The Bimodal Regulation of Epidermal Growth Factor Signaling by Human Sprouty Proteins

The bimodal regulation of epidermal growth factor signaling by human Sprouty proteins James E. Egan*, Amy B. Hall†, Bogdan A. Yatsula†, and Dafna Bar-Sagi†‡ †Department of Molecular Genetics and Microbiology and *Graduate Program in Molecular Pharmacology, State University of New York, Stony Brook, NY 11794-5222 Communicated by Joseph Schlessinger, Yale University School of Medicine, New Haven, CT, March 6, 2002 (received for review July 22, 2001) Signal transduction through epidermal growth factor receptors inhibitory effects of dSpry on DER signaling, genetic and biochem- (EGFRs) is essential for the growth and development of multicellular ical studies have shown that it also negatively regulates Drosophila organisms. A genetic screen for regulators of EGFR signaling has led tracheal branching by interfering with Branchless fibroblast growth to the identiﬁcation of Sprouty, a cell autonomous inhibitor of EGF factor (FGF) signaling (8). Although in the Drosophila tracheal signaling that is transcriptionally induced by the pathway. However, system dSpry acts non-cell-autonomously to repress FGF signaling the molecular mechanisms by which Sprouty exerts its antagonistic (8), in the Drosophila eye system, dSpry acts cell autonomously to effect remain largely unknown. Here we have used transient expres- antagonize EGFR signaling (4, 5). sion in human cells to investigate the functional properties of human The Drosophila Sprouty gene encodes a 63-kD membrane- Sprouty (hSpry) proteins. Ectopically expressed full-length hSpry1 associated protein with a unique carboxyl-terminal cysteine-rich and hSpry2 induce the potentiation of EGFR-mediated mitogen- domain that is highly conserved in the four vertebrate Sprouty activated protein (MAP) kinase activation. In contrast, truncation homologs (Spry1-4) (9, 10). The biological role of vertebrate mutants of hSpry1 and hSpry2 containing the highly conserved Sprouty seems to be evolutionarily conserved as murine Spry2 has carboxyl-terminal cysteine-rich domain inhibit EGF-induced MAP ki- been shown to interfere with the FGF-mediated processes of lung nase activation. The potentiating effect of the full-length hSpry2 morphogenesis, and murine Spry2 and Spry4 inhibit FGF-mediated proteins on EGF signaling is mediated by the amino-terminal domain bone outgrowth and limb patterning (9–11). Murine Spry4 has also and results from the sequestration of c-Cbl, which in turn leads to the been shown to inhibit vascular endothelial growth factor (VEGF)- inhibition of EGFR ubiquitination and degradation. These results mediated angiogenesis and prevent FGF- or VEGF-induced MAP indicate that hSpry2 can function both as a negative and positive kinase activation (12). In mammalian cells, EGF stimulation in- regulator of EGFR-mediated MAP kinase signaling in a domain- duces the expression of Spry2 and Spry4 (13). However, the role of dependent fashion. A dual function of this kind could provide a Sprouty proteins in EGF-mediated signaling in mammalian cells mechanism for achieving proper balance between the activation and remains unclear. In this study we have investigated the effects of repression of EGFR signaling. human Spry2 (hSpry2) and Spry1 (hSpry1) on EGF-mediated extracellular signal-regulated kinase 2 (ERK2) MAP kinase acti- eceptor tyrosine kinase-mediated signaling pathways are used vation in human cells. We demonstrate that hSpry2 can exert two Rin both invertebrates and vertebrates to control critical aspects opposing effects on EGF-induced ERK2 MAP kinase activation in of organ morphogenesis, patterning, cellular proliferation, and a domain-dependent manner. Whereas the highly conserved cys- differentiation (1). These signaling pathways are tightly regulated by teine-rich domain is able to inhibit EGF-induced MAP kinase positive and negative inputs that ensure the proper cellular fate (2). activation, the amino-terminal domain potentiates EGF-induced The development of the Drosophila compound eye is regulated MAP kinase activation by interfering with EGFR ubiquitination primarily through inductive signaling emanating from the Drosoph- and down-regulation. ila homolog of the epidermal growth factor receptor (EGFR) (DER; ref. 3). Recently, a genetic screen for modifiers of DER Materials and Methods signaling in the Drosophila eye has identified Sprouty (dSpry) as a Cell Culture and Transient Transfection. HeLa cells were cultured in negative modulator of this pathway (4). Loss-of-function dSpry DMEM supplemented with 10% FBS in a humidified incubator mutations were able to overcome the phenotype resulting from the with 5% CO2 at 37°C. Subconfluent HeLa cells were transfected overexpression of another negative regulator of EGFR signaling, with the indicated plasmids, using the standard calcium phosphate Argos, whereas the overexpression of dSpry induced a phenotype method. The precipitate was left on the cells for 10 h, and cells were similar to that exhibited by EGFR loss-of-function mutants (4). In allowed