Src Phosphorylates Grb2-associated Binder 1 upon Hepatocyte Growth Factor Stimulation*

Grb2-associated binder 1 (Gab1) is known to play an important role in hepatocyte growth factor (HGF) signaling, which rapidly becomes tyrosine-phosphorylated upon HGF stimulation. In this study, we found that the tyrosine phosphorylation of Gab1 in the cells derived from Src/Yes/Fyn null mouse embryos was ∼40% lower than that in their wild type counterparts upon HGF stimulation. Increased expression of wild-type Src enhanced HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kinase-deficient mutant or treatment of the specific Src inhibitor PP1 suppressed it. Expression of a constitutively active Src mutant (Y527F) or oncogenic v-Src led to a prominent increase in Gab1 phosphorylation independent of HGF stimulation. Moreover, Src interacted with Gab1 via both its Src homology 2 and 3 domains and was capable of phosphorylating purified Gab1 in vitro. Finally, the increased phosphorylation of Gab1 by Src selectively potentiated HGF-induced activation of ERK and AKT. Taken together, our results establish a new role for Src in HGF-induced Gab1 phosphorylation.

Hepatocyte growth factor (HGF), 1 also known as scatter factor, is a mesenchymally derived factor that elicits mitogenic, motogenic, and morphogenic activities on various cell types (1). The diverse biological effects of HGF are transmitted through activation of its transmembrane receptor encoded by the c-met proto-oncogene (2,3). In vivo, HGF/c-Met signaling plays critical roles in embryogenesis (4,5), angiogenesis (6), organ regeneration (7), and tumorigenesis (8). The Met receptor is a heterodimer composed of a 45-kDa ␣ chain that remains entirely extracellular and a 145-kDa ␤ chain that traverses the plasma membrane and contains the intracellular tyrosine kinase domain (9 -11). Upon HGF binding, the intrinsic tyrosine kinase of the receptor is activated, resulting in autophosphorylation on specific tyrosine residues in the ␤ chain (12,13). Two tyrosine residues in the COOH terminus of the ␤ chain (Tyr 1349 and Tyr 1356 ) are required for all biological activities of the receptor (14,15) and serve as docking sites for the Grb2associated binder-1 (Gab1) docking protein (16 -18) and multiple Src homology 2 (SH2) and phosphotyrosine binding domaincontaining proteins, including phosphatidylinositol 3-kinase, Src, Grb2, Shc, and STAT3 (19 -21).
Gab1 is the major phosphorylated protein downstream of the Met receptor in epithelial cells (18), which belongs to a family of docking proteins, including Gab2 and Gab3 in mammals, Dos in Drosophila melanogaster, and SOC-1 in Caenorhabditis elegans (22). Gab1 contains an NH 2 -terminal pleckstrin homology domain, a Met-binding domain, six proline-rich sequences, and 16 potential tyrosine phosphorylation sites that represent SH2-binding motifs (23,24). Upon HGF stimulation, Gab1 associates with the p85 regulatory subunit of phosphatidylinositol 3-kinase, phospholipase C␥, the tyrosine phosphatase SHP-2, and the adaptor protein Crk and acts to recruit these signaling proteins to the Met receptor (25)(26)(27)(28). Overexpression of Gab1 in epithelial cells induces ligand-independent morphogenesis characteristic of c-Met activation (23). In addition, Gab1 is also phosphorylated after stimulation of cells by several other growth factors, such as epidermal growth factor (29,30), insulin (31), nerve growth factor (32), and basic fibroblast growth factor (33) as well as by different cytokines and antigen receptors (34). The gab1 gene knockout is lethal to mouse embryos, and the fibroblasts derived from Gab1 Ϫ/Ϫ mouse embryos are defective in extracellular signal-regulated kinase (ERK) activation in response to multiple growth factors and the cytokine IL-6 (35,36).
