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At least 19 recordsLinked to original sources
PubMed1998

Codon optimization of the gene encoding a domain from human type 1 neurofibromin protein results in a threefold improvement in expression level in Escherichia coli.

An internal domain from the human type 1 neurofibromin has previously been expressed in Escherichia coli as a fusion with gluthathione S-transferase (GST). The expression level of this protein was lower than expected and so a gene was constructed using the distribution of codons found in highly expressed E. coli proteins. Codons were assigned using a Microsoft Visual Basic computer program to give a distribution similar to those found in genes which are highly expressed in E. coli. The optimized gene was then cloned back into the same GST fusion plasmid and it was found that the expression of soluble protein had increased threefold.

Base Sequence↗
PubMed2004

The interferon consensus sequence-binding protein activates transcription of the gene encoding neurofibromin 1.

Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this "cytokine hypersensitivity." In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP-/- mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hypersensitivity of ICSBP-/- myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP-/- cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders.

Animals↗
PubMed2004

Cell shape normalization, dendrite orientation, and melanin production of normal and genetically altered (haploinsufficient NF1)-melanocytes by microstructured substrate interactions.

Little is known about how functional regulation failure in genetically altered cells is influenced by topographical confinement of cells, a situation often present in tissues in vivo. We used cultured melanocytes derived from human skin samples as a model system for such investigations. Normal melanocytes have a very well defined shape consisting of a cell body and two dendrites arranged 180 degrees relative to each other. In contrast, neurofibromin 1-melanocytes (NF1-melanocytes) have up to a 50% reduction of neurofibromin 1, which results in an altered morphology that can be easily measured. NF1-melanocytes deviate from the defined structure of normal melanocytes by forming more than two dendrites per cell. We show that morphology consequences of genetically altered melanocytes can be canceled if cells interact with substrates microstructured by stripes that apply mechanophysical signals in the form of physical topography. The strength of the mechanophysical signal was varied systematically by increasing the height of the microstructures. Melanocytes respond to surface topographical features that are larger than 50 nm and have lateral confinements smaller 4 microns. The response of normal and NF1-melanocytes to different topographies was analyzed quantitatively by determining density distributions for the number of dendrites per cell, the angles between dendrites, and the orientation imprinted in the substrate. The synthesis of melanin, a pigment produced by melanocytes, differs in the case of genetically altered NF1- and normal melanocytes. In both cases, the interaction with microstripes enhanced melanin production significantly. This enhanced melanin production is speculated to be caused by the mechanical stabilization of the dendrites by substrate guidance.

Cell Size↗
PubMed2000

[Characterization of the neurofibromatosis type 1 gene and neurofibromin's role in cells].

Neurofibromatosis type 1(NF1), a pleiotrophic autosomal dominant disorder, was first described in 1882 by Friedrich Daniel von Recklinghausen. The cloning of the NF1 gene located at 17q11.2 revealed that the gene contains 60 exons and spans 350 kb of genomic DNA in 1990. The gene product of NF1 is neurofibromin. Neurofibromin is a major negative regulator of the Ras pathway in cells, which transmits mitogenic signals to the nucleus through the cascade of MAP kinase. Loss of neurofibromin in patients with NF1 leads to accumulation of activated Ras (bound to GTP), and thus increases downstream mitogenic signaling. Future understanding the neurofibromin's role will contribute to the development of agents and genetic therapies which modulate Ras-mediated signaling pathways.

Chromosomes, Human, Pair 17↗
PubMed2005

The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway.

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

Actin Depolymerizing Factors↗
PubMed1995

Expression of two new protein isoforms of the neurofibromatosis type 1 gene product, neurofibromin, in muscle tissues.

