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

We have identified, in Xenopus oocyte cytosol, a protein kinase named REKS (Ras-dependent extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) stimulator), which phosphorylates and activates recombinant ERK2 through recombinant MEK in a recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ras-dependent manner. We show here that this REKS activity is synergistically enhanced by a combination of mammalian recombinant GTP gamma S-KiRas and 14-3-3 protein purified from rat brain. 14-3-3 protein is known to activate tyrosine and tryptophan hydroxylases, to modulate the protein kinase C activity, to stimulate secretion, and to show phospholipase A2 activity per se. 14-3-3 protein did not affect the MEK activity. 14-3-3 protein neither interacted with Ki-Ras nor affected the neurofibromin activity to stimulate the GTPase activity of Ki-Ras under the conditions where the recombinant N-terminal fragment of c-Raf-1 inhibited it. These results suggest that 14-3-3 protein has an additional function in the regulation of the Ras-MEK-ERK cascade pathway through the activation of REKS.
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PMID:Synergistic activation by Ras and 14-3-3 protein of a mitogen-activated protein kinase kinase kinase named Ras-dependent extracellular signal-regulated kinase kinase stimulator. 808 86

Individuals affected with neurofibromatosis 1 (NF1) harbor increased numbers of GFAP-immunoreactive cerebral astrocytes and develop astrocytomas that can lead to blindness and death. Mice heterozygous for a targeted Nf1 mutation (Nf1+/-) were employed as a model for the human disease to evaluate the hypothesis that reduced NF1 protein (neurofibromin) expression may confer a growth advantage for astrocytes, such that inactivation of only one NF1 allele is sufficient for abnormal astrocyte proliferation. Here, we report that Nf17+/- mice have increased numbers of cerebral astrocytes and increased astrocyte proliferation compared to wild-type littermates. Intriguingly, primary Nf1+/- astrocyte cultures failed to demonstrate a cell-autonomous growth advantage unless they were cocultured with C17 neuronal cells. This C17 neuronal cell-induced Nf1+/- increase in proliferation was blocked by MEK inhibition (PD98059), suggesting a p21-ras-dependent effect. Furthermore, mice heterozygous for a targeted mutation in another GAP molecule, p120-GAP, demonstrated no increases in cerebral astrocyte number. These findings suggest that reduced NF1 expression results in a cell context-dependent increase in astrocyte proliferation that may be sufficient for the development of astrocytic growth abnormalities in patients with NF1.
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PMID:Haploinsufficiency for the neurofibromatosis 1 (NF1) tumor suppressor results in increased astrocyte proliferation. 1044 36

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive malignancies that arise within peripheral nerves. These tumors occur with increased incidence in patients with neurofibromatosis type 1 (NF1), exhibiting increased Ras activity due to loss of the NF1 gene product, neurofibromin, and abnormal expression of the epidermal growth factor receptor (EGFR). We previously found that MPNSTs express increased levels of the CD44 family of transmembrane glycoproteins that have been implicated in tumor cell invasion and metastasis. In two MPNST cell lines, we have found that elevated CD44 expression and cell invasion are dependent on Src kinase activity but are independent of mitogen-activated protein kinases (MAPK) kinase (MEK) activity. In contrast, inhibition of Src kinase activity has no influence on MPNST cell proliferation. Reduction of CD44 levels, using antisense oligonucleotides, results in reduced MPNST cell invasion in vitro, suggesting that Src contributes in part to MPNST cell invasion by increasing CD44 levels. At least some of this increased CD44 expression results from elevated EGFR levels through a Src-dependent mechanism, consistent with the notion that EGFR promotes constitutive Src activation in MPNSTs. These data indicate that Src and CD44 are putative targets for the treatment of MPNST invasion and metastasis.
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PMID:Malignant peripheral nerve sheath tumor cell invasion is facilitated by Src and aberrant CD44 expression. 1273 Sep 55

