Figure 4.

PP2Cα C314 glutathionylation activates JNK and ERK pathways by selectively altering protein–protein interactions.A, activation of JNK, ERK1/2, and MEK4 upon PP2Cα glutathionylation. MDA-MB-231 cells expressing PP2Cα WT or C314S were incubated in low or high glucose for 6 h, and phosphorylation levels of PP2Cα substrates were analyzed by western blots (n = 3). B–C, JNK activation is responsible for increased cell migration. The wound-healing migration for 24 h (n = 3) (B) and JNK phosphorylation after 6 h (n = 3) (C) in cells expressing PP2Cα WT or C314S upon the addition of JNK-inhibitor (JNK-Inh). D, PP2Cα glutathionylation induces its dissociation from JNK, ERK1/2, and MEK4. After incubation of cells in low or high glucose for 6 h, co-immunoprecipitation was used to monitor their binding interactions by western blots (n = 3). E, a model for cell migration induced by PP2Cα glutathionylation. In a nonstressed condition, PP2Cα binds to its substrates for their dephosphorylation, which suppresses cell migration. However, H₂O₂ or ROS induced by low glucose causes PP2Cα glutathionylation at C314, which dissociates PP2Cα from selected signaling substrates, such as JNK, ERK1/2, and MEK4. The dissociation increases phosphorylation levels of JNK, ERK1/2, and MEK4, which causes increased cell migration via the JNK-paxillin pathway. Data show the mean ± SD or representative of three independent experiments. The statistical difference was analyzed by one-way ANOVA with Tukey’s post hoc test (A, D) or two-way ANOVA with Turkey’s post hoc test (B), where ∗p < 0.03, ∗∗p < 0.002, ∗∗∗p < 0.0002, ∗∗∗∗p < 0.0001.