Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ras proteins function through the formation of specific complexes with Raf-1, B-raf, PI-3 kinase and RalGDS. These interactions all require Ras-GTP with an intact effector binding domain (Switch I region). We have examined the requirements of the Switch II region (amino acids 60-72) for the production of stable interactions between Ras and its downstream effectors. A point mutation at position 65 or 64 combined with additional mutations at either position 65 or 71 rendered nucleotide-free Ras protein unable to stably interact with Ras specific guanine nucleotide exchange factors. Ha-Ras containing point mutations at positions 65 and 71 possessed a twofold higher affinity for B-raf and consequently MEK1. The point mutation at 64, in combination with additional point mutations at either position 65 or 71, resulted in a protein which failed to interact with either PI-3 kinase or neurofibromin, though these Ras mutants effectively bound both Raf-1 and B-raf. An activated form of Ras, Q61L-Ras, associated with all effector proteins independent of the bound guanine nucleotide. Q61L-Ras-GDP was almost as effective as wild type Ras-GMPPNP in the in vitro activation of MEK1 and MAP kinase. Competitive studies with the catalytic domain if neurofibromin, NF1-GRD, demonstrated that its interaction with Ras-GMPPNP is mutually exclusive with both Raf-1 and B-raf. These data suggest that rasGAP and neurofibromin are unable to downregulate Ras-GTP complexed to Raf-1 or B-raf.
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PMID:Different structural requirements within the switch II region of the Ras protein for interactions with specific downstream targets. 763 Jun 28

The c-Rmil/B-raf proto-oncogene belongs to the mil/raf family encoding serine/threonine protein kinases shown to be involved in signal transduction from the membrane to the nucleus. We previously showed that the avian c-Rmil gene encodes two proteins of 94 and 95 kDa resulting from the alternative splicing of a 120 bp exon encoding 40 aminoacids (exon 10). We isolated from a mouse brain library B-raf cDNAs containing this exon 10 and a previously unidentified 36 bp insert which constitutes an additional alternatively spliced exon designated exon 8b. These two exons are located between the CR2 region and the catalytic domain of the protein. By using specific sera generated against different regions of the B-Raf protein, we identified 10 B-Raf isoforms and we defined their structure and their expression pattern in adult mouse tissues. The B-Raf proteins are mainly expressed in neural tissues and, interestingly, isoforms containing aminoacids encoded by exon 10 are specifically expressed in these tissues. We also show that several B-Raf isoforms interact with the Mek-1 protein (MAP kinase kinase) and phosphorylate this protein on serine residues 218 and 222.
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PMID:[B-raf gene encodes for multiple isoforms with Mek-1 kinase activity]. 764 69

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.
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PMID:Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf. 973 1

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.
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PMID:B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts. 1519 48

The ERK group of mitogen-activated protein kinases (MAPKs) is essential for cell proliferation stimulated by mitogens, oncogenic ras and raf (ref. 1). All MAPKs are activated by MAP3K/MEK/MAPK core pathways and the Raf proto-oncoproteins, especially B-Raf, are ERK-specific MAP3Ks (refs 1-3). Mixed lineage kinase-3 (MLK3) is a MAP3K that was thought to be a cytokine-activated, and comparatively selective, regulator of the JNK group of MAPKs (refs 1, 4-6). Here we report that silencing of mlk3 by RNAi suppressed mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocked mitogen-stimulated phosphorylation of B-Raf at Thr 598 and Ser 601, a step required for B-Raf activation. Furthermore, silencing mlk3 prevented serum-stimulated cell proliferation and the proliferation of tumour cells bearing either oncogenic Ki-Ras or loss-of-function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumour cells containing activating B-raf or raf-1 mutations was unaffected by silencing mlk3. Our results define an unexpected role for MLK3 in mitogen regulation of B-Raf, ERK and cell proliferation.
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PMID:MLK3 is required for mitogen activation of B-Raf, ERK and cell proliferation. 1530 91

Raf kinases play an integral role in the classic mitogen-activated protein (MAP) kinase (Raf/MEK/extracellular signal-related kinase [ERK]) intracellular signaling cascade, but their role in specific developmental processes is largely unknown. Using a genetic approach, we have identified a role for B-Raf during hematopoietic progenitor cell development and during megakaryocytopoiesis. Fetal liver and in vitro embryonic stem (ES) cell-derived myeloid progenitor development is quantitatively impaired in the absence of B-Raf. Biochemical data suggest that this phenotype is due to the loss of a normally occurring rise in B-Raf expression and associated ERK1/2 activation during hematopoietic progenitor cell formation. However, the presence of B-raf-/- ES cell-derived myeloid progenitors in the bone marrow of adult chimeric mice indicates the lack of an obligate cell-autonomous requirement for B-Raf in myeloid progenitor development. The lack of B-Raf also impairs megakaryocytopoiesis. Thrombopoietin (Tpo)-induced in vitro expansion of ES cell-derived megakaryocyte-lineage cells fails to occur in the absence of B-Raf. Moreover, this quantitative in vitro defect in megakaryocyte-lineage expansion is mirrored by chimeric mice data that show reduced B-raf-/- genotype contribution in megakaryocytes relative to its contribution in myeloid progenitors. Together, these data suggest that B-Raf plays a cell-autonomous role in megakaryocytopoiesis and a permissive role in myeloid progenitor development.
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PMID:A critical function for B-Raf at multiple stages of myelopoiesis. 1578 29

