kinases to regulate their activity (9,43,44). Therefore, they probably do not contribute to the Src-independent pathway, which relays the signal from G/CCK B receptors to the activation of ERK-1. Kranenburg et al. (12) also observe a regulation of the ERK pathway by GPCRs that do not involve Src and Shc proteins. Their findings suggest that a wortmannin-sensitive PI 3-kinase, whose activity is independent on Src-family kinases, links G i -coupled receptors to ERKs activation. In the present study, we demonstrate in CHO cells transfected with the G/CCK B receptor that gastrin-mediated PI 3-kinase activation is totally dependent on Src-family tyrosine kinases. Consequently, PI 3-kinase cannot play a role in the Src-independent pathway that leads to ERK-1 activation by gastrin. Various isoforms of protein kinase C are capable of directly activating Raf-1 by phosphorylation (45,46). Raf-1 subsequently phosphorylates the ERK kinases, which in turn activates the ERKs. This pathway, which requires neither the activation of tyrosine kinases nor the formation of the complex Shc⅐Grb2⅐Sos⅐Ras, may be involved in the Src-independent pathway that links G/CCK B receptors to ERK-1 activation.
Recently, several studies have reported the activation of the p85/p110 isoform of PI 3-kinase by GPCRs including chemoattractant receptors, angiotensin receptors, and adrenergic receptors (34,35,47). However, very little is known about the molecular basis of this activation. Activation of the enzyme in chemoattractant-stimulated neutrophils seems to involve direct binding of the Src-related kinase, Lyn, to the p85 subunit of PI 3-kinase. This interaction might occur via the SH3 domain (for Src-Homology 3) of Lyn and the prolinerich domain of the p85 subunit as described previously for the activation of the PI 3-kinase by B cell antigen receptor (47)(48)(49). In the present study, we describe a different mechanism for the activation of the p85/p110-PI 3-kinase by Src-family kinases, which uses IRS-1 as a docking molecule. Indeed, we demonstrate by Western blot that gastrin induces a rapid tyrosine phosphorylation of IRS-1 as well as the association of this protein with Src. We also observed an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src, phosphorylated on tyrosine residues in response to gastrin. This hypothesis was supported by the inhibition of gastrin-induced Src⅐IRS-1 association and IRS-1 phosphorylation in CHO cells transfected with Csk. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation were abolished by Csk overexpression. The tyrosine phosphorylation of IRS-1 in response to thrombin, which binds to GPCRs, has also been observed (50). However, the role of this phosphorylation remains to be determined. Angiotensin-1 receptors also belong to the family of GPCRs. They have been shown to mediate the tyrosine phosphorylation of IRS-1 via JAK2 tyrosine kinase, which associates with AT1 receptor and IRS-1 after angiotensin stimulation. Surprisingly, in this study an inhibition of the PI 3-kinase activity was observed after the association of the p85 regulatory subunit to IRS-1 (51). In contrast, our observations suggest that the tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.