The Experts below are selected from a list of 4770 Experts worldwide ranked by ideXlab platform
David H Gutmann - One of the best experts on this subject based on the ideXlab platform.
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A Conserved Circadian Function for the Neurofibromatosis 1 Gene
Elsevier, 2018Co-Authors: Lei Bai, Yool Lee, Cynthia T. Hsu, Julie A. Williams, Daniel Cavanaugh, Xiangzhong Zheng, Carly Stein, Paula Haynes, Han Wang, David H GutmannAbstract:Summary: Loss of the Neurofibromatosis 1 (Nf1) protein, Neurofibromin, in Drosophila disrupts circadian rhythms of locomotor activity without impairing central clock function, suggesting effects downstream of the clock. However, the relevant cellular mechanisms are not known. Leveraging the discovery of output circuits for locomotor rhythms, we dissected cellular actions of Neurofibromin in recently identified substrates. Herein, we show that Neurofibromin affects the levels and cycling of calcium in multiple circadian peptidergic neurons. A prominent site of action is the pars intercerebralis (PI), the fly equivalent of the hypothalamus, with cell-autonomous effects of Nf1 in PI cells that secrete DH44. Nf1 interacts genetically with peptide signaling to affect circadian behavior. We extended these studies to mammals to demonstrate that mouse astrocytes exhibit a 24-hr rhythm of calcium levels, which is also attenuated by lack of Neurofibromin. These findings establish a conserved role for Neurofibromin in intracellular signaling rhythms within the nervous system. : Bai et al. show that the gene mutated in the disease Neurofibromatosis 1 is required for maintaining levels or cycling of calcium in circadian neurons in Drosophila and in mammalian cells. These effects likely account for effects of Nf1 on circadian behavior in Drosophila and may be relevant in explaining sleep phenotypes in patients. Keywords: circadian rhythms, neurofibromatosis 1, Drosophila, peptide signaling, cycling of calcium, mouse astrocyte
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elucidating the impact of neurofibromatosis 1 germline mutations on Neurofibromin function and dopamine based learning
Human Molecular Genetics, 2015Co-Authors: Corina Anastasaki, Ludwine Messiaen, Albert S Woo, David H GutmannAbstract:Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurologic condition characterized by significant clinical heterogeneity, ranging from malignant cancers to cognitive deficits. Recent studies have begun to reveal rare genotype-phenotype correlations, suggesting that the specific germline NF1 gene mutation may be one factor underlying disease heterogeneity. The purpose of this study was to define the impact of the germline NF1 gene mutation on brain Neurofibromin function relevant to learning. Herein, we employ human NF1-patient primary skin fibroblasts, induced pluripotent stem cells and derivative neural progenitor cells (NPCs) to demonstrate that NF1 germline mutations have dramatic effects on Neurofibromin expression. Moreover, while all NF1-patient NPCs exhibit increased RAS activation and reduced cyclic AMP generation, there was a Neurofibromin dose-dependent reduction in dopamine (DA) levels. Additionally, we leveraged two complementary Nf1 genetically-engineered mouse strains in which hippocampal-based learning and memory is DA-dependent to establish that neuronal DA levels and signaling as well as mouse spatial learning are controlled in an Nf1 gene dose-dependent manner. Collectively, this is the first demonstration that different germline NF1 gene mutations differentially dictate Neurofibromin function in the brain.
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Phosphorylation of Neurofibromin by PKC is a possible molecular switch in EGF receptor signaling in neural cells.
