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First published online November 24, 2004
doi: 10.1242/10.1242/jcs.01519

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FRS2-dependent SRC activation is required for fibroblast growth factor receptor-induced phosphorylation of Sprouty and suppression of ERK activity

¹ CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK
² CR-UK Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD, UK

View larger version (30K): [in a new window]   Fig. 1. Activated FGFR phosphorylates hSpry2 Y55 in an FRS2-dependent manner. (A) hSpry2 was tyrosine-phosphorylated downstream of the active FGFR. Wild-type hSpry2 (hSpry2WT) was co-transfected into 293T cells with either constitutively active FGFR1 (FcFGFR1/FA/VT+), kinase dead FGFR1 (FcFGFR1/FD) or truncated FcFGFR1 (FcFGFR1/FT). FGFR constructs were immunoprecipitated by protein A (IP:Fc) and either immunoblotted with anti-IgG Fc or anti-phosphotyrosine antibodies. Similarly, hSpry2 was immunoprecipitated and immunoblotted with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies (IP:hSpry2). (B) Selective phosphorylation of hSpry2 on Y55 by the FGFR1/VT+ splice isoform (FcFGFR1/FA/VT+). 293T cells were either stimulated with FGF2 (30 minutes) or transfected with either FcFGFR1/FA/VT+ or VT-isoforms in the presence of either hSpry2WT or hSpry2Y55A. hSpry2 was immunoprecipitated and immunoblotted with anti-hSpry2 antibody and anti-phosphotyrosine antibodies (IP:hSpry2). FGFR constructs were immunoprecipitated by protein A and immunoblotted with anti-IgG Fc (IP:Fc).

View larger version (30K): [in a new window]   Fig. 2. SU6656 inhibits activated FGFR-mediated tyrosine phosphorylation of hSpry2. (A) Inhibition of activated FGFR-mediated tyrosine phosphorylation of hSpry2 by SU6656. hSpry2 was co-transfected with wild-type FGFR2 into 293T cells. Cells were pre-treated with various concentrations of SU6656 for 1 hour before FGF2 stimulation (30 minutes). hSpry2 was immunoprecipitated and then immunoblotted with anti-hSpry2 antibody or anti-phosphotyrosine antibodies (IP:hSpry2). Antibodies against SFKs (anti-SRC2) and active SFKs (anti-SRC pY416) were used to examine SFK expression and activity. (B) Effect of 20uM SU6656 on overexpressed FGFR2-induced tyrosine phosphorylation profile. 293T cells were transfected with FGFR2 and were either pre-treated with 20 µM SU6656 or DMSO (control) for 1 hour prior to FGF2 stimulation for 30 minutes. FGFR2 was immunoprecipitated (IP:FGFR2) and immunoblotted with both and anti-phosphotyrosine antibody anti-FGFR2 antibodies (IB:pY and IB:FGFR2, respectively).

View larger version (28K): [in a new window]   Fig. 3. hSpry2 is a substrate for activated SRC kinase induced by FGF stimulation. (A) Tyrosine-phosphorylation of hSpry2 in SYF/wtSRC cells was dependent on FGF2 stimulation. Wild-type hSpry2 was transfected into SYF cells stably expressing wild-type SRC (SYF/wtSrc) or SYF cells stably expressing kinase active SRC (SYF/Src Y527F) and cells were incubated in the presence or absence of FGF2 (30 minutes). hSpry2 was immunoprecipitated and immunoblotted with either anti-phosphotyrosine antibodies (IP:hSpry2) or anti-hSpry2 antibody (IB:pY and IB:hSpry2, respectively). Antibodies against SFKs (anti-SRC2) and active SFKs (anti-SRC pY416) were used to confirm SFK expression and activity. (B) SRC/FYN/YES deficiency resulted in loss of tyrosine-phosphorylation of hSpry2. SYF cells (deficient in SRC/FYN/YES tyrosine kinases) and NIH3T3 fibroblasts were stimulated with FGF2 (30 minutes) in either the presence or absence of transfected hSpry2WT. hSpry2 was immunoprecipitated and immunoblotted with either anti-phosphotyrosine antibodies or anti-hSpry2 antibody. Anti-SRC (SRC2) antibody was used to check SFK expression in the cells. Cell lysates were immunoblotted with anti-active-ERK1/2 to assess the activation of endogenous ERK1/2 and reprobed with anti-ERK1/2 to confirm comparable protein loading. (C) SRC and FYN restored tyrosine phosphorylation of hSpry2 in SYF cells. Wild-type hSpry2 was co-expressed with either SRC or FYN into SYF cells. SYF or SYF control cells transfected with hSpry2WT were stimulated with FGF2 (30 minutes). hSpry2 was immunoprecipitated and immunoblotted with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies. Antibodies against SFKs (anti-SRC2) and active SFKs (anti-SRC pY416) were used to confirm SFK expression and activity. Cell lysates were immunoblotted with anti-active-ERK1/2 to assess the activation of endogenous ERK1/2 and reprobed with anti-ERK1/2 to confirm comparable protein loading.

