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.