to recover for 12 h in DMEM supplemented with 10% FBS. addition, dSpry function is required for the proper development of Chinese hamster ovary (CHO) cells were cultured in Ham’s F-12 other tissues in Drosophila that require DER signaling, including medium supplemented with 10% FCS. For transfection, CHO cells embryonic chordotonal organ precursors, the wing imaginal disk, were incubated with Fugene6 (Roche Molecular Biochemicals) for midline glia, and ovarian follicle cells (5, 6). Overexpression of 24 h and then serum-deprived for 16 h. dSpry in these tissues mimics a DER loss-of-function phenotype, whereas loss of dSpry leads to a hyperactivation of the DER EGF Stimulation. Cells were serum-deprived for 2 h and then pathway. incubated with EGF stimulation medium [DMEM containing 25 The activation of DER by EGF leads to the stimulation of the mM Hepes (pH 7.4) and 10 ng͞ml recombinant human EGF] at 4°C ͞ Ras mitogen-activated protein (MAP) kinase pathway (7). In for 15 min. The stimulation medium was subsequently removed, CELL BIOLOGY Drosophila, dSpry seems to impinge on the DER pathway upstream of MAP kinase as overexpression of dSpry leads to a decrease in activated MAP kinase (6). Epistasis experiments have indicated Abbreviations: MAP, mitogen-activated protein; ERK2, extracellular signal-regulated ki- that during wing development dSpry is acting downstream of the nase 2; EGF, epidermal growth factor; EGFR, EGF receptor; HA, hemagglutinin; MBP, myelin basic protein; DER, Drosophila homolog of EGFR; CHO, Chinese hamster ovary; IP, immu- receptor and at or below the level of Raf (6). However, genetic noprecipitated͞immunoprecipitation; Wb, Western blotting. analysis of eye development has placed the level of inhibition by ‡To whom reprint requests should be addressed. E-mail: [email protected]. dSpry upstream of Ras (4). The expression of dSpry is induced by The publication costs of this article were defrayed in part by page charge payment. This DER signaling, indicating the involvement of dSpry in the negative article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. feedback control of DER signaling (4–6). In addition to the §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.052090899 PNAS ͉ April 30, 2002 ͉ vol. 99 ͉ no. 9 ͉ 6041–6046 Downloaded by guest on October 1, 2021 Fig. 1. The expression of hSpry1 and hSpry2 potentiates EGF-induced MAP kinase activation. Serum- deprived HeLa cells coexpressing HA-ERK2 and vector, HA-hSpry2 (A and B), or HA-hSpry1 (C and D) were stimulated with 10 ng͞ml of EGF for the indicated times. MAP kinase activation was measured by an immunocomplex kinase assay with MBP as a substrate. (A and C) Expression levels of hSpry proteins and ERK2 were determined by Western blotting (Wb), using anti-Spry and anti-ERK2 Abs, respectively. (B and D) Quantitation of the kinetics of MAP kinase activation by EGF in the absence (open circles) or presence (closed circles) of hSpry proteins. The amount of phosphory- lated MBP was determined with a PhosphorImager and is presented in arbitrary units as the fold activation over unstimulated vector-expressing cells. Shown are the averages of ﬁve independent experiments Ϯ SDs. and the cells were incubated with DMEM at 37°C for the indicated immunoprecipitations were kindly provided by Joseph Schless- times. To stop stimulation, cells were washed with ice-cold PBS and inger. Human recombinant EGF was obtained from GIBCO. kept on ice. Protein A Sepharose was obtained from Sigma–Aldrich. Mixed monoclonal anti-PLC-␥1 and anti-phosphotyrosine (4G10) were EGFR Ubiquitination Assay. Serum-deprived CHO cells were washed purchased from Upstate Biotechnology (Lake Placid, NY) Ab. twice with cold PBS and lysed in coimmunoprecipitation buffer (50 mM Tris, pH 7.5͞150 mM NaCl͞10% glycerol͞1% Nonidet P-40͞1 Western Blotting and Coimmunoprecipitation. HeLa cells were lysed mM EDTA͞1 mM phenylmethanesulfonyl fluoride͞1mM in 1 ml of RIPA buffer (150 mM NaCl͞10 mM Tris⅐HCl, pH ͞ ␮ ͞ ␮ ͞ ␮ ͞ ͞ ͞ ͞ NaV04 10 g/ml pepstatin 10 g/ml aprotinin 10 g/ml leupep- 7.4 1% Triton X-100 0.5% deoxycholate 0.1% SDS 1mM tin͞10 mM benzamidine). The lysates were clarified at 14,000 ϫ g EDTA͞50 mM NaF), and lysates were incubated with 2 ␮g͞ml of for 15 min and then incubated with anti-EGFR mAb for1hat4°C. the indicated Abs for 45 min, followed by a 45-min incubation with Subsequently, the immune complexes were incubated with protein 60 ␮l of a 50% slurry protein A Sepharose beads at 4°C. The A Sepharose beads for 30 min. The immunoprecipitates were immunocomplexes were resolved by SDS͞PAGE, and the proteins washed 3 times in cold lysis buffer and then resuspended in SDS were transferred to nitrocellulose membranes. Membranes were sample buffer. The samples were resolved by SDS͞PAGE. incubated for 30 min at room temperature in blocking buffer containing 5% BSA. The anti-HA Ab (1:10,000), anti-ERK2 Cloning and Plasmid Construction. cDNAs encoding human Spry1 (1:1,000), anti-T7 (1:10,000), anti-hSpry (1:2,500), antiphosphoty- and Spry2 were isolated by reverse transcription–PCR.

Load more