Oncogene, 2005Co-Authors: D Mangoura, David H Gutmann, Y Sun, D. K. Singh, Adrian G. Flores, M Ahmed, G VallianatosAbstract:Children with neurofibromatosis (NF1) typically develop central nervous system (CNS) abnormalities, including aberrant proliferation of astrocytes and formation of benign astrocytomas. The NF1 gene encodes Neurofibromin, a Ras-GAP, highly expressed in developing neural cells; the mechanism of regulation of Neurofibromin as a Ras-GAP, remains however unknown. We now show that, in response to EGF, Neurofibromin is in vivo phosphorylated on serine residues by PKC-α, in human, rat, and avian CNS cells and cell lines. EGF-induced PKC phosphorylation was prominent in the cysteine/serine-rich domain (CSRD) of Neurofibromin, which lies in the N-terminus and upstream of the Ras-GAP domain (GRD), and this modification significantly increased the association of Neurofibromin with actin in co-immunoprecipitations. In addition, we show that Ras activation in response to EGF was significantly lowered when C62B cells overexpressed a construct encoding both CSRD+GRD. Moreover, when PKC-α was downregulated, the Ras-GAP activity of CSRD+GRD was significantly diminished, whereas overexpressed GRD alone acted as a weaker GAP and in a PKC-independent manner. Most importantly, functional Ras inhibition and EGF signaling shifts were established at the single cell level in C6-derived cell lines stably overexpressing CSRD+GRD, when transient co-overexpression of Ras and PKC-depletion prior to stimulation with EGF-induced mitosis. Taken together, these data provide the first evidence of a functional, allosteric regulation of GRD by CSRD, which requires Neurofibromin phosphorylation by PKC and association with the actin cytoskeleton. Our data may suggest a novel mechanism for regulating biological responses to EGF and provide a new aspect for the understanding of the aberrant proliferation seen in the CNS of children with NF1.
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The Neurofibromatosis 1 Gene Product Neurofibromin Regulates Pituitary Adenylate Cyclase-Activating Polypeptide-Mediated Signaling in Astrocytes
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2003Co-Authors: Biplab Dasgupta, Laura L. Dugan, David H GutmannAbstract: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.
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loss of Neurofibromin is associated with activation of ras mapk and pi3 k akt signaling in a neurofibromatosis 1 astrocytoma
Journal of Neuropathology and Experimental Neurology, 2000Co-Authors: Nelson Lau, David H Gutmann, Matthias M Feldkamp, Luba Roncari, Allison Loehr, Patrick Shannon, Abhijit GuhaAbstract:Neurofibromatosis 1 (NF1) is a common autosomal dominant cancer predisposition syndrome, in which 15% to 20% of affected individuals develop astrocytomas. Neurofibromin, the protein product of the NF1 gene, functions as a tumor suppressor, largely by inhibiting Ras activity. While loss of Neurofibromin has been implicated in the molecular pathogenesis of other NF1-associated tumors, there is no formal evidence demonstrating loss of Neurofibromin function in NF1-associated astrocytomas. In this report, we describe an NF1 patient from whom both astrocytoma tumor tissue as well as corresponding non-neoplastic white matter were available for analysis. Loss of Neurofibromin expression was observed in the tumor and was associated with elevated levels of Ras-GTP. However, elevated Ras-GTP levels were not the result of oncogenic Ras mutations, altered p120-GAP function, growth factor receptor activation, or abnormal p53, Rb, or p16 expression. Furthermore, increased Raf-MAPK and PI3-K/Akt activity was detected in the NF1 astrocytoma compared with the corresponding normal white matter. These results support a role for Neurofibromin as the critical GAP in the molecular pathogenesis of NF1 astrocytomas.
Yi-ping Hsueh - One of the best experts on this subject based on the ideXlab platform.
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From neurodevelopment to neurodegeneration: the interaction of Neurofibromin and valosin-containing protein/p97 in regulation of dendritic spine formation
Journal of biomedical science, 2012Co-Authors: Yi-ping HsuehAbstract:Both Neurofibromatosis type I (NF1) and inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) are autosomal dominant genetic disorders. These two diseases are fully penetrant but with high heterogeneity in phenotypes, suggesting the involvement of genetic modifiers in modulating patients' phenotypes. Although NF1 is recognized as a developmental disorder and IBMPFD is associated with degeneration of multiple tissues, a recent study discovered the direct protein interaction between Neurofibromin, the protein product of the NF1 gene, and VCP/p97, encoded by the causative gene of IBMPFD. Both NF1 and VCP/p97 are critical for dendritic spine formation, which provides the cellular mechanism explaining the cognitive deficits and dementia found in patients. Moreover, disruption of the interaction between Neurofibromin and VCP impairs dendritic spinogenesis. Neurofibromin likely influences multiple downstream pathways to control dendritic spinogenesis. One is to activate the protein kinase A pathway to initiate dendritic spine formation; another is to regulate the synaptic distribution of VCP and control the activity of VCP in dendritic spinogenesis. Since Neurofibromin and VCP/p97 also regulate cell growth and bone metabolism, the understanding of Neurofibromin and VCP/p97 in neurons may be applied to study of cancer and bone. Statin treatment rescues the spine defects caused by VCP deficiency, suggesting the potential role of statin in clinical treatment for these two diseases.