The neurofibromatosis type 1 (NF1) gene encodes a tumor suppressor protein, termed neurofibromin, which is expressed predominantly in neurons, Schwann cells, oligodendrocytes, and leukocytes. There are at least three isoforms of neurofibromin produced by the alternative use of exons 23a and 48a. Previously we described the identification of an NF1 mRNA isoform containing an additional 54 nucleotides from exon 48a (type 3 NF1) in human skeletal, cardiac and smooth muscle tissues by reverse-transcribed (RT)-PCR. To extend our initial observations, we have produced high titer chicken IgY antibodies which specifically recognize this muscle-specific neurofibromin isoform. An NF1 cDNA was generated containing human exon 48a sequences and expressed as a fusion protein in bacteria. The muscle-specific neurofibromin antibodies detected this exon 48a fusion protein by Western immunoblotting. Immunoprecipitation using these type 3 neurofibromin antibodies also specifically detected a 250 kDa protein in human and rat muscle tissues. Type 3 neurofibromin was found in rat heart and muscle, but not in liver brain, kidney or spleen with levels of expression declining after postnatal day 7. Expression of total NF1 RNA during rat embryonic development was detected at high levels in E15 heart, tongue, and limb bud. In addition, using type 2 neurofibromin-specific antibodies, the existence of a fourth isoform of neurofibromin (type 4 neurofibromin) containing both exon 23a and 48a sequences was demonstrated in rat heart muscle tissues. The identification of two muscle-specific isoforms of neurofibromin expands our definition of this important tumor suppressor protein and suggests additional roles for neurofibromin in muscle development and differentiation.

Amino Acid Sequence↗
PubMed2006

Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle.

DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.

Animals↗
PubMed1996

The differential effects of the Gly-60 to Ala mutation on the interaction of H-Ras p21 with different downstream targets.

We examined the effects of the Gly-60 to Ala mutation on the interaction of H-Ras with Ras GTPase activating protein (GAP), neurofibromin 1 (NF1), Raf-1, and ral guanine nucleotide dissociation stimulator (ralGDS), factors that interact with GTP-bound form of H-Ras. Previous study has shown that the G60A mutation perturbs GTP-induced conformational changes of H-Ras. We found that the G60A mutation decreases GTPase activity of H-Ras without significantly affecting GTP/GDP binding. The reduction in GTPase activity is most dramatic in the presence of GAP or NF1. Interestingly, the G60A mutation does not appear to alter the affinity of H-Ras for GAP or NF1. The G60A mutation moderately reduces the binding of H-Ras to Raf-1 Ras binding domain; however, the binding of H-Ras to ralGDS Ras binding domain was more significantly affected by the same mutation. These results indicate that although GAP, NF1, Raf-1, and ralGDS all interact with H-Ras in a GTP-dependent manner and they are able to compete against each other for binding to H-Ras, these factors share overlapping but not identical binding domains on H-Ras. The significance of our findings is discussed in the light of the GTP-induced conformational change model.

Alanine↗
PubMed2006

Leukemia-associated, constitutively active mutants of SHP2 protein tyrosine phosphatase inhibit NF1 transcriptional activation by the interferon consensus sequence binding protein.

Deficiency in either the interferon consensus sequence binding protein (ICSBP) or neurofibromin 1 (Nf1) increases the proliferative response of myeloid progenitor cell to hematopoietic cytokines. Consistent with this, we previously demonstrated that ICSBP activates transcription of the gene encoding Nf1 (the NF1 gene). In the studies presented here, we determine that ICSBP tyrosine phosphorylation is necessary for the activation of NF1 transcription. Since ICSBP is tyrosine phosphorylated in response to hematopoietic cytokines, these studies identify a novel pathway by which cytokine-induced posttranslational modification of ICSBP results in NF1 transcription. Nf1 subsequently inactivates cytokine-activated Ras, thereby creating a negative feedback mechanism for cytokine-induced proliferation. In these studies, we also determine that ICSBP is a substrate for SHP2 protein tyrosine phosphatase (SHP2-PTP). We find that wild-type SHP2-PTP dephosphorylates ICSBP only in undifferentiated myeloid cells. In contrast, a leukemia-associated, constitutively activated mutant form of SHP2-PTP dephosphorylates ICSBP in both myeloid progenitors and differentiating myeloid cells. Activated SHP2-PTP mutants thereby inhibit ICSBP-dependent NF1 transcription, impairing this negative feedback mechanism on cytokine-activated Ras. Therefore, these studies suggest that leukemia-associated ICSBP deficiency cooperates with leukemia-associated activating mutants of SHP2-PTP to contribute to the proliferative phenotype in myeloid malignancies.