Gab1 is recruited to the Met receptor by both direct and indirect mechanism. The minimal amino acid sequence in the Met-binding domain of Gab1 sufficient for direct binding to the Tyr 1349 and to a lesser extent to the Tyr 1356 of the Met, called the Met-binding sequence, comprises 13 amino acids (positions 487-499) and is absent in other Gab proteins (37). Because the Met-binding sequence lacks obvious sequence similarity to other SH2 and phosphotyrosine binding domains, it may represent a novel type of phosphotyrosine binding domain. However, most Gab1-Met interaction is mediated indirectly via the adaptor protein Grb2. Two of the proline-rich motifs of Gab1 mediate the binding of the COOH-terminal SH3 domain of Grb2 (18). One of these is a classical Grb2 SH3 binding site ( 342 PXXPXK 347 ), but the other ( 518 PXXXRXXKP 526 ) fails to conform to this motif (24). Grb2 also contains an SH2 domain, which targets the constitutive Grb2-Gab1 complex to the phosphorylated Tyr 1356 of the Met receptor (18).
The tyrosine kinase c-Src is activated in response to HGF (19,38) and plays a crucial role in HGF-induced cell motility and anchorage-independent growth (39,40). The SH2 domain of c-Src binds either Tyr 1349 or Tyr 1356 of the Met receptor with fast association and dissociation rates (19). In this study, we set out to examine the role of Src in HGF-induced Gab1 phosphorylation. Our study reveals that Src binds to and phosphorylates Gab1 in response to HGF, which contributes to HGFinduced activation of ERK and AKT.
Plasmids-The expression plasmid pcDNA3-HA-Gab1 was kindly provided by Dr. Toshio Hirano (Osaka University). The plasmids pEVX-cSrc, pEVX-Srckd, pEVX-SrcY527F, and pM-vSrc were obtained from Dr. David Shalloway (Cornell University, Ithaca, NY). The plasmid pMT2-Tpr-Met was provided by Dr. Paolo M. Comoglio (University of Torino, Italy). The plasmids encoding GST fusion proteins containing the SH2 domains of Src, phosphatidylinositol 3-kinase, and Grb2 and the SH3 domains of Src and Grb2 were described previously (41,42). To construct pGEX2T-Gab1, the cDNA encoding Gab1 was amplified by polymerase chain reaction with primers 5Ј-GATGAGCGGTGGTGAAG-TG-3Ј and 5Ј-TCATTTCACACTCTTCGCTG-3Ј using pcDNA3-HA-Gab1 as a template, which was then purified and cloned in frame to the SmaI site of pGEX2T vector (Amersham Biosciences). The Gab1 P449A/ P452A mutant was generated using site-directed mutagenesis by overlap extension using the polymerase chain reaction with mutagenic primers. The mutagenic primers were 5Ј-CGTCGCAATGAATGCCAA-TT-3Ј and 5Ј-AATTGGCATTCATTGCGACG-3Ј. The underlines indicate the positions of substituted bases. The flanking primers were 5Ј-CCTGGAAACTCTTGGCATTC-3Ј and 5Ј-TTACTAGTGGATCCGAGC-TC-3Ј. The polymerase chain reaction products were cloned to the EcoRV site of pBluescript II vector (Stratagene, La Jolla, CA). The desired mutations were confirmed by dideoxy-DNA sequencing. The cDNA encoding the Gab1 P449A/P452A mutant in pBluescript II vector was used to replace the corresponding fragment in the plasmid pcDNA3-HA-Gab1 using the BamHI site.
Cell Culture and Transfections-Cells used in this study were all maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. SYF (src Ϫ/Ϫ yes Ϫ/Ϫ fyn Ϫ/Ϫ ) cells, wild type control cells, and the SYF cells stably reexpressing chicken c-Src were described previously (43). Madin-Darby canine kidney (MDCK) cells stably expressing the Src kd mutant were established by co-transfection of pcDNA3 with pEVX-Src kd (in a 1:10 ratio) and selection in the medium containing 0.5 mg/ml G418. NIH3T3 cells stably expressing c-Src, SrcY527F mutant, or v-Src were established by co-transfection of pcDNA3 with pEVX-cSrc, pEVX-SrcY527F, or pM-vSrc and selection in the medium containing 0.5 mg/ml G418. For transient transfections, human embryonic kidney (HEK) 293 cells or SYF cells (5 ϫ 10 5 ) were seeded on a 6-cm culture dish. 18 h later, the cells were incubated with the mixture of plasmid (1-2 g) and LipofectAMINE (10 l) for 5 h and lysed in 1% Nonidet P-40 lysis buffer 24 h later. For HGF stimulation, cells were serum-starved for 18 h and treated with 10 ng/ml HGF for 15 min.
