Shp2 activation in focal adhesions results in transient ERK activation and ERK-dependent spreading. (A) HEK293T cells transiently expressing FAK(397)FRB-mVenus coexpressed with R32L RapR-Shp2-mVenus, CA-Shp2-mVenus, or DN-Shp2-mVenus were treated with rapamycin for indicated time and analyzed for phosphorylation of ERK1/2. Representative blots of N = 3 biological replicates. Right: Quantification of phospho-ERK signal normalized to total ERK signal and to the 0 min time point. Error bars show 90% CI. 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 (0 min). Significant difference in the means was observed (F[7,1] = 9.33, P < 0.01). *P < 0.05. (B) HeLa cells transiently expressing R32L RapR-Shp2 and FAK(397)-FRB were stained with CellMask Deep Red membrane marker and imaged for 4 h every 2 min. Cells were pretreated with Trametinib (10 nM) for 1 h prior to imaging. Rapamycin was added at 0 min. Normalized change in cell area was binned over 1 h intervals. The area of R32L RapR-Shp2 targeted to FAK(397)-FRB in the absence of trametinib (green, data from Fig. 8 C) plotted against R32L RapR-Shp2 targeted to FAK(397)-FRB with trametinib treatment (cyan, F[3,1] = 4.69, P = 0.005; N = 22 cells, three independent biological replicates). Treatment with trametinib significantly influenced cell spreading vs FAK targeting without treatment (F[3,1] = 15.69, P < 0.001). Box and whisker plots: Box indicates first through third quartile with median line, whiskers are 10–90%, and open square represents mean. To evaluate the effect of RapR-Shp2 versus each inhibitor two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point for R32L RapR-Shp2 targeted to FAK with and without Trametinib. *P < 0.05, **P < 0.01, ***P < 0.001.