Type 1 neurofibromatosis (NF1) is a common autosomal dominant disorder that results in neuroectodermal tumors. The NF1 tumor-suppressor gene encodes neurofibromin, which includes a GTPase-activating domain for Ras inactivation. Affinity purification showed N-Ras to be the predominant activated isoform of Ras in two independent neurofibrosarcoma cell lines from NF1 patients (lines ST88-14 and NF90-8). These NF1 cells also demonstrated increased constitutive activity of the extracellular signal-regulated kinases 1 and 2 (ERK1,2) mitogen-activated protein (MAP) kinases compared with a sporadic malignant schwannoma cell line that maintains neurofibromin expression (STS-26T). Thus, MAP kinase kinase (MEK) inhibitors may be a rational approach to NF1 therapy. The MEK inhibitors PD98059 [2'-amino-3'-methoxyflavone], PD184352 (also called CI-1040) [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide], and U0126 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] all produced concentration-dependent suppression of the proliferation of the three cell lines. Individual MEK inhibitors had similar effects in all three cell lines. However, only the antiproliferative effects of PD184352 correlated closely with the elimination of ERK1,2 MAP kinase activities. PD98059 was primarily cytostatic, whereas U0126 and PD184352 were cytotoxic. Only PD184352 induced apoptosis in all three lines, as indicated by morphology, activation of DEVDase, procaspase-3 cleavage, and the appearance of populations having sub-G(0)/G(1) DNA contents. The differential effects of the MEK inhibitors on cell survival were not dependent on p53 status or effects on the ERK5 pathway. PD184352 was also proapoptotic to primary rat Schwann cells. Hence, although PD184352 effectively killed neurofibrosarcoma cells, its effects on normal Schwann cells may limit its usefulness in the clinic.
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PMID:The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD184352 (CI-1040) selectively induces apoptosis in malignant schwannoma cell lines. 1623 99

The neurofibromatosis Type 1 (NF1) gene functions as a tumor suppressor gene. One known function of neurofibromin, the NF1 protein product, is to accelerate the slow intrinsic GTPase activity of Ras to increase the production of inactive rasGDP, with wide-ranging effects on p21ras pathways. Loss of neurofibromin in the autosomal dominant disorder NF1 is associated with tumors of the peripheral nervous system, particularly neurofibromas, benign lesions in which the major affected cell type is the Schwann cell (SC). NF1 is the most common cancer predisposition syndrome affecting the nervous system. We have developed an in vitro system for differentiating mouse embryonic stem cells (mESC) that are NF1 wild type (+/+), heterozygous (+/-), or null (-/-) into SC-like cells to study the role of NF1 in SC development and tumor formation. These mES-generated SC-like cells, regardless of their NF1 status, express SC markers correlated with their stage of maturation, including myelin proteins. They also support and preferentially direct neurite outgrowth from primary neurons. NF1 null and heterozygous SC-like cells proliferate at an accelerated rate compared to NF1 wild type; this growth advantage can be reverted to wild type levels using an inhibitor of MAP kinase kinase (Mek). The mESC of all NF1 types can also be differentiated into neuron-like cells. This novel model system provides an ideal paradigm for studies of the role of NF1 in cell growth and differentiation of the different cell types affected by NF1 in cells with differing levels of neurofibromin that are neither transformed nor malignant.
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PMID:A mouse embryonic stem cell model of Schwann cell differentiation for studies of the role of neurofibromatosis type 1 in Schwann cell development and tumor formation. 1759 22

Patients with the genetic disease type I neurofibromatosis (NF1) exhibit characteristic pigmentary lesions associated with loss of a single allele of NF1, encoding the 260 kDa protein neurofibromin. To understand the basis for these pigmentary problems, the properties of melanocytes haploinsufficient for the murine gene Nf1 were studied using Nf1(+/-) knockout mice. We demonstrate that neurofibromin regulates the Kit-Mitf signaling axis in vivo during melanocyte development. Primary Nf1(+/-) melanocytes were purified by FACS to measure melanogenic gene expression. We found that Nf1(+/-) melanocytes exhibit higher levels of melanogenic gene expression than their wild-type counterparts. Both prior to and following Kit stimulation, Nf1(+/-) melanocytes also exhibit increased activation of the MAP kinase pathway compared with primary cells. The melanogenic response of primary melanocytes to Mek inhibition is consistent with the changes observed with Nf1 haploinsufficiency; however, these changes differ from those observed with their immortalized counterparts. The observation that reduction of neurofibromin, either from haploinsufficiency in the case of primary melanocytes or from neurofibromin knockdown in the case of melan-a cells, enhances melanogenic gene expression suggests that neurofibromin plays a dominant role to MEK activity in controlling melanogenic gene expression in murine melanocytes.
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PMID:Neurofibromin as a regulator of melanocyte development and differentiation. 1808 49