Immunoprecipitations, Immunoblotting, and in Vitro Kinase Assays-Cells were lysed in 1% Nonidet P-40 lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, and 1 mM Na 3 VO 4 ) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitory units/ml aprotinin, and 20 g/ml leupeptin). The lysates were centrifuged for 10 min at 4°C to remove debris, and the protein concentrations were determined using the Bio-Rad protein assay. For immunoprecipitation, aliquots of lysates were incubated with l g of polyclonal anti-Gab1, anti-Met, or anti-Src (N-16) or monoclonal anti-HA for 1.5 h at 4°C. Immunocomplexes were collected on Protein A-Sepharose beads. For monoclonal antibodies, Protein A-Sepharose beads were coupled with rabbit anti-mouse IgG (1 g) before use. The beads were washed three times with 1% Nonidet P-40 lysis buffer, boiled for 3 min in SDS sample buffer, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher and Schuell). Immunoblotting was performed with appropriate antibodies using the Amersham Biosciences enhanced chemiluminescence system for detection.
To perform in vitro kinase assays, the immunoprecipitates by polyclonal anti-Met, anti-Src, or anti-C-terminal Src kinase were washed three times with 1% Nonidet P-40 lysis buffer and once in 20 mM Tris buffer. Kinase reactions were carried out in 40 l of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MnCl 2 ) containing 10 Ci of [␥-32 P]ATP (3000 Ci mmol Ϫ1 ; PerkinElmer Life Sciences) and 1 g of purified glutathione S-transferase (GST) or GST-Gab1 fusion proteins for 20 min at 25°C. Reactions were terminated by the addition of SDS sample buffer, and proteins were resolved by SDS-polyacrylamide gel electrophoresis. To prepare soluble GST-Gab1 fusion proteins, immobilized GST-Gab1 proteins on glutathione-agarose beads were eluted by 10 mM reduced glutathione in 50 mM Tris buffer (pH 8.0).
In Vitro Binding Assays-GST fusion proteins were immobilized on glutathione-agarose beads and then incubated with HEK293 cell lysates containing HA epitope-tagged Gab1 proteins in 1% Nonidet P-40 lysis buffer for 1 h at 4°C. The complexes were washed four times with 1% Nonidet P-40 lysis buffer, resolved by SDS-polyacrylamide gel electrophoresis, and analyzed by immunoblotting with anti-HA.

Deficiency in the Src Family Kinases Decreases HGF-induced
Gab1 Phosphorylation-Although Gab1 associates with activated Met and is phosphorylated on specific tyrosine residues in response to HGF stimulation, it is not clear whether Met is the only kinase responsible for HGF-induced Gab1 phosphorylation. To explore the role of the Src family kinases in this event, the tyrosine phosphorylation of Gab1 was first compared between Src/Yes/Fyn (SYF) null cells and their wild type counterparts. When the cells were grown in the medium supplemented with 10% serum, the tyrosine phosphorylation of Gab1 in SYF cells was only 20% of that in the control cells (Fig. 1A). To evaluate the relative contribution of the Src family kinases to HGF-induced Gab1 phosphorylation, SYF cells and the control cells were serum-starved and then treated with HGF for 15 min. Although HGF stimulation increased the phosphorylation of Gab1 in both SYF cells and the control cells, the Gab1 phosphorylation in SYF cells was ϳ40% lower than that in the control cells (Fig. 1B). Reexpression of c-Src into SYF cells enhanced the ability of HGF to stimulate Gab1 phosphorylation (Fig. 1C). These results together suggest that in addition to Met, Src family kinases account partially for Gab1 phosphorylation upon HGF stimulation.
