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First published online December 9, 2005
doi: 10.1242/10.1242/jcs.02711

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Efficient suppression of FGF-2-induced ERK activation by the cooperative interaction among mammalian Sprouty isoforms

Laboratory of Cell Regulation, Department of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, 1-14, Bunkyo-machi, Nagasaki 852-8521, Japan

View larger version (20K): [in a new window]   Fig. 1. Mammalian Sprouty isoforms form homo-/hetero-oligomers through their C-terminal domains. (A) Expression constructs of Sprouty1- 4, either Flag-tagged at their N-terminus (F1-F4) or Myc-tagged at their C-terminus (1M-4M), are illustrated. Hatched boxes indicate cysteine-rich C-terminal domains. (B) 293T cells were co-transfected with two expression plasmids (0.5 µg each), one encoding one of the Flag-tagged Sprouty isoforms and the other encoding one of the Myc-tagged Sprouty isoforms as indicated. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-Myc/anti-Flag antibody, followed by immunoblotting (IB) with anti-Flag/anti-Myc antibody. Rabbit non-immune IgG was used as a control. (C) F1, F1n, and F1c represent constructs encoding Flag-tagged full-length, N-terminal domain (residues 1-174), and C-terminal domain (residues 175-313) of Sprouty1, respectively. Expression of these constructs in 293T cells was assured by immunoblot analysis with anti-Flag antibody. (D) 293T cells were co-transfected with two plasmids (0.5 µg each), one encoding one of the Myc-tagged Sprouty isoforms and the other encoding Flag-tagged full-length, N-terminal domain or C-terminal domain of Sprouty1 as indicated. Cell lysates (500 µg protein) were subjected to immunoprecipitation using anti-Flag/anti-Myc antibody, followed by immunoblotting with anti-Myc/anti-Flag antibody. Data shown in B and D are representative of three separate experiments that gave essentially the same results.

View larger version (33K): [in a new window]   Fig. 2. Specific association between Grb2 and Sprouty1 or Sos1 and Sprouty4. (A) 293T cells were transfected with the expression plasmid encoding either Flag-tagged Sprouty1, Sprouty2, Sprouty3 or Sprouty4 (0.5 µg). After 24 hours, cells were serum-starved for 6 hours and then mock-treated (C) or treated with 20 ng/ml EGF (E) or 20 ng/ml FGF-2 (F) for 15 minutes. The cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-Grb2 antibody, anti-Sos1 antibody, or anti-RasGAP antibody, followed by immunoblotting with anti-Flag antibody (for Sprouty proteins), anti-Grb2 antibody, anti-Sos1 antibody or anti-RasGAP antibody. Rabbit non-immune IgG was used as a control. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of Sprouty proteins. (B) 293T cells transfected with the expression plasmid encoding Myc-tagged Sprouty1 or Sprouty2 were treated with 20 ng/ml EGF for 15 minutes. Cell lysates (500 µg protein) were subjected to a pull-down assay using Grb2-agarose beads (Grb2) or glutathione-sepharose 4B beads (Glut), followed by immunoblotting with anti-Myc antibody (for Sprouty1/2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Myc antibody to show the expression levels of exogenous Sprouty1/2 (Lys). (C) 293T cells were co-transfected with expression plasmids encoding Myc-tagged Sprouty1 and each of either Flag-tagged Sprouty2, Sprouty3 or Sprouty4, or co-transfected with expression plasmids encoding Myc-tagged Sprouty4 and each of either Flag-tagged Sprouty1, Sprouty2 or Sprouty3 as indicated (0.5 µg each). After 24 hours, the cells were serum-starved for 6 hours and then mock-treated (C) or treated with 20 ng/ml EGF (E) or 20 ng/ml FGF-2 (F) for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation using anti-Grb2 antibody or anti-Sos1 antibody, followed by immunoblotting with anti-Flag antibody, anti-Myc antibody, anti-Grb2 antibody, or anti-Sos1 antibody. Similar results were obtained in three independent experiments.

View larger version (22K): [in a new window]   Fig. 3. Co-expression of Sprouty1 and Sprouty4 efficiently suppresses ERK activation induced by FGF-2. (A) 293T cells were co-transfected with expression plasmids encoding HA-tagged ERK2 (0.5 µg) and each of either vector control (–), Flag-tagged Sprouty1, Sprouty2 or Sprouty4 (0.5 µg). After 24 hours, cells were serum-starved for 6 hours and then mock-treated (C) or treated with 20 ng/ml EGF, 20 ng/ml FGF-2 or 10 ng/ml PMA for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-HA antibody, followed by immunoblotting (IB) with anti-ppERK1/2 antibody. An anti-HA blot demonstrates equal amounts of immunoprecipitated ERK2. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/2/4. (B) 293T cells were co-transfected with expression plasmids encoding HA-tagged ERK-2 (0.5 µg) and vector control (–), Flag-tagged Sprouty1 (1 µg), Flag-tagged Sprouty2 (1 µg), Flag-tagged Sprouty4 (1 µg), Flag-tagged Sprouty1 and Sprouty2 in combination (0.5 µg each), Flag-tagged Sprouty1 and Sprouty4 in combination (0.5 µg each), or Flag-tagged Sprouty2 and Sprouty4 in combination (0.5 µg each) as indicated. ERK activation induced by FGF-2 or EGF was analyzed as described above. The relative intensity of phosphorylated HA-ERK2 band, as compared with that of the respective HA-ERK2 signal, was determined by using the Multi Gauge software, version 3.0 (Fuji Photo Film, Tokyo), and normalized to 1.00 for control cells without exogenous Sprouty protein(s) (ppERK2/ERK2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/2/4. Similar results were obtained in three independent experiments.

