Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).
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PMID:A quantitative method for measuring protein phosphorylation. 1257 52

Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a 'halo' SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low-level microsatellite instability at one locus, D10S214. The low rate of RAS-RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS-RAF-MEK-ERK-MAPK pathway. Germline DNA from members of 111 multiple-case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.
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PMID:Low prevalence of RAS-RAF-activating mutations in Spitz melanocytic nevi compared with other melanocytic lesions. 1751 71

Tumor cells often aberrantly reexpress molecules that mediate proper embryonic development for advantageous growth or survival. Here, we report that ankyrin repeat-rich membrane spanning (ARMS), a transmembrane protein abundant in the developing and adult neural tissues, is overexpressed in melanoma, a tumor ontogenetically originating from neural crest. Immunohistochemical study of 79 melanocytic lesions showed significantly increased expression of ARMS in primary malignant melanomas (92.9%) and metastatic melanoma (60.0%) in comparison with benign nevocellular nevi (26.7%). To investigate the role of ARMS in melanoma formation, murine B16F0 melanoma cells with stable knockdown of ARMS were established by RNA interference. Down-regulation of ARMS resulted in significant inhibition of anchorage-independent growth in soft agar and restrictive growth of melanoma in severe combined immunodeficient mice. Importantly, depletion of ARMS facilitated UVB-induced apoptosis in melanoma cells through inactivation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK. Addition of MEK inhibitor PD98059 further sensitized ARMS-depleted melanoma cells to UVB-induced apoptosis, whereas constitutively active MEK rescued ARMS-depleted cells from apoptosis. We further showed that BRAF, a downstream signaling molecule of ARMS in ERK pathway, is not mutated as a constitutively active form in acral lentiginous melanoma; in contrast, BRAF(T1799A) mutation, which leads to constitutive activation of ERK signaling, was detected in 57.1% of superficial spreading melanoma. Our study suggests that overexpression of ARMS per se serves as one mechanism to promote melanoma formation by preventing stress-induced apoptotic death mediated by the MEK/ERK signaling pathway, especially in acral lentiginous melanoma, most of which does not harbor BRAF mutation.
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PMID:ARMS depletion facilitates UV irradiation induced apoptotic cell death in melanoma. 1808 83

Expression of an oncogene in a primary cell can, paradoxically, block proliferation by inducing senescence or apoptosis through pathways that remain to be elucidated. Here we perform genome-wide RNA-interference screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we find a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated upregulation of BNIP3L, a proapoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi, and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.
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PMID:Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. 1855 73

Constitutive ERK activation is a common finding in human cancer and is often the result of activating mutations of BRAF and RAS. BRAF missense mutations occur in approximately 8% of human tumors, most frequently in melanoma, papillary thyroid cancer and colon cancer. Mutations in BRAF have been found predominantly in tumors in which RAS is commonly mutated but concurrent mutations of both BRAF and RAS are extremely rare. Though over 40 different kinase domain mutations in BRAF have been identified, a single base-pair substitution in exon 15 at codon 600 (V600E) is found in over 80% of cases. These mutations cluster in the glycine-rich loop and activation segments of the kinase and are predicted to induce kinase activation by disrupting the inhibitory glycine-rich loop/activation segment interaction which characterizes the inactive conformation. The majority of mutations identified cause constitutive kinase activation with the V600E mutation demonstrating approximately 500-fold greater kinase activity than wild-type BRAF. Supporting its classification as an oncogene, V600E BRAF stimulates ERK signaling, induces proliferation and is capable in model systems of promoting transformation. However, BRAF mutations are common in nevi and colon polyps suggesting that BRAF mutation alone is insufficient for tumorigenesis and additional mutations are required for cancer development. Though such data suggest that BRAF mutation is likely an early initiating event in tumors such as melanoma and colon cancer, preclinical studies suggest that tumors with V600E BRAF mutation remain dependent upon BRAF for proliferation and survival. Given its frequent occurrence in human cancer and the continued requirement for BRAF activity in tumors with BRAF mutation, efforts are underway to develop targeted inhibitors of BRAF and its downstream effectors. The first generation of RAF inhibitors, including sorafenib, were notable for their lack of specificity and potency for RAF and these agents have shown limited efficacy in tumors with a high incidence of BRAF mutation such as melanoma. Novel inhibitors of the pathway with greater selectivity for BRAF and MEK are now in Phase 1 and 2 clinical trials with promising early results. To maximize the likelihood of success with these agents, clinical trials enriched with patients whose tumors possess BRAF and RAS mutations have been proposed.
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PMID:Therapeutic strategies for targeting BRAF in human cancer. 1847 97

