guanosine-triphosphate and Neurofibromatosis-1

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder whose major feature is the occurrence of multiple neurofibromas, which are benign tumors of the nerve sheath. It affects an estimated one in 3000 to 4000 individuals. In addition to neurofibromas, there are many other clinical manifestations, including malignant tumors such as gliomas or malignant peripheral nerve sheath tumors, and nontumor effects such as skeletal dysplasia and learning disability. Diagnosis is established on the basis of clinical criteria. Molecular genetic testing is feasible, but the large size of the gene and wide range of pathogenic mutations have so far impeded the development of a clinical diagnostic test. Insights into pathogenesis have followed from identification of the NF1 gene and the development of animal models. The major function of the gene product appears to be regulation of the ras protein. Tumors are believed to arise by the loss of function of the NF1 protein, suggesting that NF1 behaves as a tumor suppressor gene. Heterozygous effects on some cell types are also likely, however. The role of ras in the pathogenesis of tumors in NF1 has suggested an approach to treatment using ras inhibitors, some of which are likely to begin in clinical trials in NF1 patients in the near future.

Topics: Animals; Brain Neoplasms; Cafe-au-Lait Spots; Cell Transformation, Neoplastic; Female; Genes, Dominant; Genes, Neurofibromatosis 1; Glioma; Guanosine Triphosphate; Humans; Hypertension; Learning Disabilities; Leukemia; Male; Mice; Mice, Knockout; Neurofibroma; Neurofibromatosis 1; Neurofibromin 1; Protein Structure, Tertiary; ras Proteins; Rhabdomyosarcoma; Scoliosis

Proteins that bind and hydrolyse GTP are being discovered at a rapidly increasing rate. Each of these many GTPases acts as a molecular switch whose 'on' and 'off' states are triggered by binding and hydrolysis of GTP. Conserved structure and mechanism in myriad versions of the switch--in bacteria, yeast, flies and vertebrates--suggest that all derive from a single primordial protein, repeatedly modified in the course of evolution to perform a dazzling variety of functions.

Topics: Animals; Biological Transport; Cysteine; Endocytosis; Exocytosis; GTP Phosphohydrolase-Linked Elongation Factors; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Neurofibromatosis 1; Peptide Elongation Factors; Peptide Initiation Factors; Pheromones; Protein Processing, Post-Translational; Proto-Oncogene Proteins p21(ras); Saccharomyces cerevisiae; Signal Transduction

Sprouty-related, EVH1 domain-containing (SPRED) proteins negatively regulate RAS/mitogen-activated protein kinase (MAPK) signaling following growth factor stimulation. This inhibition of RAS is thought to occur primarily through SPRED1 binding and recruitment of neurofibromin, a RasGAP, to the plasma membrane. Here, we report the structure of neurofibromin (GTPase-activating protein [GAP]-related domain) complexed with SPRED1 (EVH1 domain) and KRAS. The structure provides insight into how the membrane targeting of neurofibromin by SPRED1 allows simultaneous interaction with activated KRAS. SPRED1 and NF1 loss-of-function mutations occur across multiple cancer types and developmental diseases. Analysis of the neurofibromin-SPRED1 interface provides a rationale for mutations observed in Legius syndrome and suggests why SPRED1 can bind to neurofibromin but no other RasGAPs. We show that oncogenic EGFR(L858R) signaling leads to the phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. The structural, biochemical, and biological results provide new mechanistic insights about how SPRED1 interacts with neurofibromin and regulates active KRAS levels in normal and pathologic conditions.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Cafe-au-Lait Spots; Catalytic Domain; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Guanosine Triphosphate; HEK293 Cells; Humans; K562 Cells; Neurofibromatosis 1; Neurofibromin 1; Oncogenes; Phosphorylation; Point Mutation; Protein Binding; Protein Domains; Protein Interaction Maps; Proto-Oncogene Proteins p21(ras); Signal Transduction

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Topics: Action Potentials; Animals; Capsaicin; Disease Models, Animal; Guanosine Triphosphate; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neurofibromatosis 1; Neurofibromin 1; Potassium Channels; Proto-Oncogene Proteins p21(ras); Sensory Receptor Cells; Sensory System Agents; Sodium Channels; Tetrodotoxin

