1 Center for Craniofacial Biology, Departments of Surgery and Pediatrics, and the Developmental Biology Program, The Childrens Hospital Los Angeles Research Institute, University of Southern California Schools of Dentistry and Medicine, Los Angeles, California 90023; and 2 Equipe Morphogenesis Cellulaire et Tumorale, Institut Curie-Unité Mixte de Recherche 144 Centre National de la Recherche Scientifique, 75248 Paris Cedex 05, France
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ABSTRACT |
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Murine Sprouty2 (mSpry2) is a conserved ortholog of Drosophila Sprouty, a gene that inhibits several tyrosine kinase receptor pathways, resulting in net reduction of mitogen-activated protein (MAP) kinase activation. However, the precise mechanism mediating mSpry2 function as a negative regulator in tyrosine kinase growth factor pathways that regulate diverse biological functions remains incompletely characterized. Fibroblast growth factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis and induces epithelial expression of mSpry2 adjacent to mesenchymal sites of FGF10. Herein, we demonstrate that FGF10 stimulation of mouse lung epithelial cells (MLE15) overexpressing mSpry2 results in both mSpry2 tyrosine phosphorylation and differential binding of mSpry2 to several key upstream target proteins in the MAP kinase-activating pathway. Thus FGF receptor (FGFR) activation results in increased association of mSpry2 with growth factor receptor-binding protein 2, suc-1-associated nuerotrophic factor target 2, and Raf but decreased binding to protein tyrosine phosphatase 2 and GTPase-activating protein 1, resulting in a net reduction of MAP kinase activation. mSpry2 also spatially translocates to the plasma membrane and intracellular membrane structures in response to FGF10 stimulation. Our data demonstrate novel intracellular mechanisms mediating mSpry2 function as a negative regulator of uncontrolled FGF-induced MAP kinase signaling.
fibroblast growth factor 10 stimulation; murine Sprouty2; differentially binding; mouse lung epithelial cells
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INTRODUCTION |
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THE FIBROBLAST GROWTH FACTOR (FGF) family and their cognate receptors (FGFR) affect a wide variety of biological events, including cell growth, differentiation, neurite outgrowth, embryogenesis, angiogenesis, and wound healing (6). There are at least 24 known FGF ligands, which bind and induce transmembrane dimerization of four major classes of tyrosine kinase FGFR, leading to autophosphorylation and recruitment of intracellular signaling molecules (10). FGFR dimerization occurs either as homodimers or heterodimers. This is facilitated by multivalent interactions between heparin sulfate proteoglycans and the extracellular domain of the FGFR (3). FGFRs contain an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular tyrosine kinase domain. Ligand binding to the receptors is determined through the extracellular domain, leading to ligand-induced receptor dimerization. The extracellular domain contains three immunoglobulin-like domains (12). Alternative splicing of the mRNA results in several different forms of the FGFR extracellular sequence, with the b isoforms expressed primarily in epithelial tissues and the c isoforms expressed in mesenchymal tissues. The intracellular juxtamembrane domain is connected to the extracellular domain by a single transmembrane stretch (4). Various substrates containing Src homology 2 (SH2) domains bind to specific FGFR phosphorylation sites, activating downstream signal transduction.
FGFRs activate the Ras/mitogen-activated protein (MAP) kinase-signaling pathway via several different upstream target proteins. Binding of cognate ligand to the FGFRs leads to receptor dimerization and activation of tyrosine kinase activity, followed by autophosphorylation of numerous tyrosines. Unlike other tyrosine kinase receptors, FGFRs do not directly bind growth factor receptor-binding protein (Grb) 2 but require adapter or docking proteins to recruit the Grb2/son of sevenless (Sos) complex upon stimulation, leading to downstream activation of the Ras/MAP kinase signaling pathway. The suc-1-associated neurotrophic factor target (FRS2) functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGFR activation. Upon FGFR activation, FRS2 is tyrosine phosphorylated and translocates to the plasma membrane where it recruits the Grb2/Sos complex (11). The Grb2/Sos complex catalyzes GDP conversion to GTP on Ras, which is required for Raf (serine/threonine kinase) activation. The reaction is terminated by the hydrolysis of GTP to GDP by GTPase-activating proteins (Gap). The linking of FGF receptor with MAP kinase by FRS2 is essential for neuronal growth and differentiation (7).