Animals↗
PubMed2007

PU.1, interferon regulatory factor (IRF) 2, and the interferon consensus sequence-binding protein (ICSBP/IRF8) cooperate to activate NF1 transcription in differentiating myeloid cells.

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.

Cell Differentiation↗
PubMed2005

[Neurofibromatosis: the most frequent hereditary tumor predisposition syndrome].

With an incidence of one in 3000 to 4000 individuals, neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant genetic diseases and very likely the most frequent disorder with increased cancer risk. Approximately fifty percent of all patients are familial cases and the remaining half consists of sporadic cases with no affected parent. The hallmark clinical features present in over 90% of all patients are café-au-lait spots and neurofibromas. However, the disorder should not be underestimated as a "mere cosmetic problem", since NF1 patients are at increased risk to also develop malignant tumours, such as malignant peripheral nerve sheath tumours (MPNST), juvenile myelomonocytic leukaemia (JMML), optic glioma and pheochomocytoma. Renovascular disease represents an additional risk factor for NF1 patients. The NF1 gene is a classic example for a tumour suppressor gene. It functions as a negative regulator of the protooncogene Ras. This function explains well its involvement in tumour formation. During the last 15 years, since the cloning of the gene, enormous progress has been made towards a better understanding of the natural history of the disorder. However, it cannot be said if and when a cure of the disorder will be possible. Great advantages have been achieved in the monitoring and management of several NF1 complications, for instance in the treatment of tibia pseudarthrosis and optic gliomas. Owing to the technical improvements of the approaches applied to identify NF1-mutations molecular-genetic testing with high mutation detection rates may help nowadays in patients in which the clinical diagnosis may not readily be established, such as in young children or atypical cases. A greater awareness of the complications and the different expression forms of NF1 and NF2 on the part of all types of physicians will further help to offer all patients adequate and timely counselling and treatment. The establishment of multi-disciplinary counselling and treatment centres for neurofibromatosis could be an important step towards a better management of NF1 and NF2 patients.

Adolescent↗
PubMed1996

Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase.

The critical function of the neurofibromatosis type 1 (NF1) gene product (neurofibromin) is not well defined except that neurofibromin has homology with a family of the GTPase-activating proteins (GAPs). In this study, we confirmed that neurofibromin is constitutively phosphorylated and detected kinase activities which specifically phosphorylate the cysteine/serine-rich domain and the C-terminal domain of the neurofibromin in cell lysate. In vitro and in-gel kinase assays strongly indicated that cAMP-dependent protein kinase (PKA) is a candidate for the neurofibromin kinase. THe biological significance of the phosphorylation of neurofibromin is unclear at present, but we speculate that neurofibromin plays a crucial role in cellular function since it links the two major cellular pathways which are the GAP-ras and PKA-associated signals.

Base Sequence↗
PubMed1993

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules.

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways.

3T3 Cells↗
PubMed1993

Modulation of the neurofibromatosis type 1 gene product, neurofibromin, during Schwann cell differentiation.

Neurofibromin, the product of the neurofibromatosis type 1 (NF1) gene, is a approximately 250 kDa protein expressed predominantly in cortical neurons and oligodendrocytes in the central nervous system (CNS) and sensory neurons and Schwann cells in the peripheral nervous system (PNS). To gain insight into the biological role of neurofibromin in Schwann cells, the modulation of NF1 gene expression in a Schwann cell line (MT4H1) stimulated to either proliferate or differentiate in response to agents that elevate intracellular cAMP was examined. Untreated cells and cells exposed to mitogenic doses of forskolin (1-10 microM) or 8-bromo-cAMP (0.1 mM) expressed low levels of NF1 mRNA and the protein was barely detectable. High doses of forskolin (100 microM) or 8-bromo-cAMP (1 mM) induced the expression of both myelin P0 protein and neurofibromin with an identical time course. Although NF1 mRNA levels peaked within 1-6 hr, the rise in neurofibromin was not apparent until 24-48 hr and peaked 72 hr after treatment. P0 and neurofibromin were also coinduced by cell-cell contact in high density, untreated cultures. Moreover, differentiation initiated by either cAMP stimulation or high density culture conditions was associated with predominant expression of the type 2 NF1 mRNA isoform. In contrast, type 1 NF1 mRNA isoform expression was observed in untreated Schwann cells or those stimulated with mitogenic doses of forskolin or 8-bromo-cAMP. A switch from the type 1 neurofibromin that can efficiently downregulate p21-ras to the type 2 isoform with reduced activity may facilitate a p21-ras signaling pathway associated with Schwann cell differentiation.