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valosin containing protein and Neurofibromin interact to regulate dendritic spine density
Journal of Clinical Investigation, 2011Co-Authors: Hsiao Fang Wang, Yu Tzu Shih, Chiung Ya Chen, Hsu Wen Chao, Yiping Hsueh, Yi-ping HsuehAbstract:Inclusion body myopathy with Paget disease of bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder characterized by progressive myopathy that is often accompanied by bone weakening and/or frontotemporal dementia. Although it is known to be caused by mutations in the gene encoding valosin-containing protein (VCP), the underlying disease mechanism remains elusive. Like IBMPFD, neurofibromatosis type 1 (NF1) is an autosomal dominant disorder. Neurofibromin, the protein encoded by the NF1 gene, has been shown to regulate synaptogenesis. Here, we show that Neurofibromin and VCP interact and work together to control the density of dendritic spines. Certain mutations identified in IBMPFD and NF1 patients reduced the interaction between VCP and Neurofibromin and impaired spinogenesis. The functions of Neurofibromin and VCP in spinogenesis were shown to correlate with the learning disability and dementia phenotypes seen in patients with IBMPFD. Consistent with the previous finding that treatment with a statin rescues behavioral defects in Nf1+/– mice and providing further support for our hypothesis that there is crosstalk between Neurofibromin and VCP, statin exposure neutralized the effect of VCP knockdown on spinogenesis in cultured hippocampal neurons. The data presented here demonstrate that there is a link between IBMPFD and NF1 and indicate a role for VCP in synapse formation.
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Neurofibromin interacts with CRMP-2 and CRMP-4 in rat brain.
Biochemical and biophysical research communications, 2008Co-Authors: Yi-ling Lin, Yi-ping HsuehAbstract:Neurofibromin, encoded by the neurofibromatosis type 1 (NF1) gene, regulates the Ras and cAMP pathways and plays a role in proliferation and neuronal morphogenesis. The details of the molecular mechanism of Neurofibromin action in these processes are still unclear. In this study, immunoprecipitation and proteomics were used to identify novel proteins from rat brain that interact with Neurofibromin. Mass spectrometry analysis showed that two proteins, the collapsin response mediator protein-2 (CRMP-2) and propionyl-CoA carboxylase alpha chain (PCCA), associated with Neurofibromin. Immunoprecipitation-immunoblotting analysis confirmed the interactions between Neurofibromin and CRMP-2 and CRMP-4, but not CRMP-1, in rat brain. CDK5, a kinase that regulates CRMP-2 in axonal outgrowth, was required for the interaction between Neurofibromin and CRMP-2. Since both Neurofibromin and CRMP proteins are involved in proliferation and axonal morphogenesis, these results suggest that the interaction with CRMPs contributes to the function of Neurofibromin in tumorigenesis and neuronal morphogenesis.
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syndecan 2 induces filopodia and dendritic spine formation via the Neurofibromin pka ena vasp pathway
Journal of Cell Biology, 2007Co-Authors: Yi-ling Lin, Yating Lei, Chenjei Hong, Yi-ping HsuehAbstract:Syndecan-2 induced filopodia before spinogenesis; therefore, filopodia formation was used here as a model to study the early downstream signaling of syndecan-2 that leads to spinogenesis. Screening using kinase inhibitors indicated that protein kinase A (PKA) is required for syndecan-2–induced filopodia formation in both human embryonic kidney cells and hippocampal neurons. Because Neurofibromin, a syndecan-2–binding partner, activates the cyclic adenosine monophosphate pathway, the role of Neurofibromin in syndecan-2–induced filopodia formation was investigated by deletion mutant analysis, RNA interference, and dominant-negative mutant. The results showed that Neurofibromin mediates the syndecan-2 signal to PKA. Among actin-associated proteins, Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) were predicted as PKA effectors downstream of syndecan-2, as Ena/VASP, which is activated by PKA, induces actin polymerization. Indeed, when the activities of Ena/VASP were blocked, syndecan-2 no longer induced filopodia formation. Finally, in addition to filopodia formation, Neurofibromin and Ena/VASP contributed to spinogenesis. This study reveals a novel signaling pathway in which syndecan-2 activates PKA via Neurofibromin and PKA consequently phosphorylates Ena/VASP, promoting filopodia and spine formation.
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Neurofibromin signaling and synapses.
