Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf

Brummer, Tilman; Naegele, Heike; Reth, Michael; Misawa, Yukiko

doi:10.1038/sj.onc.1207185

Cited by 105 publications

(100 citation statements)

References 67 publications

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“…As we have shown previously (Brummer et al, 2003), expression of B-Raf wt , but not of B-Raf K483W , resulted in the differentiation of 10-20% of transfectants ( Figure 2). In line with its high signalling activity (Figure 1b), approximately 50% of PC12 transfectants expressing B-Raf V600E displayed prominent neurites, a percentage that has been also observed upon expression of other Raf oncogenes (MacNicol et al, 2000;Dhillon et al, 2003) or the constitutively active enhanced green fluorescent protein (EGFP)-DB-Raf protein (Figure 2; Brummer et al 2003). In comparison with B-Raf wt transfectants, those expressing B-Raf R188L displayed a 3-4-fold reduction in neurite outgrowth, indicating that B-Raf must interact with Ras-GTPases to exhibit its full biological activity (Figure 2b).…”

Section: V600esupporting

confidence: 80%

“…It should be noted that the recently revised human B-Raf cDNA (Wellbrock et al, 2004a) as well as the chicken B-Raf cDNA used in this study (Brummer et al, 2003) represent the same splice variant (lacking the neuron-specific exon 10) and are both one amino acid longer than the previously reported human cDNA. Consequently, amino-acid (aa) residues like R187, S364, S445, V599 and S728, which were mentioned throughout earlier publications, correspond in fact to R188, S365, S446, V600 and S729 in both chicken B-Raf and the recently revised human B-Raf (for a review, see Wellbrock et al (2004a)).…”

Section: Plasmid Constructsmentioning

confidence: 83%

“…To this end, we introduced the V600E mutation into the pFlu/B-Raf vector encoding N-terminal, hemagglutinine (HA)-tagged chicken B-Raf wt , which is 93% identical with human B-Raf at the amino-acid level ( Figure 1a; Brummer et al, 2003). To investigate the effect of the V600E mutation on Ras-binding deficient B-Raf, we also introduced this mutation into a vector encoding Ras-GTP binding-deficient B-Raf R188L .…”

Section: Resultsmentioning

confidence: 99%

“…The pFlu/B-Raf vector encoding HA-tagged bursal chicken BRaf has been described (Brummer et al, 2003). It should be noted that the recently revised human B-Raf cDNA (Wellbrock et al, 2004a) as well as the chicken B-Raf cDNA used in this study (Brummer et al, 2003) represent the same splice variant (lacking the neuron-specific exon 10) and are both one amino acid longer than the previously reported human cDNA.…”

Section: Plasmid Constructsmentioning

confidence: 90%

“…PC12 cells were transfected with the indicated pFlu/B-Raf plasmids. As a positive and negative control for differentiation, cells were transfected with an expression vector encoding for a constitutively active enhanced green fluorescent protein (EGFP)-DB-Raf fusion protein or catalytically inactive B-Raf K483W , respectively (Brummer et al, 2003). Transfectants were visualized by immunofluorescence using an anti-hemaggluttinine (HA) antibody or GFP fluorescence.…”

