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Frs2α Attenuates FGF Receptor Signaling by Grb2-Mediated Recruitment of the Ubiquitin Ligase

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Frs2α Attenuates FGF Receptor Signaling by Grb2-Mediated Recruitment of the Ubiquitin Ligase Corrections APPLIED BIOLOGICAL SCIENCES. For the article ‘‘In vitro and in vivo BIOPHYSICS. For the article ‘‘CLOUDS, a protocol for deriving a studies of a VEGF121/rGelonin chimeric fusion toxin target- molecular proton density via NMR,’’ by Alexander Grishaev and ing the neovasculature of solid tumors,’’ by Liesbeth M. Miguel Llina´s,which appeared in number 10, May 14, 2002, of Veenendaal, Hangqing Jin, Sophia Ran, Lawrence Cheung, Proc. Natl. Acad. Sci. USA (99, 6707–6712), Eq. 3 appeared Nora Navone, John W. Marks, Johannes Waltenberger, Philip incorrectly due to a printer’s error. The correct equation appears Thorpe, and Michael G. Rosenblum, which appeared in number below. 12, June 11, 2002, of Proc. Natl. Acad. Sci. USA (99, 7866–7871), on page 7866, in the last sentence of the Introduction, ‘‘Caprice’s N 6J ϩ 3J ϩ J sarcoma cells’’ should read ‘‘Kaposi’s sarcoma cells.’’ ϭ ␳self ϩ ␳leak ϩ ͫ 2 1 oͬ ͸ ␴ fi i Ϫ ij [3] 6J2 Jo j i www.pnas.org͞cgi͞doi͞10.1073͞pnas.162346299 www.pnas.org͞cgi͞doi͞10.1073͞pnas.162295699 BIOCHEMISTRY. For the article ‘‘FRS2␣ attenuates FGF receptor signaling by Grb2-mediated recruitment of the ubiquitin ligase Cbl,’’ by Andy Wong, Betty Lamothe, Arnold Li, Joseph Schlessinger, and Irit Lax, which appeared in number 10, May 14, 2002, of Proc. Natl. Acad. Sci. USA (99, 6684–6689; First Published May 7, 2002; 10.1073͞pnas.052138899), the authors note that the author Arnold Li should read Arnold Lee. The online journal has been corrected. www.pnas.org͞cgi͞doi͞10.1073͞pnas.162346399 CORRECTIONS www.pnas.org PNAS ͉ August 6, 2002 ͉ vol. 99 ͉ no. 16 ͉ 10941 Downloaded by guest on September 28, 2021 FRS2␣ attenuates FGF receptor signaling by Grb2- mediated recruitment of the ubiquitin ligase Cbl Andy Wong*, Betty Lamothe, Arnold Lee†, Joseph Schlessinger‡, and Irit Lax Department of Pharmacology, Sterling Hall of Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520 Contributed by Joseph Schlessinger, March 8, 2002 Attenuation of growth factor signaling is essential for the regu- for recruitment of multiprotein complexes that are responsible lation of developmental processes and tissue homeostasis in most for both signal activation and attenuation. organisms. The product of Cbl protooncogene is one such regula- tor, which functions as an ubiquitin ligase that ubiquitinates and Materials and Methods promotes the degradation of a variety of cell signaling proteins. Cell Culture, Abs, and Other Reagents. Cells were cultured in the Here, we demonstrate that Grb2 bound to tyrosine-phosphory- presence of DMEM containing 10% FBS, 2 mM L-glutamine, lated FRS2␣ forms a ternary complex with Cbl by means of its Src and penicillin͞streptomycin. PC12 cells were grown in DMEM homology 3 domains resulting in the ubiquitination of fibroblast supplemented with 10% FCS and 10% heat-inactivated horse growth factor (FGF) receptor and FRS2␣ in response to FGF stim- serum. Generation of FRS2␣Ϫ͞Ϫ cells expressing wild-type or ulation. These observations highlight the importance of FRS2␣ in FRS2␣ mutants were performed as described (3). Transient the assembly of both positive (i.e., Sos, phosphatidylinositol 3- transfections of HEK293 and HeLa S3 cells were performed kinase) and negative (i.e., Cbl) signaling proteins to mediate a with Lipofectamine (GIBCO) according to the manufacturer’s balanced FGF signal transduction. However, the partial inhibition protocols. ,of FGF receptor down-regulation in FRS2␣؊͞؊ cells indicates that All retrovirus plasmids were constructed in pBABE͞puro the attenuation of signaling by FGF receptor is regulated by whereas plasmids used in transient expression experiments were redundant or multiple mechanisms. constructed in pRK5. FRS2␣ point mutants were generated with the QuickChange Site-directed Mutagenesis kit from Stratagene he docking proteins, FRS2␣ and FRS2␤, play a critical role (1, 2). Abs against FRS2␣, Grb2, phosphotyrosine (pTyr), and Tin mediating the intracellular signals that are generated at EGF receptor have been described (1–4). Abs against Myc, Cbl, the cell surface by activation of the fibroblast growth factor Sos1, and horseradish peroxidase (HRP)-conjugated anti-mouse (FGF), nerve growth factor (NGF), or glial-derived neurotro- Abs were purchased from Santa Cruz Biotechnology. Anti-FLAG phic factor receptors. Both FRS2␣ and FRS2␤ contain myristyl was purchased from Sigma. HRP anti-hemagglutinin (HA) Abs anchors and phosphotyrosine-binding domains in their N ter- were obtained from Roche Molecular Biochemicals. HRP mini and multiple tyrosine phosphorylation sites in their C- protein-A was obtained from Jackson ImmunoResearch. terminal tails that serve as binding sites for the adaptor protein, Grb2, and for the Src homology (SH) 2 domain containing In Vitro Binding Experiments, Immunoprecipitation, and Immunoblot- protein tyrosine phosphatase, Shp2 (1, 2). In response to FGF or