Due to elaborate control mechanisms, in benign tumors the activation of oncogenes primarily induces senescence, associated with cessation of cellular proliferation; for example, melanocytic nevi expressing mutant B-Raf. These mechanisms include the RB and/or the p53 pathway. The current model of melanomagenesis postulates that progression to immortal melanoma cells requires inactivating aberrations in signaling cascades controlling senescence. Thus, melanoma cells carrying mutant B-Raf should be resistant to mitogen-activated protein kinase (MAPK) pathway-induced senescence. Here, we demonstrate that hyperactivation of the MAPK pathway following activation of an inducible form of oncogenic C-Raf induces a senescence-like proliferation arrest in B-Raf mutant melanoma cells. This Raf-induced senescence is initially strictly dependent on MEK signaling, but seems to be independent of MAPK signaling after prolonged continuance. It is associated with reduced levels of RB phosphorylation and an increase in p21 expression, but is independent of p16(Ink4a) and p53. These data argue against the existence of fundamental changes in melanoma cells completely precluding senescence.
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PMID:Proliferation arrest in B-Raf mutant melanoma cell lines upon MAPK pathway activation. 1865 Aug 48

Recent studies have indicated an increasing incidence of melanoma worldwide. Although UV signature mutations are found rarely in melanoma cells, there is some evidence that intense intermittent exposure to sunlight can induce melanocyte tumorigenesis, and this is also observed after UV irradiation in some animals. The purpose of this paper is to review some of the most important mechanisms involved in the pathogenesis of this tumor. Genetic studies showed the familiar melanoma is linked to the mutation or deletion of the suppressor gene CDKN2A, and perhaps to CDK4. Studies showed that BRAF mutation is frequent in primary and metastatic melanoma cells but also in naevocytic nevi. This mutation activates the RAF/MEK pathway. Exposure to UV radiation induces immunosuppression. Recent investigations showed that chemokines, angiogenesis, metalloproteinases can play a role in the mechanism of metastasis. In spite of these advances the initiating events are still not completely understood. In conclusion, the pathogenesis of melanoma is very complex because numerous genetic and epigenetic factors are implicated in its development and progression, but some of the showed mechanisms can be targets for new therapies.
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PMID:Molecular and genetic mechanisms in melanoma. 1913 18

Rare reports indicate that the frequency of BRAFV600E mutations is high in atypical Spitz nevi. The purpose of this study was to ascertain the utility of the RAF/RAS mutational status as a diagnostic adjunct in lesions with histologic features that deviate from a typical Spitz nevus and, to examine expression of Insulin growth factor binding protein 7 (IGFBP7), a tumor suppressor acting through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling, in the same. Genomic DNA for genotyping was isolated from 6 regular Spitz nevi and 14 atypical spitzoid nevomelanocytic proliferations (including 1 melanoma with spitzoid histomorphology). NRAS1, NRAS2 and KRAS were analyzed, in addition to BRAFV600E. A mutation in BRAFV600E was noted in only one case-that of a regular Spitz nevus. IGFBP7 expression appeared to be maintained in this case, but was absent in 7/17 cases, which included 5 atypical spitzoid nevomelanocytic proliferations. Lack of expression of IGFBP7 in atypical spitzoid nevomelanocytic proliferations with histologically concerning features but BRAF-WT indicates that the evolutionary path in atypical spitzoid nevomelanocytic proliferations is genetically distinct from that of IGFBP7-negative BRAF-positive melanoma. From an oncogenic BRAF perspective, our findings suggest that the majority of 'atypical' spitzoid nevomelanocytic proliferations are probably no different from conventional Spitz nevi.
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PMID:Oncogenic BRAF and the tumor suppressor IGFBP7 in the genesis of atypical spitzoid nevomelanocytic proliferations. 1978 44