Journal of biomedical science, 2007Co-Authors: Yi-ping HsuehAbstract:Neurofibromin, encoded by the Neurofibromatosis type I (NF1) gene, has been shown to regulate the Ras and cAMP signaling pathways. The signaling functions of Neurofibromin may account for tumor formation in patients with NF1, as well as influencing neuronal function. Learning defects have been documented in NF1 mutant mice, and in NF1 patients, learning disabilities are common. In this review, the recent studies related to the role of Neurofibromin in neuronal morphogenesis will be discussed, which may partly explain why the patients with NF1 have learning defects.
Klaus Scheffzek - One of the best experts on this subject based on the ideXlab platform.
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Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.
Cell reports, 2020Co-Authors: Wupeng Yan, Frank Mccormick, Klaus Scheffzek, Matthew Drew, Evan Markegard, Srisathiyanarayanan Dharmaiah, Anatoly Urisman, Dominic Esposito, Dwight V. Nissley, Dhirendra K. SimanshuAbstract: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.
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Restoring functional Neurofibromin by protein transduction
Scientific reports, 2018Co-Authors: Kevin Mellert, S. Lechner, M. Lüdeke, Markus Lamla, Peter Möller, Ralf Kemkemer, Klaus Scheffzek, Dieter KaufmannAbstract:In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular Neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP Neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated Neurofibromin replacement in cultured human fibroblasts showing reduced amount of Neurofibromin. Full length Neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of Neurofibromin. The transduced Neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant Neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.
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NMR resonance assignments of the EVH1 domain of Neurofibromin's recruitment factor Spred1.
Biomolecular NMR assignments, 2017Co-Authors: Sebastian Führer, Klaus Scheffzek, Theresia Dunzendorfer-matt, Linda Ahammer, Angela Ausserbichler, Martin TollingerAbstract:Neurofibromin and Sprouty-related EVH1 domain-containing protein 1 (Spred1) both act as negative regulators of the mitogen-activated protein kinase pathway and are associated with the rare diseases Neurofibromatosis type 1 and Legius syndrome, respectively. Spred1 recruits the major GTPase activating protein (GAP) Neurofibromin from the cytosol to the membrane in order to inactivate the small G protein Ras. These functions are dependent on the N-terminal EVH1 domain and the C-terminal Sprouty domain of Spred1 whereas the former specifically recognizes the GAP related domain of Neurofibromin and the latter is responsible for membrane targeting. Within the GAP domain, Spred1 binding depends on the GAPex portion which is dispensable for Ras inactivation. In a first step towards the characterization of the Neurofibromin Spred1 interface in solution we assigned backbone and side chain 1H, 13C, and 15N chemical shifts of the Spred1 derived EVH1 domain. Our chemical shift data analysis indicate seven consecutive β-strands followed by a C-terminal α-helix which is in agreement with the previously reported crystal structure of Spred1(EVH1). Our data provide a framework for further analysis of the function of patient-derived mutations associated with rare diseases.
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The Neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Theresia Dunzendorfer-matt, Frank Mccormick, Ellen L. Mercado, K. Maly, Klaus ScheffzekAbstract:Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein Neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with Neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, Neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with Neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of Neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of Neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of Neurofibromin that is present in all known canonical RasGAPs.
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Neurofibromin: Protein Domains and Functional Characteristics
Neurofibromatosis Type 1, 2012Co-Authors: Klaus Scheffzek, Stefan WeltiAbstract:The tumor suppressor gene NF1 encodes the giant signal regulator Neurofibromin (320 kDa) that is nonfunctional in NF1 patients due to gene alterations. Neurofibromin is a Ras-specific GTPase activating protein (RasGAP) which downregulates the biological activity of activated Ras via a central GAP-related domain (GRD). Adjacent to its carboxy terminal end, a bipartite glycerophospholipid binding module has been discovered that comprises a Sec14- and a pleckstrin homology (PH)-like domain. While a number of interaction partners of Neurofibromin have been reported, the RasGAP activity currently appears to represent the only clearly defined biochemical function of this giant protein. This chapter is focused on current knowledge about Neurofibromin domains that are structurally validated. Functional and regulatory features will be briefly addressed and the potential impact of non-truncating NF1 mutations detected in patients will be discussed in the light of available three-dimensional structural information.
