Neurofibromatosis Type I Tumor Suppressor Neurofibromin Regulates Neuronal Differentiation via Its GTPase-activating Protein Function toward Ras

Abstract

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominantnegative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance. Neurofibromatosis type 1 (NF1)1 is one of the most common autosomal dominantly inherited disorders, with an incidence of about 1 in 3500 individuals (1). The NF1 hallmark is the development of benign tumors of the peripheral nervous system and the increased risk of developing malignancies. The NF1 phenotype is highly variable; it affects several organ systems, including bones, skin, irises, and central nervous system manifested as gliomas and learning disabilities. The NF1 gene lies on chromosome 17q11.2 and encodes neurofibromin, a large 2818-amino acid protein (2). Since the majority of NF1 gene mutations frequently found in NF1 patients prevent intact neurofibromin expression, functional disruption of neurofibromin is potentially relevant to the expression of some or all of the multiple abnormalities that occur in NF1 patients (3).A region centered around the 360 amino acids encoded by the NF1 gene shows significant homology to the known catalytic domains of mammalian Ras GTPase-activating protein (p120GAP) and is also similar to yeast IRA1/2 proteins, which interact with Ras and mediate hydrolysis of Ras-bound GTP to GDP, resulting in Ras protein inactivation. The GAP-related domain of the NF1 gene product (NF1-GRD) also stimulates Ras GTPase and consequently inactivates Ras protein (4, 5).Two different isoforms, type I and type II, which are formed by alternative splicing, have been identified in the NF1-GRD region. Type II contains an additional 63-bp insertion (exon 23a) that encodes 21 amino acids in the center of NF1-GRD type I (6). The specific expression patterns of the two isoforms have been studied in several organs and cells (7,8) and pro...

BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas

“…While NF1-associated pilocytic astrocytomas show loss and inactivation of NF1 alleles, sporadic pilocytic astrocytomas lack NF1 mutations or reduced NF1 expression (39). Neurofibromin, the gene product of NF1, is involved in the repression of MAPK signaling by its GTPase domain (12). Interestingly, while not detecting BRAF gene dupli- cations, Jones et al (10) identified a small copy number gain of the RAF1 locus at 3p25 in 1 pilocytic astrocytoma (10).…”

“…Neurofibromin, the gene product of the NF1 gene, physiologically contributes to growth arrest of astrocytic cells and neuronal differentiation by downregulation of the MAPK signaling pathway via its GTPase-activating domain. Loss of neurofibromin expression conversely leads to increased Ras activity and astrocyte proliferation (12). Mutations of other components of the MAPK pathway cause several other inherited diseases, such as Noonan syndrome, LEOPARD syndrome, cardio-facio-cutaneous (CFC) syndrome, and Costello syndrome (13)(14)(15)(16)(17)(18).…”

The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

“…In addition, as we have shown previously, neurofibromin regulates neuronal differentiation via its GAP function. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after nerve growth factor stimulation and were correlated with the down-regulation of Ras activity during neurite extension (6). Although cell migration and neurite extension are known to be regulated by cytoskeletal reorganization in cells, the molecular mechanisms by which neurofibromin is involved in these cytoskeletal dynamics are understood poorly.…”

Activation of EGF receptor endocytosis and ERK1/2 signaling by BPGAP1 requires direct interaction with EEN/endophilin II and a functional RhoGAP domain

“…Although GAPs usually function to inactivate their cognate GTPase substrates, some are believed to function as effectors that mediate downstream signalingfor example, the RasGAP neurofibromatosis 1 (Yunoue et al, 2003) and TcGAP (Chiang et al, 2003). By contrast, some enhance the effect of the protein they interact with, such as the RhoGAP domain in the regulatory subunits of phosphoinositide 3-kinase, p85 (Zheng et al, 1994).…”