Increased Expression of c-Src Enhances HGF-induced Gab1 Phosphorylation, and, in Contrast, Expression of Src Kinasedeficient Mutant Suppresses It-To examine the effect of c-Src overexpression on HGF-induced Gab1 phosphorylation, HAepitope-tagged Gab1 (HA-Gab1) was transiently co-expressed with c-Src or its kd mutant in HEK293 cells ( Fig. 2A). In the absence of HGF stimulation, increased expression of c-Src by itself was not sufficient to induce Gab1 phosphorylation. Upon HGF stimulation, elevated expression of c-Src substantially promoted HGF-induced phosphorylation of Gab1, and, in contrast, expression of the Src kd mutant suppressed it. To exclude the possibility that the effect of c-Src or its kd mutant on Gab1 phosphorylation was indirectly through its modulation on the activity of Met, thereby leading to alteration on Gab1 phosphorylation, the activation of Met was measured by immunoblot-ting with an antibody specific for the phosphorylation of two tyrosine residues (Tyr 1234 and Tyr 1235 ) in the activation loop of Met. Clearly, neither the activity nor the expression of Met was affected by transient expression of c-Src or its kd mutant in HEK293 cells ( Fig. 2A). Consistently, stable expression of Src kd mutant in MDCK cells significantly suppressed the total Src activity and Gab1 phosphorylation without an effect on Met activation (Fig. 2B). Accordingly, HGF-induced Gab1 phosphorylation was ϳ60% inhibited by the specific Src inhibitor PP1 in MDCK cells (Fig. 2C). Notably, PP1 substantially inhibited the activating phosphorylation of Src at Tyr 416 but had no effect on Met activation induced by HGF stimulation (Fig. 2C).
Oncogenic Src Persistently Induces Gab1 Phosphorylation Independent of HGF Stimulation-To examine the effect of oncogenic Src on Gab1 phosphorylation, HA-Gab1 was co-expressed with the activated c-Src mutant (Y527F) or v-Src in HEK293 cells (Fig. 3A). After 18 h of serum starvation, the tyrosine phosphorylation of Gab1 in the control cells was hardly detected, whereas it remained high in the cells express-ing SrcY527F mutant or v-Src independent of HGF stimulation. The level of Gab1 phosphorylation induced by SrcY527F mutant or v-Src was comparable with that induced by the oncogenic form of Met (i.e. Tpr-Met), which was at least 10-fold higher than that induced by HGF treatment (Fig. 3A). Accordingly, NIH 3T3 cells transformed by SrcY527F mutant or v-Src had a prominent increase in Gab1 phosphorylation even in the absence of HGF stimulation (Fig. 3B). These results indicate that oncogenic Src persistently leads to Gab1 phosphorylation independent of HGF stimulation. It is noteworthy that Met was activated by the expression of SrcY527F mutant or v-Src, rendering it likely that the ability of oncogenic Src to induce such profound phosphorylation on Gab1 may be partially through its activation toward Met.
Src Directly Phosphorylates and Associates with Gab1-Experiments to this point clearly indicate that Src is involved in HGF-induced Gab1 phosphorylation. To examine whether Src directly phosphorylates Gab1, GST-Gab1 fusion proteins were purified and added to in vitro kinase assays as a substrate (Fig.  4A). The result showed that GST-Gab1, but not GST alone, was phosphorylated by Met and c-Src. C-terminal Src kinase was used as a negative control to demonstrate the specificity of the assay, which did not phosphorylate Gab1. Next, we performed co-immunoprecipitation to demonstrate the in vivo association of Src with Gab1. In the SYF cells stably reexpressing Src, the Src-Gab1 association was enhanced upon HGF stimulation (Fig. 4B). In vitro, the SH2 domain of Src bound to HA-Gab1 transiently expressed in HEK293 cells in a manner dependent on HGF (Fig. 4C), in accord with the above findings that HGF promotes the in vivo association of Src with Gab1. To examine whether the Met-mediated Gab1 phosphorylation provides docking site(s) for the SH2 domain of Src, Gab1 in SYF cells was examined for its binding to the SH2 domain of Src (Fig.  4D). The result showed that more Gab1 from HGF-treated SYF cells bound to the SH2 domain of Src than that from serumstarved SYF cells, supporting the possibility that Met-mediated phosphorylation of Gab1 may initially provide binding site(s) for the SH2 domain of Src.
