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 Ser(338). 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 Ser(338) to Ala or Tyr(340)-Tyr(341) 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/4beta-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 Pak1 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 (338)SSYY(341); 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.
Zang
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Diabetes and Metabolism Research Unit, Endocrinology Section, Evans Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Waelde
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CA
Xiang
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Rana
A
A
Wen
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Luo
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D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D008870:Microtubules / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016335:Zinc Fingers; D018631:ras Proteins / Q000378:metabolism*
D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D013755:Tetradecanoylphorbol Acetate; D015739:Nocodazole