Stimulation of phosphatidylinositol 3-kinase by fibroblast growth factor receptors is mediated by coordinated recruitment of multiple docking proteins S. H. Ong*, Y. R. Hadari†, N. Gotoh†, G. R. Guy*, J. Schlessinger†‡, and I. Lax† †Department of Pharmacology and The Skirball Institute, New York University, Medical School, New York, NY 10016; and *Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609 Contributed by J. Schlessinger, March 7, 2001 The docking protein FRS2 is a major downstream effector that links cific docking proteins such as IRS1 and Gab1, respectively fibroblast growth factor (FGF) and nerve growth factor receptors (19–22). In EGF signaling, PI3-kinase is recruited by hetero- with the Ras͞mitogen-activated protein kinase signaling cascade. oligomerization with ErbB3 and by tyrosine phosphorylation of In this report, we demonstrate that FRS2 also plays a pivotal role Gab1; a docking protein that upon tyrosine phosphorylation in FGF-induced recruitment and activation of phosphatidylinositol recruits and activates PI3-kinase (18, 21, 23). Moreover, PI3- 3-kinase (PI3-kinase). We demonstrate that tyrosine phosphoryla- kinase activity recruited through Gab1 was shown to be essential tion of FRS2␣ leads to Grb2-mediated complex formation with the for NGF-induced protection from apoptosis induced by serum docking protein Gab1 and its tyrosine phosphorylation, resulting in deprivation, as well as for neurite outgrowth in PC12 cells the recruitment and activation of PI3-kinase. Furthermore, Grb2 (24–26). Gab1 was originally cloned as a Grb2-associated protein bound to tyrosine-phosphorylated FRS2 through its SH2 domain that is tyrosine phosphorylated in cells upon stimulation with interacts primarily via its carboxyl-terminal SH3 domain with a EGF or insulin (20). Gab1 possesses an N-terminal pleckstrin proline-rich region in Gab1 and via its amino-terminal SH3 domain homology (PH) domain, a Met-binding domain (MBD) that with the nucleotide exchange factor Sos1. Assembly of mediates interactions with the Met receptor and other receptor FRS2␣:Grb2:Gab1 complex induced by FGF stimulation results in tyrosine kinases, and a large C-terminal portion containing activation of PI3-kinase and downstream effector proteins such as multiple tyrosine residues (20, 27). Tyrosine-phosphorylated the S͞T kinase Akt, whose cellular localization and activity are Gab1 is capable of recruiting several SH2 domain-containing regulated by products of PI3-kinase. These experiments reveal a signaling molecules including Grb2, Shp2, PLC␥, Crk1, PI3- unique mechanism for generation of signal diversity by growth kinase, and Nck (20, 24, 28, 29). Recently, a highly homologous factor-induced coordinated assembly of a multidocking protein isoform of Gab1, termed Gab2, was isolated (30, 31). Unlike the complex that can activate the Ras͞mitogen-activated protein ki- broad expression pattern of Gab1, Gab2 is predominantly ex- nase cascade to induce cell proliferation and differentiation, and pressed in hematopoietic cells (30). Several reports have shown PI3-kinase to activate a mediator of a cell survival pathway. that the Gab family of docking proteins are involved in the activation of PI3-kinase in response to growth factors or cyto- ibroblast growth factors (FGFs) play key roles in diverse kines stimulation and by activation of the B cell antigen receptor Fcellular processes including mitogenesis, differentiation, mi- (20, 21, 24, 25, 27, 28, 30, 32–35). gration, and survival (reviewed in refs. 1 and 2). We have In this report, we show that in response to FGF stimulation, FRS2␣ and Gab1 associate indirectly via Grb2 resulting in recently identified a major downstream mediator of signaling tyrosine phosphorylation of Gab1 and activation of the PI3- through the activated FGF receptors (FGFRs), termed FRS2, kinase͞Akt survival pathway. These experiments reveal a mech- that was redesignated FRS2␣ when a highly homologous iso- anism for activation of multiple signaling pathways by coordi- form, FRS2␤, was identified (3, 4). The FRS2 proteins are nated assembly of docking proteins. targeted to the plasma membrane by myristylation at the N terminus and contain a phosphotyrosine-binding domain that Materials and Methods mediates direct interaction with FGF or nerve growth factor Antibodies. Antibodies for FGFR1, FRS2, Grb2, Shc, Sos1, and (NGF) receptors (4–8). The C-terminal region of FRS2 contains pTyr were previously described (3, 4, 36). Anti-Shp2 antibodies multiple tyrosine residues that are phosphorylated by activated were from Transduction Laboratories (Lexington, KY). Anti- FGF or NGF receptors, serving as recognition motifs for the Gab1 antibodies were from Upstate Biotechnology (Lake