It is already known that the COOH-terminal SH3 domain of Grb2 binds to Gab1 with high affinity (18,24). Our result showed that the SH3 domain of Src bound to Gab1 as efficiently as the SH3 domain of Grb2 in vitro. Moreover, although the SH2 and SH3 domains of Src could bind to Gab1, the SH3 domain of Src appeared to be more efficient than its SH2 domain in this aspect (Fig. 4C). Gab1 contains six proline-rich sequences, two of which have been demonstrated to serve as the binding sites for the COOH-terminal SH3 domain of Grb2 (24). Because the preferential binding sequences for the SH3 domains of Src and Grb2 were suggested to be different (44), we reasoned that the SH3 domain of Src might bind to proline-rich sequences of Gab1, to which the SH3 domain of Grb2 does not bind. On this basis, a Gab1 mutant (P449A/P452A) was generated by substitutions of two proline residues with alanine in the proline-rich sequence of Gab1, 449 PMNPNSPPR 457 , which displays the class II SH3 ligand PXXPXR motif. The Gab1 P449A/P452A mutant had a marked (ϳ50%) decrease in association with Src (Fig. 4E), supporting the significance of the SH3 domain-mediated interaction between Src and Gab1 in vivo.
Src-stimulated Gab1 Phosphorylation Potentiates HGF-induced Activation of ERK and AKT-Gab1 is known to be important for HGF-induced activation of ERK and AKT (36). We found that, in HEK293 cells, increased expression of Gab1 alone only slightly increased HGF-induced activation of ERK and AKT (Fig. 5A). However, simultaneous expression of Gab1 and Src selectively potentiated HGF-induced activation of ERK FIG. 1. Deficiency in the Src family kinases decreases HGFinduced Gab1 phosphorylation. A, SYF cells and their wild type counterparts (control) were grown in the medium supplemented with 10% serum. To analyze the tyrosine phosphorylation of Gab1, Gab1 proteins were immunoprecipitated (IP) by anti-Gab1 and subjected to immunoblotting (IB) with anti-phosphotyrosine (PY) or anti-Gab1. The tyrosine phosphorylation of Gab1 was quantified and expressed as -fold relative to that of the control cells. Whole cell lysates (WCL) were analyzed by immunoblotting with anti-Src to verify the expression of Src. B, the cells as described for A were serum-starved and treated with (ϩ) or without (Ϫ) 20 ng/ml HGF for 15 min. The tyrosine phosphorylation of Gab1 was analyzed as described for A and expressed as -fold relative to that of the serum-starved control cells. C, the SYF cells reexpressing c-Src (SYF/Src) and the control SYF cells were serumstarved and stimulated with HGF for 15 min. The tyrosine phosphorylation of Gab1 was analyzed as described for A.

FIG. 2. Effect of c-Src wild type (wt), its kd mutant, or the specific Src inhibitor PP1 on HGF-induced Gab1 phosphorylation.