Animals↗
PubMed2003

The neurofibromatosis 1 gene product neurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediated signaling in astrocytes.

Individuals with the neurofibromatosis 1 (NF1)-inherited tumor predisposition syndrome develop low-grade astrocytomas. The NF1 tumor suppressor gene product neurofibromin exhibits GTPase-activating activity (GAP) toward RAS, such that loss of neurofibromin expression leads to high levels of activated RAS and increased cell proliferation. Previous work has demonstrated that Nf1 inactivation in astrocytes leads to increased cell proliferation in vitro and in vivo, accompanied by increased RAS pathway activation. Studies on Nf1 mutant Drosophila have suggested that neurofibromin might also regulate cAMP signaling. Because intracellular cAMP levels have profound effects on astrocyte growth control, we sought to determine the contribution of neurofibromin to astrocyte cAMP regulation. In this report, we demonstrate that Nf1 inactivation in astrocytes results in reduced cAMP generation in response to PACAP and attenuated calcium influx and Rap1 activation. Based on the differential effects of forskolin and dibutyryl-cAMP on Nf1-/- astrocytes, neurofibromin likely functions at the level of adenylyl cyclase activation. Last, the reintroduction of a fragment of neurofibromin containing residues sufficient for restoring RAS-GAP function in Nf1-/- cells resulted in only partial restoration of neurofibromin-mediated cAMP regulation. These results demonstrate that neurofibromin positively influences cAMP generation and activation of cAMP growth regulatory targets in astrocytes and expands the role of the NF1 gene in astrocyte growth regulation.

Adenylyl Cyclases↗
PubMed1993

Neurofibromatosis type 1. Beyond positional cloning.

Recent advances in molecular genetics have enabled researchers to more rapidly identify human disease genes. The identification of these genes by positional cloning has opened the door to a better understanding of such diseases through a more complete appreciation of the molecular biologic processes that underlie them. In this review, the approaches taken to dissect the function of the gene for von Recklinghausen neurofibromatosis are presented. These general approaches involve identification of the protein product, determination of its relation to other known proteins, analysis of its distribution in tissues, within cells, and over development, and dissection of its role in producing the disease phenotype. Last, the insights gained from studying the molecular biology of the neurofibromatosis type 1 gene have direct impact on other biologic processes, such as neoplasia, cellular differentiation, and growth factor-mediated signal transduction as well as potential application to improved treatments for neurofibromatosis type 1 and cancer.

Cloning, Molecular↗
PubMed1997

Do NF1 gene deletions result in a characteristic phenotype?

Neurofibromatosis-1 (NF1) is an autosomal dominant disorder with marked variability of expression. Analysis of the NF1 gene (NF1) has detected a variety of mutations without any clear correlation with phenotype. However, deletions which remove all of NF1 have been reported in a small number of patients who have minor facial abnormalities, mental retardation, learning disabilities, and early or excessive burden of cutaneous or plexiform neurofibromas. The purpose of this study was to determine whether these phenotypic traits are associated with whole gene deletions. Out of 406 of our NF1 patients, 70 patients had manifestations previously associated with gene deletions. Thirty-five of these patients from 26 families were available for study. By fluorescence in situ hybridization (FISH) analysis, 4 were found to have deletions of the entire gene, including 2 sporadic cases, 1 familial case, and 1 case where family history could not be verified. In addition, the mother of the familial case was found to be mosaic for the deletion. Our results suggest that although large NF1 deletions occur with relatively high frequency in patients with certain findings, the presence of a deletion cannot be predicted solely on the basis of clinical phenotype.

Abnormalities, Multiple↗