Regulation of cell spreading and migration by Shp2 activation in different signaling complexes. (A) Schematic of RapRTAP system to specifically target and activate different Shp2 signaling complexes. RapRTAP-Shp2 can be independently targeted to FRB-Gab1 (purple), FRB-Gab2 (orange) or FAK-FRB (green). (B–E) HeLa cells transiently expressing R32L RapR-Shp2-Cerulean-flag and the FRB fused target protein were stained with CellMask Deep Red membrane dye and imaged live. Rapamycin was added at 0 min (gray line in B and D). CellMask images were used for analysis. (B and C) Changes in the area of cells coexpressing R32L RapR-Shp2 and FRB fusion proteins: FRB-GFP-Gab1 (Y628F/Y660F) (N = 38 cells, five separate biological replicates), FRB-GFP-Gab2 (Y604F/Y634F) (N = 57 cells, five separate biological replicates), and FAK-FRB-mVenus (N = 43 cells, four separate biological replicates). (C) Changes in cell area (over 1 h intervals) induced by activation of Shp2 targeted to Gab1 (purple, F[3,1] = 16.69, P < 0.001), Gab2 (orange, F[3,1] = 17.05, P < 0.001), and FAK (green, F[3,1] = 67.18, P < 0.001). (D) Changes in Protrusive and Retraction activity induced by R32L RapR-Shp2 targeted to Gab1 (purple), Gab2 (orange), and FAK (green). (E) Changes in cell migration (shown as path length over 1 h intervals) induced by activation of Shp2 targeted to Gab1 (purple, F[3,1] = 19.68, P < 0.001), Gab2 (orange, F[3,1] = 23.03, P < 0.001), and FAK (green, F[3,1] = 2.63, P = 0.053). (C and E) Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, open square represents mean. Analysis of statistical significance at the 0.05 level was determined using one-way repeated measures ANOVA with Dunnett’s correction post hoc relative to basal (−1 h). *P < 0.05, **P < 0.01, ***P < 0.001.