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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.
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