Microtubule integrity regulates Pak leading to Ras-independent activation of Raf-1: Insights into mechanisms of Raf-1 activation

Growth factors activate Raf-1 by engaging a complex program, which requires Ras binding, membrane recruitment, and phosphorylation of Raf-1. The present study employs the microtubule-depolymerizing drug nocodazole as an alternative approach to explore the mechanisms of Raf activation. Incubation of cells with nocodazole leads to activation of Pak1/2, kinases downstream of small GTPases Rac/Cdc42, which have been previously indicated to phosphorylate Raf-1 Ser³³⁸. Nocodazole-induced stimulation of Raf-1 is augmented by co-expression of small GTPases Rac/Cdc42 and Pak1/2. Dominant negative mutants of these proteins block activation of Raf-1 by nocodazole, but not by epidermal growth factor (EGF). Thus, our studies define Rac/ Cdc42/Pak as a module upstream of Raf-1 during its activation by microtubule disruption. Although it is Ras-independent, nocodazole-induced activation of Raf-1 appears to involve the amino-terminal regulatory region in which the integrity of the Ras binding domain is required. Surprisingly, the Raf zinc finger mutation (C165S/C168S) causes a robust activation of Raf-1 by nocodazole, whereas it diminishes Ras-dependent activation of Raf-1. We also show that mutation of residues Ser³³⁸ to Ala or Tyr³⁴⁰-Tyr³⁴¹ to Phe-Phe immediately amino-terminal to the catalytic domain abrogates activation of both the wild type and zinc finger mutant Raf by both EGF/4β -12-O-tetradecanoylphorbol-13-acetate and nocodazole. Finally, an in vitro kinase assay demonstrates that the zinc finger mutant serves as a better substrate of Pakl than the wild type Raf-1. Collectively, our results indicate that 1) the zinc finger exerts an inhibitory effect on Raf-1 activation, probably by preventing phosphorylation of ³³⁸SSYY³⁴¹; 2) such inhibition is first overcome by an unknown factor binding in place of Ras-GTP to the amino-terminal regulatory region in response to nocodazole; and 3) EGF and nocodazole utilize different kinases to phosphorylate Ser ³³⁸, an event crucial for Raf activation.