Margaret R. Wallace - One of the best experts on this subject based on the ideXlab platform.
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Neurofibromin level directs ras pathway signaling and mediates sensitivity to targeted agents in malignant peripheral nerve sheath tumors
Oncotarget, 2018Co-Authors: Elliot Kahen, Andrew S Brohl, Darcy Welch, Christopher L Cubitt, Jae K Lee, Yunyun Chen, Sean J Yoder, Jamie K Teer, Yonghong O Zhang, Margaret R. WallaceAbstract:Malignant peripheral nerve sheath tumor (MPNST) is a type of soft-tissue sarcoma strongly associated with dysfunction in Neurofibromin; an inhibitor of the RAS pathway. We performed high-throughput screening of an array of FDA approved and promising agents in clinical development both alone and in combination at physiologically achievable concentrations against a panel of established MPNST cell line models. We found that drugs targeting a variety of factors in the RAS pathway can effectively lead to cell death in vitro with considerable drug combination synergy in regimens that target MEK or mTOR. We observed that the degree of relative sensitivity to chemotherapeutic agents was associated with the status of Neurofibromin in these cell line models. Using a combination of agents that target MEK and mTORC1/2, we effectively silenced RAS/PI3K/MEK/mTOR signaling in vitro. Moreover, we employed RNAi against NF1 to establish that MPNST drug sensitivity is directly proportional to relative level of intracellular Neurofibromin. Thus, two-drug combinations that target MEK and mTORC1/2 are most effective in halting the RAS signaling cascade, and the relative success of this and related small molecule interventions in MPNSTs may be predicated upon the molecular status of Neurofibromin.
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microrna 10b regulates tumorigenesis in neurofibromatosis type 1
Cancer Science, 2010Co-Authors: Guolin Chai, Ning Liu, Janet L. Oblinger, Agasanur K. Prahalad, Meng Gong, Long-sheng Chang, Margaret R. Wallace, David Muir, Abhijit GuhaAbstract:MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low Neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed Neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored Neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of Neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting Neurofibromin and RAS signaling.
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MicroRNA‐10b regulates tumorigenesis in neurofibromatosis type 1
Cancer science, 2010Co-Authors: Guolin Chai, Ning Liu, Janet L. Oblinger, Agasanur K. Prahalad, Meng Gong, Long-sheng Chang, Margaret R. Wallace, David MuirAbstract:MicroRNAs (miRNAs) are frequently deregulated in human tumors, and play important roles in tumor development and progression. The pathological roles of miRNAs in neurofibromatosis type 1 (NF1) tumorigenesis are largely unknown. We demonstrated that miR-10b was up-regulated in primary Schwann cells isolated from NF1 neurofibromas and in cell lines and tumor tissues from malignant peripheral nerve sheath tumors (MPNSTs). Intriguingly, a significantly high level of miR-10b correlated with low Neurofibromin expression was found in a neuroectodermal cell line: Ewing's sarcoma SK-ES-1 cells. Antisense inhibiting miR-10b in NF1 MPNST cells reduced cell proliferation, migration and invasion. Furthermore, we showed that NF1 mRNA was the target for miR-10b. Overexpression of miR-10b in 293T cells suppressed Neurofibromin expression and activated RAS signaling. Antisense inhibition of miR-10b restored Neurofibromin expression in SK-ES-1 cells, and decreased RAS signaling independent of Neurofibromin in NF1 MPNST cells. These results suggest that miR-10b may play an important role in NF1 tumorigenesis through targeting Neurofibromin and RAS signaling.
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Neurofibromin physically interacts with the N-terminal domain of focal adhesion kinase.
Molecular carcinogenesis, 2009Co-Authors: Frederick Kweh, Margaret R. Wallace, Min Zheng, Elena Kurenova, Vita M. Golubovskaya, William G. CanceAbstract:The NF1 gene that is altered in patients with type 1 neurofibromatosis (NF1) encodes a Neurofibromin protein that functions as a tumor suppressor. In this report, we show for the first time physical interaction between Neurofibromin and focal adhesion kinase (FAK), the protein that localizes at focal adhesions. We show that Neurofibromin associates with the N-terminal domain of FAK, and that the C-terminal domain of Neurofibromin directly interacts with FAK. Confocal microscopy demonstrates colocalization of NF1 and FAK in the cytoplasm, perinuclear and nuclear regions inside the cells. Nf1+/+ MEF cells expressed less cell growth during serum deprivation conditions, and adhered less on collagen and fibronectin-treated plates than Nf1−/− MEF cells, associated with changes in actin and FAK staining. In addition, Nf1+/+ MEF cells detached more significantly than Nf1−/− MEF cells by disruption of FAK signaling with the dominant-negative inhibitor of FAK, C-terminal domain of FAK (FAK-CD). Thus, the results demonstrate the novel interaction of Neurofibromin and FAK and suggest their involvement in cell adhesion, cell growth, and other cellular events and pathways. © 2009 Wiley-Liss, Inc.