Section: V600ementioning

confidence: 99%

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Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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The BRAF V600E mutation is found in approximately 6% of human cancers and mimics the phosphorylation of the kinase domain activation segment. In wild-type B-Raf (B-Raf wt ), activation segment phosphorylation is thought to cooperate with negative charges within the N-region for full activation. In contrast to Raf-1, the N-region of B-Raf is constitutively negatively charged owing to the presence of residues D447/D448 and the phosphorylation of S446. Therefore, it has been suggested that this hallmark predisposes B-Raf for oncogenic activation. In this study, we demonstrate that neutralizing mutations of these residues (in particular S446 and S447), or uncoupling of B-Raf from Ras-guanine 5 0 -triphosphate (GTP), strongly reduce the biological activity of B-Raf in a PC12 cell differentiation assay. We also confirm that S365 is a 14-3-3 binding site, and determine that mutation of this residue rescues the impaired biological activity of B-Raf proteins with a neutralized N-region, suggesting that the N-region opposes a 14-3-3-mediated transition into an inactive conformation. However, in the case of B-Raf V600E , although complete N-region neutralization resulted in a 2.5-fold reduction in kinase activity in vitro, this oncoprotein strongly induced PC12 differentiation or transformation and epithelial-mesenchymal transition of MCF-10A cells regardless of its N-region charge. Furthermore, the biological activity of B-Raf V600E was independent of its ability to bind Ras-GTP. Our analysis identifies important regulatory differences between B-Raf wt and B-Raf V600E and suggests that B-Raf V600E cannot be inhibited by strategies aimed at blocking S446 phosphorylation or Ras activation. Keywords: Ras; 14-3-3 proteins; b-Catenin; E-cadherin; mammary epithelial cells IntroductionThe Ras/Raf/mitogen-activated/extracellular-regulated kinase (MEK)/extracellular signal regulated kinase (ERK) pathway plays a pivotal role in control of proliferation and differentiation and, owing to its role as a gatekeeper of this pathway, Raf appears an attractive therapeutic target (O'Neill and Kolch, 2004;Wilhelm et al., 2004). The Raf-kinase family comprises the A-Raf, B-Raf and Raf-1 isoforms in vertebrates as well as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. B-Raf, the major ERK activator in vertebrates, is required for the maintenance of basal ERK activity and displays the most potent transforming activity (Papin et al., 1998;Brummer et al., 2002;Mercer and Pritchard, 2003). All isoforms share three highly conserved regions (CRs; Figure 1a): the N-terminal CR1 contains the Ras-guanine 5 0 -triphosphate (GTP)-binding domain (RBD), which initiates the interaction with Ras-GTP through a conserved arginine residue (R188 in B-Raf) that is required for the recruitment and activation of Raf at the plasma membrane. Consequently, mutation of this residue prevents Ras/Raf interaction and renders D-Raf, B-Raf and Raf-1 unresponsive to most extracellular signals (Hou et al., 1995;Marais et al., 1997;Brummer et al., 2002). The ...

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Section: V600esupporting

confidence: 80%

Section: Plasmid Constructsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Plasmid Constructsmentioning

confidence: 90%

Section: V600ementioning

confidence: 99%

See 3 more Smart Citations

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

et al. 2006

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Regulation of GCL activity and cellular glutathione through inhibition of ERK phosphorylation

et al. 2008

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Extracellular signal-regulated protein kinase (ERK), one of the mitogen-activated protein kinase, has been known to be involved in diverse cellular functions. In this work, we found that basically inhibition of this kinase in cultured cells markedly increased the gamma-glutamate-cysteine ligase (GCL; EC 6.3.2.2) activity, but without any considerable induction of the GCL genes. The increased GCL activity consequently elevated the cellular GSH level and eventually enhanced the cellular antioxidant capacity. Genetic inhibition of B-Raf, the upstream of ERK, also resulted in increased GCL activity and GSH level. Recent evidence also suggests that chronic pro-oxidant exposure results in the loss of ERK phosphorylation in vivo. Therefore, the findings in the present study suggest that inhibition of B-Raf/MEK/ERK pathway might be a promising physiological approach to up-regulate GCL activity and GSH. This study would also help us to understand the comprehensive role of the Raf/MEK/ERK pathway in overall physio/pathological conditions.

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Targeting ERK beyond the boundaries of the kinase active site in melanoma

Sammons

Ghose

Tsai

et al. 2019

Molecular Carcinogenesis

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Extracellular signal‐regulated kinase 1/2 (ERK1/2) constitute a point of convergence for complex signaling events that regulate essential cellular processes, including proliferation and survival. As such, dysregulation of the ERK signaling pathway is prevalent in many cancers. In the case of BRAF‐V600E mutant melanoma, ERK inhibition has emerged as a viable clinical approach to abrogate signaling through the ERK pathway, even in cases where MEK and Raf inhibitor treatments fail to induce tumor regression due to resistance mechanisms. Several ERK inhibitors that target the active site of ERK have reached clinical trials, however, many critical ERK interactions occur at other potentially druggable sites on the protein. Here we discuss the role of ERK signaling in cell fate, in driving melanoma, and in resistance mechanisms to current BRAF‐V600E melanoma treatments. We explore targeting ERK via a distinct site of protein‐protein interaction, known as the D‐recruitment site (DRS), as an alternative or supplementary mode of ERK pathway inhibition in BRAF‐V600E melanoma. Targeting the DRS with inhibitors in melanoma has the potential to not only disrupt the catalytic apparatus of ERK but also its noncatalytic functions, which have significant impacts on spatiotemporal signaling dynamics and cell fate.

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Identification of novel ERK-mediated feedback phosphorylation sites at the C-terminus of B-Raf

Cited by 105 publications

References 67 publications

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Functional analysis of the regulatory requirements of B-Raf and the B-RafV600E oncoprotein

Regulation of GCL activity and cellular glutathione through inhibition of ERK phosphorylation

Targeting ERK beyond the boundaries of the kinase active site in melanoma

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