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

doi:10.1074/jbc.m802855200

Mengwei Zang, Jun Gong + Show 10 more

Open Access

https://doi.org/10.1074/jbc.m802855200

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Abstract

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.

Highlights

The Raf family of serine/threonine protein kinases consists of three isoforms, A-Raf, B-Raf, and Raf-1 (C-Raf), all of which serve as immediate downstream effectors of Ras [1]
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
We made a series of mutations to mimic or disable phosphorylation in two sets of sites, Ser338 and Tyr341, the negative region between the amino-terminal regulatory and carboxyl kinase domains, and Ser471/Ser494, in the activation segment, and tested the impacts of phosphorylation of these sites on Raf kinase activity

Summary

EXPERIMENTAL PROCEDURES

Transfections, Immunoprecipitation, and Western Blot—Human embryonic kidney 293 cells (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cell lysates containing equal amounts of specific proteins were incubated with protein A/G-agarose (Santa Cruz Biotechnology) pre-absorbed with specific antibodies at 4 °C overnight. For purification of recombinant GST-Raf, the lysates were incubated with GSH beads and washed as immunoprecipitation. Raf-1 Kinase Assay—Raf-1 kinase activity was measured by a coupled enzyme assay in which bacterially expressed recombinant GST-MEK1 and a kinase-dead mutant of ERK2 were sequentially added to the Raf preparation in the presence of [␥-32P]ATP (100 ␮M, 4000 cpm/pmol), as described previously [41]. The radiolabeled ERK2 bands were excised and quantified by liquid scintillation counting

RESULTS

Raf variants increased as more Raf

DISCUSSION

Phosphorylation has been known to play an important role in