A, HA epitope-tagged Gab1 (HA-Gab1) was co-expressed with wild type (wt) Src or its kinase-deficient (kd) mutant in HEK293 cells. 24 h after transfection, the cells were serum-starved and treated with (ϩ) or without (Ϫ) HGF for 15 min. To analyze the tyrosine phosphorylation of HA-Gab1, HA-Gab1 proteins were immunoprecipitated by anti-HA and analyzed by immunoblotting with anti-PY or anti-HA. The tyrosine phosphorylation of HA-Gab1 was measured and expressed as -fold relative to that of the serum-starved cells. Whole cell lysates were analyzed by immunoblotting with anti-Src, anti-phospho-Met, or anti-Met. B, MDCK cells stably expressing the Src kd mutant were serum-starved and treated with or without HGF for 15 min. The tyrosine phosphorylation of Gab1 was analyzed as described in the legend to Fig. 1A. Whole cell lysates were analyzed by immunoblotting with anti-Src or anti-phospho-Met. The activity of Src was measured by an in vitro kinase assay as described under "Experimental Procedures." C, MDCK cells were treated with or without HGF for 15 min in the presence or absence of the specific Src inhibitor PP1 at 10 M. The tyrosine phosphorylation of Gab1 was analyzed as described in the legend to Fig. 1A. Whole cell lysates were analyzed by immunoblotting with anti-phospho-Src, anti-Src, or anti-phospho-Met. and AKT, but not c-Jun N-terminal kinase, concomitant with increased tyrosine phosphorylation of Gab1. To further confirm the contribution of Src-mediated Gab1 phosphorylation to ERK activation, Src was transiently expressed in SYF cells, and its effects on Gab1 phosphorylation and ERK activation were measured (Fig. 5B). Our result showed that HGF-induced phosphorylation of Gab1 was enhanced along with increased expression of Src, correlated with increased activation of ERK upon HGF stimulation. These results together suggest that Src may play a role in HGF-induced activation of ERK and AKT through its phosphorylation on Gab1. DISCUSSION Although it is generally accepted that Met is the kinase responsible for HGF-induced Gab1 phosphorylation, it remains possible that other kinase(s) may participate in this event. Because both Src and Gab1 are recruited by activated Met, we speculated that Src might be able to phosphorylate Gab1 upon HGF stimulation. In this study, we provide several lines of evidence to support a role for Src to phosphorylate Gab1 in response to HGF stimulation. First, the extent of HGF-induced Gab1 phosphorylation in the Src family kinase-deficient cells is 40% lower than that in the wild type control cells (Fig. 1B). Second, increased expression of Src enhances HGF-induced Gab1 phosphorylation (Fig. 2A). Third, the expression of Src kd mutant, which serves as a dominant negative version of Src, significantly (ϳ60%) inhibits HGF-induced Gab1 phosphorylation (Fig. 2, A and B). Fourth, the ability of HGF to stimulate Gab1 phosphorylation is partially (ϳ60%) inhibited by the specific Src inhibitor PP1 (Fig. 2C). Fifth, Src directly phosphorylates purified Gab1 in vitro (Fig. 4A). These data together strongly suggest that Src plays a crucial role in HGF-induced Gab1 phosphorylation. However, no matter what approaches we employed in this study, involving the use of SYF cells, Src kd mutant, and the Src inhibitor PP1, the HGF-induced Gab1 phosphorylation could only be partially inhibited by 40 -60%, suggesting that in addition to Src, other kinase(s), most likely Met, is important for HGF-induced Gab1 phosphorylation.
We show in this study that in the absence of HGF stimulation, an elevated level of c-Src by itself is not sufficient to induce Gab1 phosphorylation ( Figs. 2A and 3B), and, conversely, expression of oncogenic v-Src leads to persistent phosphorylation of Gab1 independent of HGF stimulation (Fig. 3). In addition, we found that the expression of c-Src or its kd mutant has no effect on HGF-stimulated activation of Met (Fig.  2), indicating that the effects of c-Src and its kd mutant on FIG. 4. Src directly phosphorylates and associates with Gab1. A, Met and Src were transiently expressed in HEK293 cells and immunoprecipitated by anti-Met and anti-Src, respectively. C-terminal Src kinase (Csk) was immunoprecipitated by anti-C-terminal Src kinase from NIH3T3 cells. The immunocomplexes were subjected to in vitro kinase assays in the presence of [ 32 P]ATP and purified GST or GST-Gab1. The 32 P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. B, SYF cells stably reexpressing Src (SYF/Src) and control SYF cells were serum-starved and treated with or without HGF. The anti-Gab1 immunoprecipitates were analyzed by immunoblotting with anti-Src or anti-Gab1. C, in vitro binding assay. HA-Gab1 was transiently expressed in HEK293 cells. 24 h after transfection, the cells were serum-starved and treated with or without HGF for 15 min before lysis. Bacterially expressed GST fusion proteins were purified and immobilized on glutathione-agarose beads and incubated with HEK293 cell lysates containing HA-Gab1. After washing, the beads were analyzed by immunoblotting with anti-HA. The GST fusion proteins on the nitrocellulose membrane were visualized by staining with Ponceau S solution. SrcSH2, the SH2 domain of Src; SrcSH3, the SH3 domain of Src; Grb2SH2, the SH2 domain of Grb2; Grb2CSH3, the COOH-terminal SH3 domain of Grb2. D, HA-Gab1 was transiently expressed in SYF cells. Cell lysates from SYF cells treated with or without HGF were incubated with immobilized GST fusion proteins containing the Src SH2 domain. The bound HA-Gab1 was analyzed by immunoblotting with anti-HA. E, HA-Gab1 or HA-Gab1 P449A/P452A mutant was transiently expressed in HEK293 cells. 24 h after transfection, the cells were serum-starved for 18 h and then lysed. Src was immunoprecipitated by anti-Src and subjected to immunoblotting with anti-HA or anti-Src. Whole cell lysates were analyzed by immunoblotting with anti-HA to verify the expression of HA-Gab1 and HA-Gab1 P449A/P452A mutant.
