Figure 7.
Role of N-SH2 domain of Shp2 in mediating ERK-independent migration and protrusive activity through a parallel ROCK II pathway. (A) Activation of ERK by Shp2 signaling. HEK293T cells transiently coexpressing indicated constructs and mCherry-FRB were treated with rapamycin for 1 h and cell lysates were analyzed by immunoblotting. A representative blot of N = 4 independent experiments is shown. (B–F) HeLa cells transiently expressing R32L (N = 27 cells, four separate biological replicates) or R138L (N = 23 cells, four separate biological replicates) mutants of RapR-Shp2-Cerulean-flag and mVenus-ipep-FRB were stained with CellMask Deep Red membrane marker and imaged live. Rapamycin was added at 0 min (gray line in C, E, and F). (B) Normalized area of cells expressing R32L (red, F[3,1] = 2.97, P = 0.083), R138L (blue, F[3,1] = 0.66, P = 0.57), or FRB alone (brown, data from Fig. 5) binned over 1 h intervals. Pairwise comparison of R32L or R138L vs FRB: R32L (F[3,1] = 6.40, P < 0.001], R138L (F[3,1] = 0.24, P = 0.87) (C) Changes in cell area in response to R32L (red, dashed line) or R138L (blue, solid line) RapR-Shp2 activation. Shaded area represents 90% CI. (D) Path length of cell centroid over 1 h intervals for R32L (red, F[3,1] = 0.52, P = 0.670) or R138L (blue, F[3,1] = 10.43, P < 0.001) RapR-Shp2, FRB control (brown, Data from Fig. 4) or R138L RapR-Shp2 pretreated with 10 µM Y-27632 (ROCK II inhibitor, N = 20 cells, three separate biological replicates) for 1 h (teal, F[3,1] = 1.07, P = 0.370). Only R138L RapR-Shp2 exhibited a significant effect vs FRB: R32L (F[3,1] = 0.77, P = 0.51), R138L (F[3,1] = 5.92, P = 0.001), R138L + Y-27632 (F[3,1] = 0.78, P = 0.51). (E and F) Changes in Protrusive Activity (E) and Retraction activity (F) of analyzed in C. Shaded area represents 90% CI. (B and D) 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 relative to the basal (−60 to 0 min or −1 h) time-frame post hoc. To evaluate the effect of each RapR-Shp2 SH2 domain mutant vs FRB two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point. *P < 0.05, **P < 0.01, ***P < 0.001.

Role of N-SH2 domain of Shp2 in mediating ERK-independent migration and protrusive activity through a parallel ROCK II pathway. (A) Activation of ERK by Shp2 signaling. HEK293T cells transiently coexpressing indicated constructs and mCherry-FRB were treated with rapamycin for 1 h and cell lysates were analyzed by immunoblotting. A representative blot of N = 4 independent experiments is shown. (B–F) HeLa cells transiently expressing R32L (N = 27 cells, four separate biological replicates) or R138L (N = 23 cells, four separate biological replicates) mutants of RapR-Shp2-Cerulean-flag and mVenus-ipep-FRB were stained with CellMask Deep Red membrane marker and imaged live. Rapamycin was added at 0 min (gray line in C, E, and F). (B) Normalized area of cells expressing R32L (red, F[3,1] = 2.97, P = 0.083), R138L (blue, F[3,1] = 0.66, P = 0.57), or FRB alone (brown, data from Fig. 5) binned over 1 h intervals. Pairwise comparison of R32L or R138L vs FRB: R32L (F[3,1] = 6.40, P < 0.001], R138L (F[3,1] = 0.24, P = 0.87) (C) Changes in cell area in response to R32L (red, dashed line) or R138L (blue, solid line) RapR-Shp2 activation. Shaded area represents 90% CI. (D) Path length of cell centroid over 1 h intervals for R32L (red, F[3,1] = 0.52, P = 0.670) or R138L (blue, F[3,1] = 10.43, P < 0.001) RapR-Shp2, FRB control (brown, Data from Fig. 4) or R138L RapR-Shp2 pretreated with 10 µM Y-27632 (ROCK II inhibitor, N = 20 cells, three separate biological replicates) for 1 h (teal, F[3,1] = 1.07, P = 0.370). Only R138L RapR-Shp2 exhibited a significant effect vs FRB: R32L (F[3,1] = 0.77, P = 0.51), R138L (F[3,1] = 5.92, P = 0.001), R138L + Y-27632 (F[3,1] = 0.78, P = 0.51). (E and F) Changes in Protrusive Activity (E) and Retraction activity (F) of analyzed in C. Shaded area represents 90% CI. (B and D) 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 relative to the basal (−60 to 0 min or −1 h) time-frame post hoc. To evaluate the effect of each RapR-Shp2 SH2 domain mutant vs FRB two-way repeated measures ANOVA with Holm-Bonferroni correction was performed for pairwise analysis of each time point. *P < 0.05, **P < 0.01, ***P < 0.001.

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