Characterization of Ser338 phosphorylation for Raf-1 activation

Mengwei Zang, Jun Gong, Lingqi Luo, Jing Zhou, Xiaoqin Xiang, Wei Huang, Qiren Huang, Xixi Luo, Martin Olbrot, Yihong Peng, Changyan Chen, Zhijun Luo

Research output: Contribution to journalArticlepeer-review

56 Scopus citations


Raf kinases are essential for regulating cell proliferation, survival, and tumorigenesis. However, the mechanisms by which Raf is activated are still incompletely understood. Phosphorylation plays a critical role in Raf activation in response to mitogens. The present study characterizes phosphorylation of Ser338, a crucial event for Raf-1 activation. Here we report that mutation of Lys375 to Met diminishes phosphorylation of Ser 338 on both wild type Raf-1 in cells treated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and a constitutively active mutant in which Tyr340/Tyr341 are replaced by 2 aspartic acids, a conserved substitution present in natural B-Raf. The loss of Ser338 phosphorylation in these Raf mutants is not engendered by a mutation-induced conformational change, inasmuch as mutation of another site (Ser471 to Ala) in the activation segment also abolishes Ser 338 phosphorylation, whereas both the kinase-dead mutants of Raf-1 are phosphorylated well by active Pak1. Furthermore, our data demonstrate that EGF-stimulated phosphorylation of Ser338 is inhibited by Sorafenib, a Raf kinase inhibitor, but not by the MEK inhibitor U0126. Interestingly, a kinase-dead mutation and Sorafenib also markedly reduce phosphorylation of Ser445 on B-Raf, a site equivalent to Raf-1 Ser338. Finally, our data reveal that Ser338 is phosphorylated on inactive Raf-1 by an active mutant of Raf-1 when they are dimerized in cells and that artificial dimerization of Raf-1 causes Ser338 phosphorylation, accompanied by activation of ERK1/2. Altogether, our data suggest that Ser 338 on Raf-1 is autophosphorylated in response to mitogens.

Original languageEnglish (US)
Pages (from-to)31429-31437
Number of pages9
JournalJournal of Biological Chemistry
Issue number46
StatePublished - Nov 14 2008
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry
  • Cell Biology


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