Placid, SH2 domains of Grb2 and Shp2 (3, 9). Recruitment of both Grb2 NY). Anti-Akt and anti-pS473-Akt antibodies were from New and Shp2 are required for FGF-mediated mitogen-activated England Biolabs. Anti-FLAG (M2) antibodies were from Sigma. protein kinase (MAPK) activation, proliferation of fibroblasts, and neuronal differentiation of PC12 cells (3, 9). Cell Lines. Swiss 3T3, NIH 3T3, 293, COS-1, SH-SY5Y, and Although FGFs and neurotrophins are strongly implicated in L6-FGFR1 cells were cultured in DMEM supplemented with cell survival, the intracellular signaling machinery and mecha- nisms involved are not clear (1, 10, 11). Numerous studies have provided evidence that the PI3-kinase͞Akt (or PKB) signaling Abbreviations: PI3-kinase, phosphatidylinositol 3-kinase; FGFR, fibroblast growth factor cascade activated by multiple growth factors and cytokines receptor; NGF, nerve growth factor; wt, wild type; MBD, Met-binding domain; PH, Pleck- results primarily in cell survival (12–14), as opposed to the strin homology; KD, kinase inactive mutant; MAPK, mitogen-activated protein kinase; ͞ PDGF, platelet-derived growth factor; EGF, epidermal growth factor; GST, glutathione Ras MAPK pathway, which mainly signals to control cell pro- S-transferase; SH2 and 3, Src homology 2 and 3; GDNF, glial-derived neurotrophic factor. liferation and differentiation (15, 16). Although some receptors ‡To whom reprint requests should be addressed. E-mail: [email protected]. such as the platelet-derived growth factor (PDGF) and ErbB3 The publication costs of this article were defrayed in part by page charge payment. This recruit PI3-kinase directly (17, 18), other receptors such as the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. insulin and epidermal growth factor (EGF) receptors use spe- §1734 solely to indicate this fact. 6074–6079 ͉ PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.111114298 Downloaded by guest on September 24, 2021 10% FBS, 10 mM L-glutamine, and 100 ␮g each of penicillin and streptomycin͞ml, all from GIBCO͞BRL. PC12 cells were grown in DMEM supplemented with 10% FCS and 10% heat- inactivated horse serum (GIBCO͞BRL). Expression Constructs. The FGFR1 expression vectors for wild- type (wt) or kinase-inactive (KD) mutant were previously de- scribed (36). FRS2␣ vectors for wt or the 6F-FRS2␣ mutant were described in refs. 3 and 4. Mouse Gab1 cDNA was kindly provided by W. Birchmeier (Max Delbru¨ck Center for Molecular Medicine, Berlin, Germany). Gab1 mutants consisting of the PH domain or the MBD or point mutations were generated by standard PCR and subcloning procedures. The Akt plasmid was from P. Cohen (University of Dundee, Dundee, U.K.). Transient transfection of cells was performed by using Lipofectamine (GIBCO͞BRL). Glutathione S-transferase (GST) fusion pro- teins of Grb2 (amino acids 1–217), Grb2-SH2 (amino acid 54–164), Grb2-N-SH3 (amino acid 1–68), Grb2-C-SH3 (amino acid 156–199), Gab1 (amino acids 1–695), and Gab1-MBD (amino acids 450–532) were expressed in Escherichia coli and purified with glutathione-conjugated agarose beads (Sigma) according to established procedures. Immunoprecipitations, Protein Binding Studies, and PI3-Kinase As- says. The protocols for immunoprecipitations, binding studies Fig. 1. Complex formation between Grb2 and Gab1 in FGF-stimulated cells. with GST fusion proteins and in vitro PI3-kinase assays were as (A) Quiescent Swiss 3T3 cells were unstimulated or stimulated with FGF1 and described previously (3, 4, 17). heparin (100 ng͞ml and 5 ␮g͞ml, respectively) for 10 min. Lysates were immunoprecipitated with anti-Grb2 antibodies and followed by SDS–PAGE Results and immunoblotting with anti-pTyr antibodies. (B) Swiss 3T3 cells were un- stimulated or stimulated with FGF1 and heparin (100 ng͞ml and 5 ␮g͞ml, In a survey of tyrosine phosphoproteins involved in FGF sig- respectively) for 10 min. Cell lysates were incubated with immobilized GST naling, we have identified proteins that coimmunoprecipitate fusion proteins of the N-terminal SH3, SH2, or C-terminal SH3 domains of Grb2. with Grb2 upon FGF stimulation. Lysates from FGF-stimulated Bound proteins were eluted and resolved by SDS–PAGE and then followed by Swiss 3T3 fibroblasts were subjected to immunoprecipitation immunoblotting with anti-pTyr (Upper) or anti-Gab1 antibodies (Lower). (C) with anti-Grb2 antibodies followed by SDS–PAGE and immu- Gab1 binds to the C-terminal SH3 domain of Grb2 through the MBD. 293 cells noblotting with anti-pTyr antibodies. FGF induced the associa- were transfected with the expression vector for Flag-tagged MBD of Gab1. tion of multiple tyrosine-phosphorylated proteins with Grb2 The lysates were incubated with immobilized GST fusion proteins of the SH2,