Different Effects of Point Mutations within the <b>
                     <i>B-Raf</i>
                  </b> Glycine-Rich Loop in Colorectal Tumors on Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase and Nuclear Factor κB Pathway and Cellular Transformation

Ikenoue, Tsuneo; Hikiba, Yohko; Kanai, Fumihiko; Aragaki, Jun; Tanaka, Yasuo; Imamura, Jun; Imamura, Takaaki; Ohta, Miki; Ijichi, Hideaki; Tateishi, Keisuke; Kawakami, Takayuki; Matsumura, Masaru; Kawabe, Takao; Omata, Masao

doi:10.1158/0008-5472.can-03-3591

Cited by 70 publications

(78 citation statements)

References 28 publications

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“…Similar to Mason et al (1999), we also observed a reduction of the activity of B-Raf wt proteins with a partial or neutralized N-region in some experiments, although this result could not be reproduced in all experiments as indicated by the comprehensive analysis in Figure 4a. As reported previously, B-Raf V600E displayed a robust MEK kinase activity, which was about 10-fold higher than that of B-Raf wt , a differential that has also been observed by other laboratories (Davies et al, 2002;Ikenoue et al, 2003Ikenoue et al, , 2004Grbovic et al, 2006). As expected from the aforementioned experiments in HEK293 and PC12 cells, this high activity was not considerably influenced by the R188L or S446/7A mutations.…”

Section: Regulation Of B-raf Signalling T Brummer Et Alsupporting

confidence: 87%

“…Expression of B-Raf V600E has been shown to transform fibroblasts and melanocytes as well as to induce haematopoietic dysplasia in mice and invasive melanomas in p53À/À zebrafish (Davies et al, 2002;Ikenoue et al, 2004;Wellbrock et al, 2004b;Mercer et al, 2005;Patton et al, 2005). Likewise, suppression of BRAF V600E expression in melanoma lines abrogates their transformed phenotype (Hingorani et al, 2003;Karasarides et al, 2004).…”

Section: Introductionmentioning

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: Regulation Of B-raf Signalling T Brummer Et Alsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

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

et al. 2006

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“…Evidence presented herein strongly suggests that oncogenic BRAF plays an important role in the maintenance of constitutive NF-kB activity in human melanoma cells as well as in their survival. These results are in agreement with the data obtained from forced expression of BRAF mutants in COS or NIH 3T3 cells (Ikenoue et al, 2003(Ikenoue et al, , 2004. Given that BRAF mutations tend to occur early in melanomogenesis and are often found in benign nevi (reviewed by Smalley, 2003;Smalley and Herlyn, 2005), it is tempting to speculate about the role of oncogenic BRAF-mediated NF-kB induction in the progression of malignant melanoma.…”

supporting

confidence: 89%

“…Previous reports indicated that forced expression of BRAF mutants in COS and NIH 3T3 cells leads to NF-kB activation, which parallels the activation of ERK and which can be inhibited by non-degradable IkB super-repressor (Ikenoue et al, 2003(Ikenoue et al, , 2004. Pretreatment of Melan-A cells that harbor the BRAF VE :ER T2 fusion protein with 4-OHT led to an accelerated degradation of IkBa (measured by a cycloheximide (CHX) chase) that was not seen in parental mouse Melan-A cells (Figure 2a).…”

mentioning

confidence: 99%

Oncogenic BRAF regulates β-Trcp expression and NF-κB activity in human melanoma cells

Liu

Kumar

et al. 2006

Oncogene

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Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAFdependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor jB (NF-jB) transcription factor activation triggered by proteolysis of its inhibitor IjB. IjB degradation is dependent upon its phosphorylation by the IjB kinase (IKK) complex and subsequent ubiquitination facilitated by b-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAF V600E exhibit enhanced b-Trcp expression, increased IKK activity and a concomitant increase in the rate of IjBa degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor or by selective knock-down of BRAF V600E expression by RNA interference in human melanoma cells leads to decreased IKK activity and b-Trcp expression, stabilization of IjB, inhibition of NF-jB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-jB activity and to increased survival of melanoma cells.

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“…In fact, mutation of G55 shows multiple effects on kinase activity. Replacement of G55 with valine or arginine decreases activity in INSR (G1035) ( Table S3) (18), whereas substitutions of G55 by alanine or serine increase activity in BRAF (19) or leave activity unaffected in PKA (20).…”

Section: N-lobementioning

confidence: 99%

Congenital disease SNPs target lineage specific structural elements in protein kinases

Torkamani

Kannan²,

Taylor

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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The catalytic domain of protein kinases harbors a large number of disease-causing single nucleotide polymorphisms (SNPs) and common or neutral SNPs that are not known or hypothesized to be associated with any disease. Distinguishing these two types of polymorphisms is critical in accurately predicting the causative role of SNPs in both candidate gene and genome-wide association studies. In this study, we have analyzed the structural location of common and disease-associated SNPs in the catalytic domain of protein kinases and find that, although common SNPs are randomly distributed within the catalytic core, known disease SNPs consistently map to regulatory and substrate binding regions. In particular, a buried side-chain network that anchors the flexible activation loop to the catalytic core is frequently mutated in disease patients. This network was recently shown to be absent in distantly related eukaryotic-like kinases, which lack an exaggerated activation loop and, presumably, are not regulated by phosphorylation.allostery ͉ cancer ͉ conservation ͉ evolution ͉ mutation

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Different Effects of Point Mutations within the B-Raf Glycine-Rich Loop in Colorectal Tumors on Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase/Extracellular Signal-Regulated Kinase and Nuclear Factor κB Pathway and Cellular Transformation

Cited by 70 publications

References 28 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

Oncogenic BRAF regulates β-Trcp expression and NF-κB activity in human melanoma cells

Congenital disease SNPs target lineage specific structural elements in protein kinases

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