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Fig. 4. Laminin activation of JNK is dependent on Rac and Cdc42 activation. (A) C2 cells were transfected with FLAG epitope-tagged JNK alone or in combination with expression plasmids encoding T7-epitope-tagged RacN17 or RacV12. Myotubes were treated for 15 minutes with laminin (10 nM), where specified, and the transfected JNK was immunoprecipitated from cell lysates with anti-FLAG antibody. The immunopurified JNK was incubated with [-³²P]ATP and GST-c-Jun as a substrate. Levels of transfected Rac and JNK expression were determined by western blotting with anti-T7 and anti-JNK1 primary antibodies, respectively. GST-c-Jun phosphorylation was visualized by autoradiography. As quantitated by a PhosphorImager, cells transfected with JNK alone showed a threefold increase in c-Jun phosphorylation when treated with laminin. By contrast, cells that were cotransfected with JNK plus RacN17 showed no JNK activation when treated with laminin. (B) C2 cells were cotransfected with FLAG epitope-tagged JNK and myc epitope-tagged Cdc42N17 or Cdc42V12. Immunoprecipitated JNK was incubated with [-³²P]ATP and GST-c-Jun. As quantitated by PhosphorImager, the threefold increase in phosphorylation induced by laminin was eliminated by the dominant negative Cdc42 mutant. Constitutively active mutants of Rac (RacV12) and Cdc42 (Cdc42V12) serve as positive controls for JNK activation. The fold-increases in activity in this figure were consistent over at least five separate experiments.

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