Activation of Raf-1 is a complex process in which phosphorylation of Ser338–Tyr341 is a critical step. Previous studies have shown that Pak1/2 is implicated in both Ras-dependent and -independent activation of Raf-1 by phosphorylating Raf Ser338. The present study explores the structural basis of Raf-1 phosphorylation by Pak1. We found that Pak directly associates with Raf-1 under both physiological and overexpressed conditions. The association is greatly stimulated by 4β-12-O-tetradecanoylphorbol-13-acetate and nocodazole and by expression of the active mutants of Rac and Ras. The active forms of Pak generated by mutation of Thr423 to Glu or truncation of the amino-terminal moiety exhibit a greater binding to Raf than the wild type, whereas the kinase-dead mutant Pak barely binds Raf. The extent of binding to Raf-1 is correlated with the ability of Pak to phosphorylate Raf and induce mitogen-activated protein kinase activation. Furthermore, the Raf-1 binding site is defined to the carboxyl terminus of the Pak catalytic domain. In addition, our results suggest that the amino-terminal regulatory region of Raf inhibits the interaction. Taken together, the results indicate that the interaction depends on the active conformations of Pak and Raf. They also argue that Pak1 is a physiological candidate for phosphorylation of Raf Ser338 during the course of Raf activation.
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Published, JBC Papers in Press, November 30, 2001, DOI 10.1074/jbc.M110000200
This work was supported by National Institutes of Health Grant GM 57959 (to Z. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
