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J. Biol. Chem., Vol. 280, Issue 16, 16244-16253, April 22, 2005

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B-Raf and Raf-1 Are Regulated by Distinct Autoregulatory Mechanisms^*

Nancy H. Tran, Xiaochong Wu, and Jeffrey A. Frost

From the Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030

B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser³³⁸. We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser⁴⁴⁵ was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser⁴⁴⁵ phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser⁴⁴⁵ primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.

Received for publication, February 1, 2005

^* This work was supported by American Cancer Society Research Grant RSG TBE-104121. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Integrative Biology and Pharmacology, University of Texas Health Science Center, 6431 Fannin St., Houston, TX 77030. Tel.: 713-500-6319; Fax: 713-500-7444; E-mail: jeffrey.a.frost{at}uth.tmc.edu.

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