View larger version (39K): [in a new window]   Fig. 4. SRC-dependent phosphorylation of hSpry2 on Y55 is required for hSpry2-mediated inhibition of MAP Kinase. (A) Phosphorylation of hSpry2 on Y55 was an FGFR activation-dependent event. NIH3T3 cells were transfected with either hSpry2WT or hSpry2Y55A and then incubated in the absence or presence of FGF2 (10 minutes). hSpry2 was immunoprecipitated and immunoblotted with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies. Endogenously expressed FGFR2 was immunoprecipitated and immunoblotted with either anti-phosphotyrosine antibodies or anti-FGFR2 antibody (Bek c-17). (B) SRC-dependent phosphorylation of hSpry2 is absent in hSpry2Y55A. hSpry2WT or hSpry2Y55A were transfected into SYF cells stably expressing either wild-type SRC (SYF/wtSrc) or a constitutively active SRC (SYF/Src Y527F) before stimulation with FGF2 (30 minutes). hSpry2 was immunoprecipitated and immunoblotted with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies. Cell lysates were immunoblotted with anti-active-ERK1/2 to assess the activation of endogenous ERK1/2 and reprobed with anti-ERK1/2 to confirm comparable protein loading.

View larger version (17K): [in a new window]   Fig. 5. hSpry2 is a direct substrate for SRC kinase. Five micrograms of the N-terminal region of hSpry2WT (amino acids 1-172) was used as a substrate for an in-vitro kinase assay. Reaction conditions included either the absence or presence of 100 µM ATP and/or 200ng of active SRC. Tyrosine-phosphorylation was confirmed using anti-phosphotyrosine antibodies. Equal substrate concentration was confirmed by anti-hSpry2 antibody.

View larger version (21K): [in a new window]   Fig. 6. Tyrosine-phosphorylated hSpry2 is associated with SRC. (A) hSpry2 was associated with SRC. hSpry2WT was transfected into SYF cells alone or SYF cells stably expressing wtSRC (SYF/wtSrc), kinase active SRC (SYF/Src Y527F) or a kinase dead SRC (SYF/Src KD). Cells were subsequently stimulated with FGF2 (30 minutes). SRC was immunoprecipitated with a monoclonal anti-SRCMab327 antibody and immunoblotted with anti-SRC (SRC2) antibody and hSpry2 association was examined using anti-hSpry2 antibody. Relative expression and phosphorylation of hSpry2 was confirmed with either anti-hSpry2 antibody or anti-phosphotyrosine antibodies. (B) Preferential pY55-mediated association of SRC with hSpry2. Peptides derived from hSpry2 that included Y55 in both its phosphorylated (lanes 1) and non-phosphorylated (lanes 2) form were used to isolate potential binding partners. Peptide-associated proteins were resolved on SDS-PAGE gel, and the presence of SFKs revealed utilizing anti-phosphotyrosine antibodies and anti-SFK (SRC2) antibody.