Protein tyrosine phosphatases (Shp) function as both negative and positive regulators in the Ras/MAP kinase signaling pathways (16). The NH2-terminal SH2 domain of Shp2 binds tyrosine-phosphorylated FRS2, and this binding is direct. Binding of FRS2 to Shp2 in PC-12 cells is required for sustained MAP kinase activation and potentiation of FGF-induced neuronal differentiation (3). Shp2 potentiates signaling through the MAP kinase pathway, is required for gastrulation, and is involved in mouse limb outgrowth (3, 14). Thus Shp2 functions as a positive regulator in the MAP kinase signaling pathway.
The role of inhibitory regulators in the formation of FGFR-activated signaling complexes remains incompletely characterized. In the Drosophila tracheae, Branchless (Bnl, Fgf ortholog) induces expression of its own antagonist Sprouty (2). Sprouty not only functions downstream in the Bnl signaling pathway but also functions downstream of several other receptor tyrosine kinase pathways, including epidermal growth factor receptor (EGFR) and Torso.
Sprouty is localized to intracellular membrane compartments. In vitro coprecipitation studies suggest that Sprouty binds to Gap1 and downstream receptor kinase (Drk; a Grb2 ortholog; see Ref. 1), resulting in inhibition of the Ras-MAP kinase pathway. However, other studies have suggested that Sprouty may function as an extracellular FGF ligand antagonist, either by competing with Bnl for the breathless (Btl) receptor or by signaling through a putative cognate Sprouty receptor (2). Consequently, the exact biochemical function of Drosophila Sprouty remains inconclusive.
Mice and men possess several Sprouty genes [mSpry1-4, human (h) SPRY1-3]. mSpry2 is the gene that is most closely related to Drosophila Sprouty and is also 97% homologous to hSPRY2 (15). hSPRY2 translocates to the cell membrane in response to epidermal growth factor (EGF), independent of phosphatidylinositol 3-kinase activation (8), and binds to casitas B-lineage lymphoma (c-Cbl), possibly modulating receptor-mediated endocytosis (18). Thus the intracellular function of mammalian SPRY is beginning to be elucidated.
The intracellular function of mSpry2 as a negative regulator of several tyrosine kinase pathways is just beginning to be investigated. We aimed to determine a mechanism by which Spry2 negatively regulates FGF10 signaling in immortalized distal respiratory epithelial cell lines (MLE15; see Ref. 17). The use of MLE15 cells has allowed us to initially assess the mechanism by which Spry2 negatively regulates FGF10 signaling. We found that mSpry2 is tyrosine phosphorylated upon FGF10 stimulation, differentially binds FRS2/Grb2/Raf, and is released from Gap1 and Shp2, resulting in net repression of MAP kinase activation. Our results also show that mSpry2 spatially translocates to the plasma membrane and to intracellular membrane compartments upon growth factor stimulation. We propose that the Sprouty family proteins function to inhibit the formation of specific signaling complexes downstream of tyrosine kinase receptors. Thus we speculate that Sprouty negatively modulates FGF signaling to coordinate cell growth and differentiation during organogenesis, injury, and repair.
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METHODS |
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Cell culture. MLE15, a gift from Jeffrey Whittset, were grown in DMEM with 10% FCS and 40 µg/ml gentamicin in a humidified atmosphere at 37°C and 5% CO2.