View larger version (48K): [in a new window]   Fig. 4. Co-expression of Sprouty1 and Sprouty4 efficiently inhibits the FGF-2-induced association of Grb2 with FRS2. 293T cells were co-transfected with expression plasmids encoding HA-tagged Grb2 (0.5 µg) and vector control (–), Flag-tagged Sprouty1 (1 µg), Flag-tagged Sprouty4 (1 µg), or Flag-tagged Sprouty1 and Sprouty4 in combination (0.5 µg each) as indicated. After 24 hours, cells were serum-starved for 6 hours and then mock-treated (Control) or treated with 20 ng/ml EGF or 20 ng/ml FGF-2 for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-HA antibody, followed by immunoblotting with anti-FRS2 antibody, anti-Shc antibody, anti-Grb2 antibody or anti-Flag antibody (for Sprouty1/4). The relative intensity of FRS2 band, as compared with that of the respective Grb2 signal, was determined by using the Multi Gauge software, and normalized to 1.00 for control cells without exogenous Sprouty protein(s) (FRS2/Grb2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/4. Similar results were obtained in three independent experiments.

View larger version (17K): [in a new window]   Fig. 5. Hetero-oligomer formation of Sprouty1/2 and Sprouty4 in FGF-2-stimulated Swiss 3T3 cells. (A) Swiss 3T3 cells were serum-starved for 24 hours and then stimulated with 20 ng/ml of FGF-2 for the indicated periods of time. In some experiments, the cells were pretreated with 10 µM PD184352 (PD) for 30 minutes and then stimulated with FGF-2. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Sprouty1 antibody, anti-Sprouty2 antibody, anti-Sprouty4 antibody, anti-ppERK1/2 antibody, anti-ERK1/2 antibody, anti-Sos1 antibody or anti-Grb2 antibody. (B) Swiss 3T3 cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for the indicated periods of time. Cell lysates (500 µg protein) were then subjected to immunoprecipitation (IP) using the anti-Sprouty4 antibody (2 µg each of sc-18607 and sc-18609), following by immunoblotting with anti-Sprouty1 antibody, anti-Sprouty2 antibody, anti-Sprouty4 antibody (sc-18607), anti-Sos1 antibody, or anti-Grb2 antibody. Similar results were obtained in three independent experiments.

View larger version (30K): [in a new window]   Fig. 6. siRNA knockdown of Sprouty1/Sprouty4 induces a prolonged activation of ERK1/2 in FGF-2-stimulated Swiss 3T3 cells. (A) Swiss 3T3 cells were transfected with Sprouty1 siRNA (1), Sprouty4 siRNA (4), Sprouty1 siRNA and Sprouty4 siRNA in combination (1+4), or matched scrambled RNAs to Sprouty1 siRNA and Sprouty4 siRNA in combination (C). After 24 hours, cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for 1 hour (for the analysis of Sprouty1 expression) or 2 hours (for the analysis of Sprouty4 expression). Total RNA was isolated and RT-PCR (27 cycles) was performed for Sprouty1 or Sprouty4 to generate 372 bp fragment or 443 bp fragment, respectively. A portion of GAPDH was co-amplified as an internal control (250 bp). The relative intensity of Sprouty1/4 band, as compared with that of the respective GAPDH signal, was determined by using the Multi Gauge software, and normalized to 1.00 for the respective scramble RNA-transfected control cells stimulated with FGF-2 for 0.5 hours. M, DNA ladder markers. (B) Swiss 3T3 cells were transfected with Sprouty1 siRNA (Sprouty1), Sprouty4 siRNA (Sprouty4), Sprouty1 siRNA and Sprouty4 siRNA in combination (Sprouty1 + Sprouty4), or matched scrambled RNAs to Sprouty1 siRNA and Sprouty4 siRNA in combination (Control). After 24 hours, cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for the indicated periods of time. Total cell lysates (10 µg protein) were subjected to immunoblot analysis with anti-ppERK1/2 antibody or anti-ERK1/2 antibody. The relative intensity of phosphorylated ERK1 and ERK2 bands, as compared with that of the respective ERK1 and ERK2 signals, was determined by using the Multi Gauge software, and normalized to 1.00 for the respective Swiss 3T3 cells stimulated with FGF-2 for 1 hour (ppERKs/ERKs). Similar results were obtained in two independent experiments.

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