FIG. 3.

LS mutants impair dephosphorylation of GAB1 PI3K-binding sites. (A) WT, NS, or LS fibroblasts were stimulated with EGF for the indicated times and then immunoprecipitated with an anti-GAB1 antibody and probed for p85 and GAB1. (B) Quantification of p85 precipitation using ImageJ. Only significant differences versus WT cells for the corresponding time are indicated (***, P < 0.001; n = 3). The error bars indicate SEM. (C) HEK293 cells were transfected with Myc-tagged GAB1 and the indicated V5-SHP2 constructs and then subjected to a PI3K affinity precipitation assay (pull-down) using a GST-PI3K (GST-p85) fusion protein. The amount of precipitated GAB1-Myc was analyzed with an anti-Myc antibody (top gel). Aliquots of corresponding lysates were probed with anti-Myc and anti-V5 antibodies (middle and bottom gels). (D) Analysis of recombinant LS and NS SHP2 mutants by in vitro phosphatase assays. (Left) Two micrograms of the indicated recombinant SHP2 proteins was analyzed by SDS-PAGE and Coomassie blue staining. (Right) Phosphatase assays were performed by incubating the indicated recombinant SHP2 proteins with Src-pY530 or GAB1-pY589 phosphopeptide as a phosphatase substrate. The assays were performed in the presence or absence of an SHP2-activating peptide, as indicated.