Topics: Adenylyl Cyclases; Animals; Animals, Genetically Modified; Drosophila; Drosophila Proteins; Enzyme Activation; Guanosine Triphosphate; Humans; Insect Proteins; Learning; Learning Disabilities; Nerve Tissue Proteins; Neurofibromatosis 1; Neurofibromin 1; Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction

Individuals with neurofibromatosis type 1 (NF1) are predisposed to certain cancers including juvenile chronic myelogenous leukaemia (JCML). The NF1 tumour-suppressor gene encodes a protein (neurofibromin) that accelerates GTP hydrolysis on Ras proteins. Here we show that primary leukaemic cells from children with NF1 show a selective decrease in NF1-like GTPase activating protein (GAP) activity for Ras but retain normal cellular GAP activity. Leukaemic cells also show an elevated percentage of Ras in the GTP-bound conformation. JCML cells are hypersensitive to granulocyte-macrophage colony stimulating factor (GM-CSF), and we observed a similar pattern of aberrant growth in haematopoietic cells from Nf1-/- mouse embryos. These data define a specific role for neurofibromin in negatively regulating GM-CSF signaling through Ras in haematopoietic cells and they suggest that hypersensitivity to GM-CSF may be a primary event in the development of JCML.

Topics: Animals; Cell Division; Cells, Cultured; Child; Genes, Neurofibromatosis 1; Granulocyte-Macrophage Colony-Stimulating Factor; GTPase-Activating Proteins; Guanosine Triphosphate; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mice; Neurofibromatosis 1; Neurofibromin 1; Proteins; ras GTPase-Activating Proteins; ras Proteins; Signal Transduction

As derivatives of the neural crest, epidermal melanocytes are supposed to be clinically affected by NF1 gene defects. The NF1 gene shares sequence homology with the p120 GTPase activating protein (p120-GAP) and neurofibromin has been shown to participate in Ras-regulation. By immunoprecipitation and Western blotting, neurofibromin was found to be expressed in melanocytes from the unaffected skin and café au lait macules of NF1 patients, but the intensity of the neurofibromin band was decreased compared to control cultures. The Ras-GTP/Ras-GDP ratios of NF1 derived melanocyte cultures were comparable to those derived from healthy donors. Furthermore, the total GAP-activity of cell lysates was not altered in NF1 melanocyte cultures compared to controls. However, lysates of proliferating melanocytes, both from NF1 patients and from healthy donors, showed an about 2-fold higher GAP-activity than poorly growing cells. Neurofibromin contributed approximately one third of total GAP-activity, in both control and NF1 melanocytes, indicating that it is not the major regulator of Ras in these cells. These results suggest that the function of neurofibromin in melanocytes is not limited to regulation of Ras activity.

Topics: Adult; Base Sequence; Blotting, Western; Cell Division; Cells, Cultured; Child; Genes, ras; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Melanocytes; Molecular Sequence Data; Nerve Tissue Proteins; Neurofibromatosis 1; Neurofibromin 1; Polymerase Chain Reaction; Precipitin Tests; Protein Biosynthesis; Proteins; ras GTPase-Activating Proteins; Skin; Tubulin

Topics: Cells, Cultured; Enzyme Activation; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Hydrolysis; In Vitro Techniques; Kinetics; Mutation; Neurofibromatosis 1; Neurofibromin 1; Proteins; ras GTPase-Activating Proteins; ras Proteins