Raf kinases are downstream effectors of Ras and upstream activators of the MEK-ERK cascade. Ras and MEK-ERK signaling play roles in learning and memory (L&M) and neural plasticity, but the roles of Raf kinases in L&M and plasticity are unclear. Among Raf isoforms, B-raf is preferentially expressed in the brain. To determine whether B-raf has a role in synaptic plasticity and L&M, we used the Cre-LoxP gene targeting system to derive forebrain excitatory neuron B-raf knockout mice. This conditional knockout resulted in deficits in ERK activation and hippocampal long-term potentiation (LTP) and impairments in hippocampus-dependent L&M, including spatial learning and contextual discrimination. Despite the widespread expression of B-raf, this mutation did not disrupt other forms of L&M, such as cued fear conditioning and conditioned taste aversion. Our findings demonstrate that B-raf plays a role in hippocampal ERK activation, synaptic plasticity, and L&M.
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PMID:Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory. 1634 20

The Ras-Raf-MAPK pathway has been implicated in lung carcinogenesis and, potentially, the maintenance of the malignant phenotype in these tumors. Mutations in ras and B-raf genes have been described in lung cancer, representing one of the few examples of tandem mutations in a signaling cascade. As a result, numerous approaches to inhibiting this pathway in lung cancer have been explored in the past decade. The most promising approach to date appears to be the inhibition of mitogen-activated ERK kinase or MEK. In this review, the potential utility of MEK inhibitors in the therapy of lung cancer is discussed.
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PMID:The role of mitogen-activated ERK-kinase inhibitors in lung cancer therapy. 1635 19

The kinases of the Raf family have been intensively studied as activators of the mitogen-activated protein kinase kinase/extra-cellular signal-regulated kinase (ERK) module in regulated and deregulated proliferation. Genetic evidence that Raf is required for ERK activation in vivo has been obtained in lower organisms, which express only one Raf kinase, but was hitherto lacking in mammals, which express more than one Raf kinase. Ablation of the two best studied Raf kinases, B-Raf and Raf-1, is lethal at midgestation in mice, hampering the detailed study of the essential functions of these proteins. Here, we have combined conventional and conditional gene ablation to show that B-Raf is essential for ERK activation and for vascular development in the placenta. B-Raf-deficient placentae show complete absence of phosphorylated ERK and strongly reduced HIF-1alpha and VEGF levels, whereas all these parameters are normal in Raf-1-deficient placentae. In addition, neither ERK phosphorylation nor development are affected in B-raf-deficient embryos that are born alive obtained by epiblast-restricted gene inactivation. The data demonstrate that B-Raf plays a nonredundant role in ERK activation during extraembyronic mammalian development in vivo.
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PMID:Essential role of B-Raf in ERK activation during extraembryonic development. 1643 25

Activation of the Ras/Raf/MEK/ERK pathway is frequently observed in animal and human tumors. In our study, we analyzed B-raf codon 637 (formerly 624) and Ha-ras codon 61 mutations in liver tumors from C3H, B6C3F1 and C56BL mice which differ considerably with regard to their susceptibility to hepatocarcinogenesis. In total, 73% (102/140) of tumors induced by a single application of N-nitrosodiethylamine or 7,12-dimethylbenz[a]anthracene contained either B-raf or Ha-ras mutations and only <3% (4/140) were mutated in both genes. In addition, B-raf mutations were present in 76% (19/25) of early precancerous liver lesions. The prevalence of Ha-ras mutated tumors was significantly higher in the susceptible C3H and B6C3F1 mouse strains (39-50%) than in the comparatively resistant C57BL mouse (7%). B-raf mutated tumors, by contrast, were more frequent in C57BL mice (68%) than in the other two strains (17-45%). Taken together, our findings indicate that alterations affecting the Ras/Raf/MEK/ERK signalling pathway are a hallmark of carcinogen-induced liver tumors in mice. Moreover, our results show that mutational activation of B-raf in liver tumors of different mouse strains is, by contrast to Ha-ras, inversely related to their susceptibility to hepatocarcinogenesis. Although activated Ras and Raf proteins are assumed to have similar biological effects because they feed into the same signalling pathway, there seem to be subtle strain-specific differences in selection processes favouring the preferential outgrowth of either B-raf or Ha-ras mutated tumor populations in mouse liver.
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PMID:Differential selection for B-raf and Ha-ras mutated liver tumors in mice with high and low susceptibility to hepatocarcinogenesis. 1792 10


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