Characterization of Ser338 phosphorylation for Raf-1 activation.
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 Ser338 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 Ser338 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 Ser338 on Raf-1 is autophosphorylated in response to mitogens.
Zang
Mengwei
M
Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Gong
Jun
J
Luo
Lingqi
L
Zhou
Jing
J
Xiang
Xiaoqin
X
Huang
Wei
W
Huang
Qiren
Q
Luo
Xixi
X
Olbrot
Martin
M
Peng
Yihong
Y
Chen
Changyan
C
Luo
Zhijun
Z
D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006801:Humans; D009154:Mutation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D055503:Protein Multimerization; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D054462:p21-Activated Kinases / Q000378:metabolism
D010768:Phosphoserine; D004815:Epidermal Growth Factor; D019908:Proto-Oncogene Proteins c-raf; D054462:p21-Activated Kinases
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