Role of MLK3-mediated Activation of p70 S6 Kinase in Rac1 Transformation

The signaling pathways that mediate the transforming activity of the Rac1 GTPase remain to be determined. In the present study, we used effector domain mutants of the constitutively activated Rac(61L) mutant that display differential transforming activities and differential activation of downstream effector pathways to investigate the contribution of p70 S6 kinase (p70 S6K ) to Rac1 transformation and to decipher the signaling pathways leading from Rac1 to p70 S6K . First, we found that Rac1 transforming activity could be dissociated from Rac1 activation of p70 S6K . A weakly transforming Rac1 mutant retained the ability to activate p70 S6K , whereas some potently transforming effector mutants were impaired in their ability to activate p70 S6K . These data suggest that p70 S6K is not necessary to promote full Rac1 transforming activity. We also found a strong correlation between the ability of the Rac(61L) effector mutants to activate p70 S6K and their ability to activate the JNK mitogen-activated protein kinase. We found that the MLK3 serine/threonine kinase activated JNK and p70 S6K , whereas activation of p70 S6K by Rac(61L) was significantly inhibited by dominant-negative MLK3. Additionally, the ability of the Rac(61L) effector mutants to activate MLK3 correlated well with their ability to activate p70 S6K and JNK. Taken together, these results provide evidence that Rac1 coordinately activates p70 S6K and JNK via MLK3 activation. Finally, we found that co-expression of wild type, but not kinase-dead, MLK3 significantly inhibited Rac1 transforming activity. These results suggest that MLK3 may be a negative regulator of the growth-promoting and transforming properties of Rac1.