Antibodies and growth factors. Antiphosphotyrosine (RC20), anti-extracellular signal-regulated kinase (Erk) 1 (MAP kinase), and anti-Grb2 were purchased from Transduction Laboratories. Anti-Sos, anti-FRS2, anti-Shp2, anti-Raf, anti-Gap1, and anti-FGFR2b were purchased from Santa Cruz Biotechnology, and anti-hemagglutinin (HA) was purchased from Roche Diagnostics. Anti-active MAP kinase was from Sigma. FGF10 was purchased from Research and Development. 4',6-Diamidino-2-phenylindole (DAPI) nucleic acid stain was purchased from Molecular Probes, and desmoplakin 1 and 2 were from Research Diagnostics. Texas red anti-mouse was purchased from Jackson ImmunoResearch Laboratories.
Generation of expression vectors. Full-length mSpry2 cDNA with an HA-epitope sequence fused to the COOH terminus was cloned into pGREEN LANTERN-1 (pGLV-1; Life Technologies). For immunofluorescence, full-length mSpry2 was fused to pEGFP-N1 [mSpry2-green fluorescent protein (GFP); Clonetech].
Immunoblotting and immunoprecipitation. MLE15 cells were transfected at 80% confluency with either pGLV-1 expression vector alone or pGLV-1 containing the mSpry2-HA construct using Lipofectamine (Life Technologies) reagent. After 24 h, cells were starved in DMEM for 18 h before FGF10 stimulation. Cells were either left untreated or stimulated with FGF10 (250 ng/ml) for 10 min followed by three washes of cold 1× PBS and the addition of RIPA lysis buffer [1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 1 mM orthovanadate, and protease inhibitor cocktail].
Clarified cell lysates were immunoprecipitated with the indicated antibodies overnight at 4°C followed by three washes with lysis buffer. SDS-sample buffer was added, and the immune complexes were boiled for 5 min. The proteins were separated on 10% polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked with 1% blocking solution (Roche Molecular Biochemicals) and incubated overnight at 4°C with the appropriate primary antibody at a 1:2,500 dilution. Unbound antibody was washed 4 × 10 min followed by incubation with either goat anti-rabbit IgG-horseradish peroxidase (1:20,000) or goat anti-mouse IgG-horseradish peroxidase (1:10,000) secondary antibody for 30 min. Membranes were washed 4 × 15 min, and binding was detected using the enhanced chemiluminescence method (Roche Molecular Biochemicals) and XAR-5 film (Eastman Kodak, Rochester, NY). Band size and intensity were quantitated by densitometry. All experiments were repeated at least three to four times, with similar results obtained within and between repetitive experiments.Detection of active MAP kinase. MLE15 cells were transfected with the above expression vectors followed by stimulation with FGF10 (250 ng/ml), as described above. Cell lysates were separated on 10% polyacrylamide gel and transferred to a PVDF membrane. Active MAP kinase was detected by incubating the immunoblot with anti-MAP kinase active (1:5,000) and horseradish peroxidase-labeled anti-mouse IgG (1:10,000) followed by chemiluminescence. Anti-Erk1 (1:2,000) was used to determine equal loading. All experiments were repeated at least three to four times, with similar results obtained within and between repetitive experiments.
Immunofluorescence. MLE15 cells transfected with the mSpry2-GFP construct were seeded on sterile chamber slides, starved overnight, and stimulated with FGF10 (250 ng/ml). The cells were cooled on ice, washed with 1× PBS at 4°C, and fixed for 30 min in 2% paraformaldehyde/0.1% Triton X-100 on ice. After two washes with PBS at 4°C (5 min/wash), anti-desmoplakin 1 and 2 was added for 1 h at 4°C. Cells were washed four times in 4°C PBS (5 min/wash), and Texas red anti-mouse secondary antibody (1:50) was added. After incubation for 1 h with secondary antibody, slides were washed four times with PBS and incubated with DAPI (10 µM) for 4 min in the dark. Cells were washed three times with PBS, and coverslips were applied. Confocal fluorescence microscopy of fixed and immunostained cells was performed at the Childrens Hospital Los Angeles (CHLA) Imaging Core Facility. All experiments were repeated at least three to four times, with similar results obtained within and between repetitive experiments.
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RESULTS |
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mSpry2 is tyrosine phosphorylated in response to FGF10 stimulation.