Neurofibromatosis type 1 (NF1) is a developmental and cancer predisposing syndrome resulting from haploinsufficiency or alteration in neurofibromin, a multifunctional protein that acts in various signaling pathways affecting morphogenetic processes and cell proliferation. Neurofibromin deficiency deregulates Ras/Raf/MEK/ERK and Ras/PI3K/AKT/PKB/mTOR signaling networks and intersected pathways including the cAMP-dependent protein kinase A (PKA) and the Rho-cofillin which acts on actin cytoskeleton reorganization, cell motility and adhesion. As the neurofibromin-mediated pathways are associated with biological effects depending on the cell lineage, deregulation induced by NF1 mutation clearly has cell type-specific effects. This review summarizes our increasing knowledge of NF1 as a disease rooted in defective developmental mechanisms that can also influence the potential for malignant growth. The cardinal features of NF1 patients, at birth and during life involve the cardiovascular, connective/skeletal and central nervous systems, as they reflect the NF1 mutation sensitivity of cell lineages committed to specifying these systems during embryonic development. A switch to neoplastic transformation may also occur in both the prenatal and postnatal life in cancer initiating cells of defined lineages, with the cooperation of a genetically and epigenetically modified tumor microenvironment. We emphasize how much of our current knowledge of the pathomechanisms of NF1 clinical signs and cancer has come from engineered mouse models and in vitro primary cells and cell lines exposed to inhibitors of signaling molecules. Advances in our knowledge of the developmental defects primed by the loss neurofibromin should reveal further associations between given NF1 mutations and tissue-specific symptoms, thus improving the clinical management of the patients.
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PMID:Developmental abnormalities and cancer predisposition in neurofibromatosis type 1. 1960 12

The cascade comprising Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) is a therapeutic target in human cancers with deregulated Ras signalling, which includes tumours that have inactivated the Nf1 tumour suppressor. Nf1 encodes neurofibromin, a GTPase-activating protein that terminates Ras signalling by stimulating hydrolysis of Ras-GTP. We compared the effects of inhibitors of MEK in a myeloproliferative disorder (MPD) initiated by inactivating Nf1 in mouse bone marrow and in acute myeloid leukaemias (AMLs) in which cooperating mutations were induced by retroviral insertional mutagenesis. Here we show that MEK inhibitors are ineffective in MPD, but induce objective regression of many Nf1-deficient AMLs. Drug resistance developed because of outgrowth of AML clones that were present before treatment. We cloned clone-specific retroviral integrations to identify candidate resistance genes including Rasgrp1, Rasgrp4 and Mapk14, which encodes p38alpha. Functional analysis implicated increased RasGRP1 levels and reduced p38 kinase activity in resistance to MEK inhibitors. This approach represents a robust strategy for identifying genes and pathways that modulate how primary cancer cells respond to targeted therapeutics and for probing mechanisms of de novo and acquired resistance.
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PMID:Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. 1972 76

Individuals with the neurofibromatosis type 1 (NF1) inherited cancer syndrome exhibit neuronal dysfunction that predominantly affects the CNS. In this report, we demonstrate a unique vulnerability of CNS neurons, but not peripheral nervous system (PNS) neurons, to reduced Nf1 gene expression. Unlike dorsal root ganglion neurons, Nf1 heterozygous (Nf1+/-) hippocampal and retinal ganglion cell (RGC) neurons have decreased growth cone areas and neurite lengths, and increased apoptosis compared to their wild-type counterparts. These abnormal Nf1+/- CNS neuronal phenotypes do not reflect Ras pathway hyperactivation, but rather result from impaired neurofibromin-mediated cAMP generation. In this regard, elevating cAMP levels with forskolin or rolipram treatment, but not MEK (MAP kinase kinase) or PI3-K (phosphatidylinositol 3-kinase) inhibition, reverses these abnormalities to wild-type levels in vitro. In addition, Nf1+/- CNS, but not PNS, neurons exhibit increased apoptosis in response to excitotoxic or oxidative stress in vitro. Since children with NF1-associated optic gliomas often develop visual loss and Nf1 genetically engineered mice with optic glioma exhibit RGC neuronal apoptosis in vivo, we further demonstrate that RGC apoptosis resulting from optic glioma in Nf1 genetically engineered mice is attenuated by rolipram treatment in vivo. Similar to optic glioma-induced RGC apoptosis, the increased RGC neuronal death in Nf1+/- mice after optic nerve crush injury is also attenuated by rolipram treatment in vivo. Together, these findings establish a distinctive role for neurofibromin in CNS neurons with respect to vulnerability to injury, define a CNS-specific neurofibromin intracellular signaling pathway responsible for neuronal survival, and lay the foundation for future neuroprotective glioma treatment approaches.
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PMID:Defective cAMP generation underlies the sensitivity of CNS neurons to neurofibromatosis-1 heterozygosity. 2041 Jan 11


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