Malignant melanoma, a potentially lethal skin neoplasm, is characterized by a complex and heterogeneous etiology. Both incidences and deaths associated with melanoma are increasing in Caucasian populations. While exposure to ultraviolet radiation through sun-exposure is the major risk factor; the host factors including skin type and number of moles are critical in predisposition. The CDKN2A is a high penetrance melanoma susceptibility gene as carriers of the mutations are predisposed to the disease within familial settings. The gene is also somatically altered to varying degrees in sporadic melanoma. The CDK4 gene due to occurrence of activation mutations in a few families worldwide represents another melanoma susceptibility locus. The variants within the melanocortin receptor 1 (MC1R) gene, which encodes a melanocyte specific surface receptor with a key role in pigmentation, are associated with high risk phenotypes and increased risk of melanoma. Melanoma tumors are characterized by activation of the RAS-RAF-MEK-ERK pathway through either autocrine growth factor stimulation or oncogenic mutations in the B-RAF or N-RAS genes. Somatic mutations in the B-RAF gene are complemented by those in the N-RAS gene and represent the major genetic alterations. The mutations in the B-RAF gene in melanoma due to occurrence in melanocytic nevi represent early events that additionally require loss of cell cycle inhibitors like CDKN2A for melanoma progression and development. The sequence of events points to the cooperative collaboration between different genetic pathways in tumor development that can be and are being used as targets for developing specific therapeutic agents.
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PMID:Malignant melanoma--a genetic overview. 2009 96

Oncogenic BRAF as an early and fundamental feature of melanocytic neoplasia has been confirmed with its identification in both melanoma and nevi. Oncogenic BRAF has been shown to induce senescence/apoptosis by up-regulating the tumor suppressor IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling. Given the putative neoplastic potential of dysplastic nevi, our aim was to ascertain in dysplastic nevi from intermittently sun-exposed skin and of varying severity the frequency of oncogenic BRAF and NRAS and to assess expression of IGFBP7 in the same. BRAF and NRAS genotyping was performed on genomic DNA (isolated using laser capture microdissection) from dysplastic nevi ranging in severity from mild (12), to moderate (11), and to severe (11). Immunohistochemical staining for IGFBP7 was performed on all. Overall, 9 (26%) of 34 cases (2 severely atypical dysplastic nevi, 2 moderately atypical dysplastic nevi, and 5 mildly atypical dysplastic nevi) exhibited the BRAFV600E mutation (P = .22), with lack of IGFBP7 expression in 4 (44.4%) of 9 cases (1 severely atypical, 1 moderately atypical, and 2 mildly atypical); and 25 (73.5%) of 34 cases (9 severely atypical, 9 moderately atypical, and 7 mildly atypical) were BRAFWT, with enhanced IGFBP7 expression in 12 (48%) of 25 cases (6 severely atypical, 3 moderately atypical, and 3 mildly atypical). All cases were NRASWT. The disparate expression of IGFBP7 in BRAFV600E-positive dysplastic nevi (enhanced in 56% and diminished/absent in 44%) indicates that the behavior of oncogenic BRAF in dysplastic nevi, unlike that in malignant melanoma, does not appear to consistently induce senescence/apoptosis through pathways mediated by IGFBP7.
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PMID:Oncogenic BRAF-positive dysplastic nevi and the tumor suppressor IGFBP7--challenging the concept of dysplastic nevi as precursor lesions? 2023 23


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