We have previously shown that mSpry2 mRNA expression is upregulated in
response to FGF10 in mouse whole lung cultures (9). Thus
we inspected the mSpry2 amino acid sequence by Prosite analysis (Fig.
1A) for potential regulatory
sites. A single putative tyrosine phosphorylation site was detected at
Y191. We therefore investigated the possibility that mSpry2 is
phosphorylated in response to FGF10 stimulation in MLE15 cells.
Tyrosine phosphorylation in MLE15 cells was optimal with a dose of 250 ng/ml FGF10, which was determined by the dose response (data not
shown). Control cells and MLE15 transfected with a construct containing
Spry2-HA were stimulated with FGF10 (250 ng/ml) and immunoprecipitated
with anti-HA. Analysis with anti-phosphotyrosine antibodies (Fig.
1B) showed that mSpry2-HA was tyrosine phosphorylated in
response to FGF10. These results suggest that FGF10 stimulation
posttranslationally modifies mSpry2 through tyrosine phosphorylation.
The exact site at which mSpry2 is tyrosine phosphorylated is currently
under investigation.
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mSpry2 inhibits FGF10 stimulated MAP kinase activation.
Recently, it has been shown that MAP kinase activation in response to
addition of vascular endothelial growth factor (VEGF) or FGF2 to human
umbilical vein endothelial cells (endothelial) is inhibited in cells
overexpressing hSpry-1 or hSpry-2 cDNAs (5). Thus we
examined the possible role of mSpry2 as an MAP kinase inhibitor in
MLE15 cells stimulated with FGF10. MLE15 cells were transfected with
either pGLV-1 or pGLV-1 containing the mSpry2-HA construct and
stimulated with FGF10 (250 ng/ml) after serum starvation. Immunoblotting with activated MAP kinase antibody showed that overexpression of mSpry2 significantly reduced FGF10 MAP kinase activation at 10 min compared with cells alone (data not shown) or
cells transfected with pGLV-1 (Fig. 2).
To determine equal loading, the immunoblot was reprobed with Erk1
antibody, and overexpression of mSpry2-HA was confirmed by probing with
anti-HA (data not shown).
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mSpry2 binds to upstream target proteins in response to FGF10
stimulation.
Drosophila Sprouty is intracellularly located and binds to
both Gap1 and Drk (a Grb2 ortholog), inhibiting MAP kinase activation (1). Because mSpry2 inhibits MAP kinase activation in
response to FGF10, we examined the possibility that mSpry2 binds to
upstream target proteins, resulting in reduced MAP kinase activation.
MLE15 cells were transfected with mSpry2-HA and stimulated with FGF10. After serum starvation, cells were lysed and immunopercipitated with
anti-Grb2 antibody. FGF10-stimulated cells overexpressing mSpry2 showed
a significant increase in association of mSpry2 with Grb2 (54%)
compared with unstimulated cells overexpressing mSpry2 and control
cells (Fig. 3A). There was
also a decrease in association of Grb2 with FRS2 in MLE15 cells
overexpressing mSpry2 (Fig. 3A). This suggests that mSpry2
may block or interfere with the binding of Grb2 to FRS2, inhibiting
full potentiation of MAP kinase activation. The same experiment was
performed using anti-FRS2, anti-Raf, anti-Shp2, anti-Gap1, FGFR2b, and
anti-Sos. mSpry2-HA association with FRS2 increased by 83%, and Raf
(49%) also increased (Fig. 3, B, C, and F) upon
FGF10 stimulation, whereas Shp2 and Gap1 association with mSpry2-HA was
decreased by 62 and 89%, respectively, in response to FGF10 (Fig. 3,
D-F). No change was observed in the interaction between
mSpry2 and Sos or FGFR2b upon FGF10 stimulation (data not shown). These
results support the concept that mSpry2 is an intracellular protein
that binds differentially to several key upstream target proteins,
thereby suppressing their ability to completely activate the MAP kinase pathway.
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mSpry2 translocates to the cell membrane in response to FGF10.
