Figure 6.

Preferential dephosphorylation of Sprouty1 by SHP2/T507K is responsible for Ras pathway overactivation. A, SHP2/T507K is more efficient than WT SHP2 in dephosphorylating Sprouty1. TCL means total-cell lysate. Sprouty1 was immunoprecipitated by anti-Sprouty1 antibody, and the total phosphorylation of Sprouty1 (pTyr Sprouty1) was blotted with 4G10 anti-phosphotyrosine antibody. B, SHP2/T507K promotes dephosphorylation of Sprouty1/pY53. The phosphorylation of Sprouty1 at Tyr-53 in total-cell lysates was blotted with anti-pY53 Sprouty1 antibody. C, enhanced dephosphorylation of Sprouty1 decreases sequestration of Grb2. WT SHP2 and SHP2/T507K mutant were co-transfected with Flag-tagged Grb2; Sprouty1 was immunoprecipitated (IP) by anti-Sprouty1 antibody, and the co-immunoprecipitated Grb2 was blotted with anti-Flag antibody. D, Sprouty1 siRNA effectively knocks down Sprouty1, increases pERK1/2 and pAKT, and blunts the SHP2/T507K-induced overactivation effect. At each time point, the ratio of pERK/ERK (or pAKT/AKT) for WT in scrambled siRNA-expressed cells is taken as reference (labeled as ref.), then the ratios for other three lanes at the same time point were normalized to the reference. The overactivation is calculated based on the normalized values (TK/WT). The results of Western blots are representative of three independent experiments, and the star in the figure indicates a statistical significance from Student's t test (p < 0.05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.