Raf proteins and cancer: B-Raf is identified as a mutational target

Mercer, Kathryn; Pritchard, Catrin

doi:10.1016/s0304-419x(03)00016-7

Cited by 316 publications

(383 citation statements)

References 131 publications

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“…To this end, we focused our analyses on the impact of B-Raf V600E and mutants thereof on several biological end points rather than by relying exclusively on their catalytic activity measured by immunocomplex in vitro kinase assays. This is of particular importance, as the high basal activity that is displayed by B-Raf in such assays does not necessarily reflect its in vivo potential to activate the ERK pathway or other biological end points (for a discussion, see MacNicol et al, 2000;Mercer and Pritchard, 2003). Here, we show that the N-region is indeed important for the biological activity of B-Raf wt as it opposes negative regulatory constraints probably imposed by 14-3-3 binding to the CR2 domain.…”

Section: Introductionmentioning

confidence: 74%

“…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.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…The CR2 contains a negative regulatory serine residue (S259 and S365 in Raf-1 and B-Raf, respectively) that is proposed to serve as a binding site for 14-3-3 proteins, although this has only been confirmed for Raf-1 (Roy et al, 1998;Hekman et al, 2004). The catalytic domain (CR3) harbours phosphorylation sites for Raf-regulating enzymes within two segments, the N-region and the activation segment (Mercer and Pritchard, 2003). All isoforms carry a second 14-3-3 binding motif at the C-terminal end of the CR3 domain and its mutation abrogates the biological activity of B-Raf, Raf-1 and LIN-45, demonstrating also a conserved positive role of 14-3-3 proteins for the coupling of Raf to downstream effectors (MacNicol et al, 2000;Light et al, 2002;Harding et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, this knowledge will be of particular importance for the development of selective B-Raf inhibitors. Indeed, the currently unidentified S446 kinase has been suggested to represent a potential therapeutic target in neoplasms with high B-Raf activity (Mercer and Pritchard, 2003).Given the important role of the N-region for the in vitro kinase activity of wild-type B-Raf (B-Raf wt ), we asked to what extent the charged N-region contributes to the biological activity of B-Raf wt and B-Raf V600E . To this end, we focused our analyses on the impact of B-Raf V600E and mutants thereof on several biological end points rather than by relying exclusively on their catalytic activity measured by immunocomplex in vitro kinase assays.…”

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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: Introductionmentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

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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Increased Melanoma Risk in Individuals With Papillary Thyroid Carcinoma

Oakley

Curtin

Layfield

et al. 2014

JAMA Otolaryngol Head Neck Surg

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IMPORTANCE Determining the associated risk between papillary thyroid carcinoma (PTC) and cutaneous malignant melanoma (CM) and the rate of BRAF v600e mutation could help identify a common genetic component of these 2 cancers. OBJECTIVES To define the relative risk of PTC in patients with CM, and vice versa, and their first- through fifth-degree relatives and spouses by using a unique population research database; and to assess the rate of BRAF v600e mutation in a group of patients with both diagnoses. DESIGN, SETTING, AND PARTICIPANTS Retrospective rev iew using the Utah Population Database (which is linked to medical records and the Utah Cancer Registry from 1966 to 2011) and tissue analysis in a tertiary care facility. Included were 4460 patients diagnosed with PTC and 14 569 with CM in Utah between 1966 and 2011 and their first- through fifth-degree relatives and spouses. These were compared at a 5:1 ratio with matched, population-based controls. MAIN OUTCOMES AND MEASURES Statistically significant increased risk of PTC in patients with CM, and vice versa, and any first- through fifth-degree relatives and spouses; and a significantly higher rate of BRAF v600e mutation in patients with both PTC and CM than would be expected for each individual condition alone. RESULTS Patients with CM had a 2.3-fold increased risk (P < .001) of being diagnosed as having PTC compared with population-based matched controls. Conversely, patients with PTC had a 1.8-fold increased risk (P < .001) of developing CM. First- through fifth-degree relatives and spouses of patients with PTC or CM did not show a statistically significant increased risk. Eight patients with both cancer diagnoses had tissue specimens tested, of which 4 (50%) were found to be positive for the BRAF v600e mutation in either their PTC or CM specimen, and 3 (38%) were found positive in both. CONCLUSIONS AND RELEVANCE Patients with either PTC or CMhave an increased risk of developing the other cancer as a second primary malignant neoplasm. Tissue specimens from patients with both cancers show a high rate of BRAF v600e mutation. Translational studies are needed to better define the associated genetic predisposition between PTC and CM and to test the efficacy of and implementation techniques for treatment plans using BRAF mutation as a therapeutic target.

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Models of Melanoma Metastasis: Using a Transient siRNA‐Based Protein Inhibition Strategy in Mice to Validate the Functional Relevance of Pharmacological Agents

Sharma

Robertson

2007

CP Pharmacology

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While a pharmacological agent may inhibit the activity of a protein in cultured cells by triggering a particular biological process, it may function differently in intact animals. Thus, an assay is needed to rapidly assess whether a drug candidate displays the same mechanism of action in vivo as in vitro. The experimental approach described in this unit utilizes synthetic siRNA in a transient animal assay to define the action of a drug candidate when inhibiting the activity of a particular gene. Commercially available synthetic siRNA is introduced into cancer cells by nucleofection to reduce protein expression. Cells are then introduced into animals and the mechanism responsible for tumor inhibition assessed. The action of a compound identified in vitro is then compared to that noted in vivo following siRNA-mediated inhibition to determine whether it reduces tumor development in the same manner in both systems.

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Raf proteins and cancer: B-Raf is identified as a mutational target

Cited by 316 publications

References 131 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

Increased Melanoma Risk in Individuals With Papillary Thyroid Carcinoma

Models of Melanoma Metastasis: Using a Transient siRNA‐Based Protein Inhibition Strategy in Mice to Validate the Functional Relevance of Pharmacological Agents

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