Both hSpry1 and hSpry2 were found to translocate to the plasma membrane
in response to growth factor stimulation (5). Therefore, we examined the possibility that mSpry2 translocates to the cell membrane in mouse lung epithelial cells stimulated with FGF10. The
mSpry2-GFP construct was overexpressed in MLE15 cells, and confocal
microscopy was used to determine localization. In unstimulated cells,
mSpry2-GFP was clearly distributed throughout the cytoplasm (Fig. 4,
A-C). In contrast,
FGF10-stimulated cells showed that mSpry2 spatially translocated to
specific regions near the plasma membrane and granular structures
localized within the cytoplasm (Fig. 4, D-F). Thus our
data demonstrate that FGF10 directly modulates mSpry2-GFP cellular
localization.
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DISCUSSION |
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We have previously shown in whole mouse lung explants that exogenous FGF10 induces mSpry2 expression in the epithelium and that overexpression of mSpry2 decreases epithelial proliferation and branching morphogenesis (9). However, the intracellular function of mSpry2 in response to FGF10 stimulation was not examined. Herein, we provide the first evidence that stimulation of mouse lung epithelial cells with FGF10 induces tyrosine phosphorylation of mSpry2, differential binding of mSpry2 to Grb2/FRS2/Raf, disassociation of mSpry2 from Shp2 and Gap1, net reduction of MAP kinase activation, and translocation of mSpry2 to intracellular membrane compartments. Therefore, we suggest that mSpry2 functions as an intracellular inhibitor of FGF signaling, reducing MAP kinase activation, which in turn would modulate growth and differentiation.
In Drosophila, Sprouty was shown by immunopercipitation to associate with Gap1 and Drk (Grb2 ortholog) but not with Sos, Dos, Csw, Ras, Raf, or Leo (1). However, during wing development, Sprouty was reported to interfere with proteins downstream of Raf (13) and negatively regulates both EGFR and FGFR activation, leading to a reduction of MAP kinase activity. Herein, we have shown that mSpry2 associates in complexes with Grb2, FRS2, Shp2, Gap1, Raf, FGFR2b, and Sos1 in either unstimulated or FGF10-stimulated cells overexpressing mSpry2. FGFR activation induces tyrosine phosphorylation of mSpry2 and increases association of mSpry2 with Grb2, FRS2, and Raf with a decrease in binding of mSpry2 with Shp2 and Gap1, resulting in a net reduction of MAP kinase activity. MAP kinase inhibition has previously been shown to occur in endothelial cells overexpressing mammalian Sprouty2 in response to VEGF and FGF2 (5). However, a plausible mechanism for reduction in MAP kinase activity was not reported. Our results suggest that inhibition of MAP kinase activity in cells overexpressing mSpry2 is a result of differential binding of mSpry2 to several key target proteins upstream in the MAP kinase activation pathway.
FGFRs require adapter or docking proteins linking receptor activation
to the MAP kinase signaling pathway. FGFR1 activation in neuronal cells
results in tyrosine phosphorylation of FRS2, membrane translocation,
and association of FRS2 with the Grb2/Sos complex activating MAP
kinase. However, for full potentiation of MAP kinase and PC-12 cell
differentiation, the Shp2 tyrosine phosphatase must bind to the
FRS2/Grb2/Sos complex upon FGFR activation (3). Our study
shows that, upon FGFR2b activation, mSpry2 is tyrosine phosphorylated,
and there is increased association of mSpry2 with FRS2 and Grb2,
together with a decrease in association of mSpry2 with Shp2, leading to
a net reduction in MAP kinase activation. We propose that Shp2 binds to
mSpry2 under nonstimulated conditions, thereby inhibiting mSpry2
tyrosine phosphorylation, thus keeping mSpry2 in an inactive state.