HGF-induced Gab1 phosphorylation are not in an indirect fashion by modulating the activation of Met. However, the expression of SrcY527F mutant or v-Src apparently leads to Met activation (Fig. 3). Therefore, it is possible that in addition to its direct act to phosphorylate Gab1, v-Src aberrantly phosphorylates Met at Tyr 1234 and Tyr 1235 , leading to activation of Met in the absence of ligand binding, which in turn phosphorylates Gab1. Furthermore, v-Src may also phosphorylate Met at other tyrosine residues, in particular Tyr 1349 and Tyr 1356 , both of which serve as docking sites for several intracellular signaling molecules, including Src itself and Gab1 (18,19). Through this mechanism, Gab1 may be juxtaposed to v-Src, thereby facilitating its phosphorylation by v-Src. It is plausible that the aberrant phosphorylation of Gab1 by v-Src and subsequently amplified downstream signals may account at least partially for the ability of v-Src to cause cell transformation.
Gab1 becomes tyrosine-phosphorylated in response to diverse extracellular stimuli, including various cytokines and growth factors (29 -34). However, so far only four protein-tyrosine kinases have been reported to directly phosphorylate Gab1, including epidermal growth factor (EGF) receptor (30), insulin receptor (31), Met (18) (this study), and Src (this study). The in vitro phosphorylation sites of Gab1 for EGF receptor and insulin receptor have been determined (30,31), which reveals a very similar array of phosphorylation sites for these two receptors. However, among the identified phosphorylation sites on Gab1, EGF receptor has a predominant (ϳ50%) phosphorylation of Gab1 at Tyr 657 (30), a binding site for the SH2 domain of SHP2 phosphatase (45). On the other hand, insulin receptor predominantly (75%) phosphorylates Gab1 at Tyr 447 , Tyr 472 , and Tyr 619 , which are in YXXM motifs potentially for phosphatidylinositol 3-kinase binding (31). Because Met-phosphorylated sites of Gab1 have not been determined, it is not clear whether the phosphorylation sites for Met are similar to those for EGF receptor and insulin receptor. A previous study (25) using in vivo 32 P labeling and phosphopeptide mapping demonstrated that the in vivo phosphorylation sites of Gab1 in response to EGF stimulation are the same with those in response to HGF stimulation, suggesting that the phosphorylation sites of Gab1 for EGF receptor and Met may be similar, if not the same. Again, even if this is the case, Met and EGF receptor may have their own preferential phosphorylation sites among all possible ones, as discussed above for EGF receptor and insulin receptor. In this study, we demonstrate that in addition to Met, Src is another kinase responsible for HGFinduced Gab1 phosphorylation. It is not clear whether Src and Met predominantly phosphorylate Gab1 at different tyrosine residues. To clarify this, mass spectrometry will be employed to determine the phosphorylation sites of Gab1 for Src and Met, from which the relative phosphorylation intensity among phosphorylated tyrosine residues will be measured.