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Tumorigenic Properties of Neurofibromin-Deficient Neurofibroma Schwann Cells
The American journal of pathology, 2001Co-Authors: David Muir, Debbie Neubauer, Ingrid T. Lim, Anthony T. Yachnis, Margaret R. WallaceAbstract:Dermal and plexiform neurofibromas are peripheral nerve sheath tumors that arise frequently in neurofibromatosis type 1. The goal of the present study was to examine the tumorigenic properties of Neurofibromin-deficient human Schwann cells (SCs) that were found to represent a subset of SCs present in approximately half of the total neurofibromas examined. Highly enriched SC cultures were established from 10 dermal and eight plexiform neurofibromas by selective subculture using glial growth factor-2 and laminin. These cultures had low tumorigenic potential in classical in vitro assays yet several unique preneoplastic properties were frequently observed, including delayed senescence, a lack of density-limited growth, and a strong propensity to spontaneously form proliferative cell aggregates rich in extracellular matrix. Western blot analysis failed to detect full-length Neurofibromin in any of the neurofibroma SC cultures, indicating that Neurofibromin-deficient SCs had a substantial growth advantage. Immunohistochemical staining of the originating tumors showed the majority were comprised principally of Neurofibromin-negative SCs, whereas the remainder contained both Neurofibromin-negative and Neurofibromin-positive SCs. Lastly, engraftment of Neurofibromin-deficient SC cultures into the peripheral nerves of scid mice consistently produced persistent neurofibroma-like tumors with diffuse and often extensive intraneural growth. These findings indicate that Neurofibromin-deficient SCs are involved in neurofibroma formation and, by selective subculture, provide a resource for the development of an in vivo model to further examine the role of these mutant SCs in neurofibroma histogenesis.
Frank Mccormick - One of the best experts on this subject based on the ideXlab platform.
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Abstract 3765: c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction
Immunology, 2020Co-Authors: Claire Lorenzo, Anatoly Urisman, Lucy C. Young, Alexandra Tannka, Frank MccormickAbstract:Spred1 negatively regulates Ras/MAPK signaling following growth factor stimulation. Spred1 inhibits Ras by binding and localizing Neurofibromin, a RasGAP, to the plasma membrane to accelerate Ras GTPase activity. c-Kit, a receptor tyrosine kinase (RTK), is known to interact with Spred1 but the consequence of this interaction is unknown. Here we demonstrate that c-Kit signaling regulates Neurofibromin-Spred1 interaction. Stimulation with c-Kit ligand, SCF, results in a transient disruption in Neurofibromin-Spred1 binding which corresponds to increased Ras signaling, followed by restoration of Neurofibromin-Spred binding which corresponds to nearly basal levels of Ras signaling. Mass spectrometry analysis identified potential phosphorylation sites on Spred1 that correspond to the initial disruption and later restoration of Neurofibromin-Spred1 binding. Phosphomimetic and phosphodeficient mutants affect the interaction. Our findings provide a potential mechanism by which RTK signaling regulates negative feedback to allow transient activation and subsequent termination of Ras signaling. Citation Format: Claire Lorenzo, Lucy C. Young, Alexandra Tannka, Anatoly Urisman, Frank McCormick. c-Kit mediated phosphorylation of Spred1 regulates Neurofibromin-Spred1 interaction [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3765.
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Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR.
Cell reports, 2020Co-Authors: Wupeng Yan, Frank Mccormick, Klaus Scheffzek, Matthew Drew, Evan Markegard, Srisathiyanarayanan Dharmaiah, Anatoly Urisman, Dominic Esposito, Dwight V. Nissley, Dhirendra K. SimanshuAbstract: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.
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KRAS G13D sensitivity to Neurofibromin-mediated GTP hydrolysis.