Upon FGFR activation, Shp2 disassociates from mSpry2, releasing its
inhibitory influence on mSpry2 tyrosine phosphorylation, thereby
facilitating the interaction of mSpry2 with both FRS2 and Grb2. It is
possible that the increase in association of mSpry2 with Grb2 and FRS2
upon FGFR activation also interferes with the ability of FRS2 to bind
the Grb2/Sos complex, since we have shown that overexpression of mSpry2
reduces the association between Grb2 and FRS2. This in turn could also lead to a reduction of MAP kinase activation (Fig.
5). Whether this competitive interaction
occurs directly or indirectly is currently being investigated.
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Drosophila Sprouty associates with Gap1 and either interacts with Raf or downstream of Raf (1, 13), negatively regulating both EGFR and FGFR activation. We have shown that mSpry2 interacts with both Gap1 and Raf in mouse lung epithelial cells. Upon FGFR2b activation, an increase in association with Raf is observed, suggesting that mSpry2 also acts as a negative regulator downstream of Ras. The increase in association between mSpry2 and Raf upon FGFR activation could limit full activation of Raf, also resulting in a net reduction in MAP kinase activity (Fig. 5). Gap1 can also limit Ras activation by hydrolyzing GTP to GDP, resulting in decreased MAP kinase activation. Thus it is possible that the substantial decrease in association between mSpry2 and Gap1 upon FGFR activation may also play a role in negative feedback control of MAP kinase activation.
MLE15 cells overexpressing mSpry2-GFP showed that mSpry2 was diffusely localized within the cytoplasm, whereas upon FGF10 stimulation mSpry2-GFP was concentrated at the cell membrane and in intracellular membrane compartments. These results indicate a clear-cut spatial translocation of mSpry2 upon growth factor stimulation, as demonstrated by immunohistochemistry and confocal microscopy. Previously, it has been shown that endogenous hSpry1 is associated with perinuclear and vesicular structures in proliferating cells, whereas growth factor stimulation results in modulation of hSpry1 localization to the plasma membrane (5). Our results show that a similar mSpry2 translocation occurs in response to FGF10. It is interesting to note that growth factor stimulation of PC-12 cells also results in FRS2 phosphorylation and translocation of FRS2 to the plasma membrane as well as association of FRS2 with the Grb2/Sos complex (11). A similar response is noted herein with mSpry2 upon FGF10 stimulation in MLE cells. We are currently further investigating the function of mSpry2 tyrosine phosphorylation to determine whether tyrosine phosphorylation of mSpry2 induces membrane translocation and is required for binding of mSpry2 to the FRS2/Grb2/Shp2 complex, resulting in inhibition of MAP kinase activation.
In conclusion, we have shown that mSpry2 inhibits FGF10-FGFR downstream signaling in mouse lung epithelial cells by differentially binding to FRS2, Grb2, and Raf and by disassociating from Gap1 and Shp2, resulting in a net reduction of MAP kinase activity. mSpry2 also spatially translocates to the plasma membrane in response to FGF10, suggesting that, like FRS2, mSpry2 is a potential docking protein but that, unlike FRS2, mSpry2 functions to negatively regulate FGF signaling. Herein, we have proposed a working hypothesis on how mSpry2 negatively regulates the FGF10 signaling pathway. Thus we can begin to assess the function of mSpry2 in response to other FGFs and other pathways. We conclude that mSpry2 negatively modulates FGFR signaling, such that the detrimental effects of uncontrolled MAP kinase activation are held in check.
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ACKNOWLEDGEMENTS |
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We thank George Mcnamara for assistance in the CHLA Imaging Core.
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FOOTNOTES |
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This work is supported by National Heart, Lung, and Blood Institute Grants HL-44977, HL-44060, and HL-60231 (D. Warburton), by Human Frontier Science Program Grant RG0051/1999-M, and French Association pour la Recherche sur le Cancer Grant ARC no. 5214 (S. Bellusci).
Address for reprint requests and other correspondence: D. Warburton, USC Center for Craniofacial Biology, 2250 Alcazar St., CSA 103, Los Angeles, CA 90033 (E-mail: dwarburton{at}chla.usc.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 17, 2002;10.1152/ajplung.00372.2001
Received 19 September 2001; accepted in final form 25 April 2002.
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