FIGURE 4.

MLK3 is important downstream of RALA in activation of the JIP1-JNK complex. A, activation of RALA results in phosphorylation of MLK3 in its activation loop. HEK293T cells were analyzed for phosphorylation on Thr-277/Ser-281. B, MLK3 specifically binds to GTP-bound RALA. HEK293T cells were transfected with the indicated constructs, and the GST pulldown was performed as described under “Experimental Procedures.” Western blots were probed for MLK3 and GST. C, RALA interacts with the kinase domain of MLK3. HA-tagged MLK3 mutants, as depicted in the upper panel, were overexpressed, and endogenous RALA was immunoprecipitated. Western blots were analyzed for HA and RALA. Asterisks indicate background bands. D, knockdown of MLK3 inhibits ROS-induced activation of JNK on JIP1. HA-JIP1 was immunoprecipitated with JIP1 antibody. Western blots were analyzed for pJNK (Thr-183/Tyr-185) and HA (JIP1 and JNK). Scr, Scrambled; IP, immunoprecipitation; WB, Western blot.