Proceedings of the National Academy of Sciences of the United States of America, 2019Co-Authors: Dana Rabara, Frank Mccormick, Srisathiyanarayanan Dharmaiah, Dhirendra K. Simanshu, Timothy H. Tran, Robert M. Stephens, Matthew HolderfieldAbstract:KRAS mutations occur in ∼35% of colorectal cancers and promote tumor growth by constitutively activating the mitogen-activated protein kinase (MAPK) pathway. KRAS mutations at codons 12, 13, or 61 are thought to prevent GAP protein-stimulated GTP hydrolysis and render KRAS-mutated colorectal cancers unresponsive to epidermal growth factor receptor (EGFR) inhibitors. We report here that KRAS G13-mutated cancer cells are frequently comutated with NF1 GAP but NF1 is rarely mutated in cancers with KRAS codon 12 or 61 mutations. Neurofibromin protein (encoded by the NF1 gene) hydrolyzes GTP directly in complex with KRAS G13D, and KRAS G13D-mutated cells can respond to EGFR inhibitors in a Neurofibromin-dependent manner. Structures of the wild type and G13D mutant of KRAS in complex with Neurofibromin (RasGAP domain) provide the structural basis for Neurofibromin-mediated GTP hydrolysis. These results reveal that KRAS G13D is responsive to Neurofibromin-stimulated hydrolysis and suggest that a subset of KRAS G13-mutated colorectal cancers that are Neurofibromin-competent may respond to EGFR therapies.
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The Neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Theresia Dunzendorfer-matt, Frank Mccormick, Ellen L. Mercado, K. Maly, Klaus ScheffzekAbstract:Neurofibromatosis type 1 (NF1) and Legius syndrome are related diseases with partially overlapping symptoms caused by alterations of the tumor suppressor genes NF1 (encoding the protein Neurofibromin) and SPRED1 (encoding sprouty-related, EVH1 domain-containing protein 1, Spred1), respectively. Both proteins are negative regulators of Ras/MAPK signaling with Neurofibromin functioning as a Ras-specific GTPase activating protein (GAP) and Spred1 acting on hitherto undefined components of the pathway. Importantly, Neurofibromin has been identified as a key protein in the development of cancer, as it is genetically altered in a large number of sporadic human malignancies unrelated to NF1. Spred1 has previously been demonstrated to interact with Neurofibromin via its N-terminal Ena/VASP Homology 1 (EVH1) domain and to mediate membrane translocation of its target dependent on its C-terminal Sprouty domain. However, the region of Neurofibromin required for the interaction with Spred1 has remained unclear. Here we show that the EVH1 domain of Spred1 binds to the noncatalytic (GAPex) portion of the GAP-related domain (GRD) of Neurofibromin. Binding is compatible with simultaneous binding of Ras and does not interfere with GAP activity. Our study points to a potential targeting function of the GAPex subdomain of Neurofibromin that is present in all known canonical RasGAPs.
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The RasGAP proteins Ira2 and Neurofibromin are negatively regulated by Gpb1 in yeast and ETEA in humans.
Molecular and cellular biology, 2010Co-Authors: Vernon T. Phan, Vivianne W. Ding, Robert J. Chalkley, Alma L. Burlingame, Frank MccormickAbstract:The neurofibromatosis type 1 (NF1) gene encodes the GTPase-activating protein (GAP) Neurofibromin, which negatively regulates Ras activity. The yeast Saccharomyces cerevisiae has two Neurofibromin homologs, Ira1 and Ira2. To understand how these proteins are regulated, we utilized an unbiased proteomics approach to identify Ira2 and Neurofibromin binding partners. We demonstrate that the Gpb1/Krh2 protein binds and negatively regulates Ira2 by promoting its ubiquitin-dependent proteolysis. We extended our findings to show that in mammalian cells, the ETEA/UBXD8 protein directly interacts with and negatively regulates Neurofibromin. ETEA contains both UBA and UBX domains. Overexpression of ETEA downregulates Neurofibromin in human cells. Purified ETEA, but not a mutant of ETEA that lacks the UBX domain, ubiquitinates the Neurofibromin GAP-related domain in vitro. Silencing of ETEA expression increases Neurofibromin levels and downregulates Ras activity. These findings provide evidence for conserved ubiquitination pathways regulating the RasGAP proteins Ira2 (in yeast) and Neurofibromin (in humans).