ting. The expression and purification of glutathione S-transferase NGF stimulation, Grb2 can also be recruited indirectly to FRS2␣ (GST)-Grb2, SH2, N-terminal SH3 (N-SH3), C-terminal SH3 through its interaction with tyrosine-phosphorylated Shp2 mol- (C-SH3), Cbl-N, and Cbl-C fusion proteins have been described ecules bound to the docking protein (2). (4, 5). For GST pull-down assays, Ϸ5 ␮g of recombinant protein With mouse embryonic fibroblasts (MEFs) isolated from was immobilized to glutathione-agarose beads and washed ex- FRS2␣Ϫ͞Ϫ mouse embryos we have demonstrated that FRS2␣ tensively before incubation with Ϸ2 mg of cell lysate for2hat is essential for the FGF-induced mitogen-activated protein 4°C. Bound proteins were washed, separated by SDS͞PAGE, kinase (MAPK) response, phosphatidylinositol 3-kinase (PI3- and transferred to nitrocellulose membranes for immunoblot- kinase) stimulation, cell proliferation, and cell migration (3). ting. All Abs were dissolved in 1ϫ TBS͞5% BSA, with the Although the recruitment of both Grb2 and Shp2 is essential for exception of HA-horseradish peroxidase, which was dissolved in the overall effects of FGF, recruitment of Shp2 seems to play a 1ϫ TBS͞5% BSA with nonfat milk. Chemiluminescence was more prominent role in stimulation of MAPK and cell prolif- performed according to the manufacturer’s specifications. eration (3). In addition, FRS2␣Ϫ͞Ϫ MEFs have also been used to demonstrate that tyrosine phosphorylation and recruitment of Receptor Endocytosis Assays. FGF was labeled with 125Itoa the docking protein Gab1 depends on tyrosine-phosphorylated specific activity of Ϸ5 ϫ 105 cpm͞pmol based on the conven- FRS2␣. Gab1 binds constitutively to the C-terminal SH3 (C- tional chloramine T protocol (6). Heparin sulfate was added to SH3) domain of Grb2 and its assembly in complex with Grb2͞ labeled 125I-FGF at a final concentration of 5 ␮g͞ml. Optimal FRS2␣ enables tyrosine phosphorylation of Gab1, which is and specific binding were determined by ligand titration and followed by recruitment of PI3-kinase and activation of a cell competition experiments with a 300-fold excess of unlabeled survival pathway (3, 4). In this report we demonstrate that FGF-induced tyrosine phosphorylation of FRS2␣ results in complex formation with the Abbreviations: SH, Src homology; FGF, fibroblast growth factor; FGFR, FGF receptor; MEF, adaptor protein Grb2 bound to Cbl by means of its SH3 domains. mouse embryonic fibroblast; MAPK, mitogen-activated protein kinase; PI3-kinase, phos- ␣ phatidylinositol 3-kinase; C-SH3, C-terminal SH3; N-SH3, N-terminal SH3; EGF, epidermal FGF-induced ternary complex formation among FRS2 , Grb2, growth factor; pTyr, phosphotyrosine; HA, hemagglutinin; GST, glutathione S-transferase. ␣ and Cbl results in ubiquitination and degradation of FRS2 and *Present address: Department of Cellular Biochemistry and Biophysics, Memorial Sloan– FGF receptor (FGFR). Unlike the epidermal growth factor Kettering Cancer Center, New York, NY 10021. (EGF) or platelet-derived growth factor receptor, which form a †Present address: New York University Medical School, New York, NY 10016. direct complex with Cbl by way of its SH2-like domain, Grb2 ‡To whom reprint requests should be addressed. E-mail: [email protected]. functions as a link between Cbl and FRS2␣; Grb2 is bound to ␣ The publication costs of this article were defrayed in part by page charge payment. This FRS2 by means of its SH2 domain and to Cbl by means of its article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. two SH3 domains. Thus, FRS2␣ functions as a central platform §1734 solely to indicate this fact. 6684–6689 ͉ PNAS ͉ May 14, 2002 ͉ vol. 99 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.052138899 ligand (nonspecific binding accounted for less than 5% of total counts). For each experiment, cells were seeded in 24-well plates, allowed to attach, and starved overnight. Cells were incubated with5nM125I-aFGF͞heparin sulfate in DMEM͞1% BSA for 2 h at 4°C. Unbound ligand was removed by washing with ice-cold 1ϫ PBS. Prewarmed DMEM͞1% BSA was added to the cells and incubated at 37°C for the times indicated. Samples were washed with ice-cold 1ϫ PBS then acid-stripped with ice-cold 20 mM acetic acid͞2 M NaCl for 2 min. Internalized receptors were isolated by lysis in 1 M NaOH. Cell-surface-associated and internalized radioactivities were determined by gamma count- ing. The results presented represent the average of quadruplicate experiments performed in duplicate. Results In our search for proteins that interact with Grb2 in FGF- stimulated cells, we identified several proteins that migrate on SDS͞PAGE as a broad band with an apparent molecular mass of 120 kDa. We have previously demonstrated that one of these proteins is the docking protein Gab1. Gab1 is constitutively associated with the C-terminal SH3 domain of Grb2 and is recruited to the activated FGFR via Grb2 molecules bound through its SH2 domain to tyrosine-phosphorylated FRS2␣ (4). In the experiment presented in Fig. 1A, it is demonstrated that one of the 120-kDa proteins bound to Grb2 is the product of the Cbl protooncogene.
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