Neurofibromin, the protein product of the neurofibromatosis type 1 (NF1) gene, has two alternate isoforms which are generated by alternative splicing of two exons. One of these isoforms containing exon 48a is expressed at highest levels in muscle. Since neurofibromin is a p21-ras regulator and has been recently shown to be modulated during Schwann cell differentiation, we examined the expression of the NF1 gene product during in vitro muscle differentiation. Previous work demonstrated that C2C12 murine myoblast cell differentiation could be blocked by the introduction of an activated p21-ras protein. Using this model system, we demonstrate that differentiating C2C12 cells upregulate the expression of NF1 mRNA by 2 days of serum starvation concomitant with increased expression of nicotinic acetylcholine receptor mRNA. This upregulation of mRNA expression paralleled an increase in neurofibromin and N-ras levels, but no change in the relative abundance of the isoforms containing exon 23a or exon 48a was observed during in vitro myoblast differentiation. The increase in neurofibromin levels paralleled a decrease in the levels of activated p21-ras as assayed by in vivo 32P-orthophosphate incorporation into p21-ras. These results suggest that in vitro C2C12 cell differentiation is associated with a concomitant increase in NF1 gene expression and decrease in the proportion of activated p21-ras.

Topics: Animals; Blotting, Northern; Blotting, Western; Cell Differentiation; Down-Regulation; Exons; Gene Expression Regulation, Neoplastic; Guanosine Triphosphate; Isomerism; Mice; Muscles; Neurofibromatosis 1; Neurofibromin 1; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins p21(ras); RNA, Messenger; Up-Regulation

Tumor cell lines derived from malignant schwannomas removed from patients with neurofibromatosis type 1 (NF1) have been examined for the level of expression of NF1 protein. All three NF1 lines examined expressed lower levels of NF1 protein than control cells, and the level in one line was barely detectable. The tumor lines expressed normal levels of p120GAP and p21ras. Although the p21ras proteins isolated from the tumor cells had normal (nonmutant) biochemical properties in vitro, they displayed elevated levels of bound GTP in vivo. The level of total cellular GAP-like activity was reduced in extracts from the tumor line that expresses very little NF1 protein. Introduction of the catalytic region of GAP into this line resulted in morphological reversion and lower in vivo GTP binding by endogenous p21ras. These data implicate NF1 protein as a tumor suppressor gene product that negatively regulates p21ras and define a "positive" growth role for ras activity in NF1 malignancies.

Topics: Animals; Gene Expression Regulation, Neoplastic; Genes, Tumor Suppressor; Guanosine Triphosphate; Humans; Mice; Neurilemmoma; Neurofibromatosis 1; Neurofibromin 1; Protein Binding; Proteins; Proto-Oncogene Proteins p21(ras); Tumor Cells, Cultured

Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras. A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase. Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off. With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP. The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid. Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory. These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism.

Topics: Arachidonic Acid; Arachidonic Acids; Dinoprost; Gene Expression Regulation; Genes, ras; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Kinetics; Neurofibromatosis 1; Neurofibromin 1; Prostaglandins; Proteins; Proto-Oncogene Mas; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Recombinant Proteins

The ras-encoded p21ras proteins bind GTP very tightly, but catalyse hydrolysis to GDP very slowly. In humans, two genes encode proteins that stimulate this GTPase activity (GAP, or GTPase-activating proteins), one of relative molecular mass 120,000, referred to as p120-GAP, and another NF1-GAP, which is encoded by the neurofibromatosis type-1 gene. Both GAPs are widely expressed in mammalian tissues. Here we show that although they will both bind oncogenic mutants of p21ras, neither will stimulate their GTPase activity. NF1-GAP binds to the p21ras proteins up to 300 times more efficiently than p120-GAP. The two GAPs are inhibited to different extents by certain lipids: micromolar concentrations of arachidonate, phosphatidate and phosphatidylinositol-4,5-bisphosphate affect only NF1-GAP. This inhibition does not compete with p21ras, and lipid-inactivated NF1-GAP can still bind p21ras. We used the detergent dodecyl maltoside, which inhibits only NF1-GAP, to distinguish between the two activities in cell extracts and found both types present together in several mammalian cell lines. In contrast, GAP activity in extracts of Xenopus oocytes was not affected by dodecyl maltoside. By these criteria, the mammalian cells contain both GAP activities and the oocytes have only p120-like GAP activity. These results indicate that more than one GAP regulates p21ras in the same cell.

Topics: Animals; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine Triphosphate; Humans; Neurofibromatosis 1; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Signal Transduction; Species Specificity