The association of Src with Gab1 is another novel aspect of our findings. We demonstrate that both of the SH2 and SH3 domains of Src are involved in this association (Fig. 4C). Because Src and Gab1 could be co-precipitated from the cells in the absence of HGF stimulation (Fig. 4B), rendering it possible that Src is constitutively associated with Gab1 in resting cells. Mutation that disrupts one of the proline-rich sequences of Gab1 partially (ϳ50%) decreases the binding of Src to Gab1 in serum-starved cells (Fig. 4E), supporting the existence of SH3mediated interaction in vivo and suggesting that the SH3 domain of Src may bind to more than one proline-rich sequences of Gab1. Experiments are in progress to determine the prolinerich sequences of Gab1 bound to the SH3 domain of Src. Moreover, the association of Src with Gab1 is increased upon HGF stimulation, concomitant with increased Gab1 phosphorylation (Fig. 4B). It is possible that activated Met may initially phosphorylate Gab1 at certain tyrosine residues, which in turn recruit the binding of the SH2 domain of Src, leading to activation of Src and subsequent phosphorylation of Gab1 by Src. Alternatively, Src may be initially recruited and activated by Met, which in turn phosphorylates Gab1, thereby creating binding sites for its own SH2 domain. These two possibilities are not mutually excluded; however, the former one was demonstrated in Fig. 4D, which shows that the SH2 domain of Src binds to Gab1 from HGF-treated SYF cells, in which the Gab1 phosphorylation is caused by activated Met.
Recently, the Src family kinases were reported to phosphorylate Gab2, another Gab family protein, in response to EGF stimulation (46). Interestingly, the association of Gab2 with FIG. 5. Increased Gab1 phosphorylation by Src leads to activation of ERK and AKT. A, HA-Gab1 was transiently co-expressed with (ϩ) or without (Ϫ) Src in HEK293 cells. 24 h after transfection, the cells were serum-starved and treated with or without HGF for 15 min. The total Gab1 proteins were immunoprecipitated by anti-Gab1 and subjected to immunoblotting with anti-PY. An equal amount of whole cell lysates (WCL) was analyzed by immunoblotting with antibodies as indicated. B, SYF cells and control cells were transiently transfected with (ϩ, 1 g; ϩϩ, 3 g) or without (Ϫ) pEVX-c-Src. 24 h later, the cells were serum-starved and treated with or without 20 ng/ml HGF for 15 min. The tyrosine phosphorylation of Gab1 was analyzed as described in the legend to Fig. 1A. Whole cell lysates were analyzed by immunoblotting with anti-ERK, anti-phospho-ERK, or anti-Src. Src seems to be constitutive and requires the proline-rich sequences of Gab2. Simultaneous mutations of two proline-rich sequences of Gab2 completely abolishes its association with Src and significantly (more than 70%) reduces its phosphorylation in response to EGF stimulation, suggesting that direct binding between Src and Gab2 is important for Src-mediated Gab2 phosphorylation. Whether direct binding to Gab1 is required for Src to phosphorylate Gab1 remains to be examined. Moreover, Gab1 and Gab2 are expressed ubiquitously, both of which interact with activated Met (47). It is not known whether Src also participates in HGF-induced Gab2 phosphorylation. If it is the case, it will be of importance to determine whether Srcmediated phosphorylation of Gab1 and Gab2 relays unique versus redundant signals for HGF-elicited cellular functions. In this study, we found the Src-mediated phosphorylation of Gab1 selectively augments the activation of ERK and AKT upon HGF stimulation (Fig. 5).
In summary, we demonstrate in this study that Src plays a crucial role in HGF-induced Gab1 phosphorylation not only by its direct act to phosphorylate Gab1 but also by its stable association with Gab1, which may facilitate Gab1 phosphorylation by Met or Src. Our findings add a new and potentially important substrate to the list of proteins phosphorylated by Src. Because many growth factors and cytokines known to induce Gab1 phosphorylation often activate Src, it is possible that Src or its family members are generally involved in Gab1 phosphorylation in response to various extracellular stimuli. In accordance with this assumption, we have found that Src is also important for increased phosphorylation of Gab1 in response to EGF stimulation. 2