Selective disactivation of neurofibromin GAP activity in neurofibromatosis type 1

Abstract

Neurofibromatosis type 1 (NF1) is a common familial tumour syndrome with multiple clinical features such as neurofibromas, café-au-lait spots (CLS), iris Lisch nodules, axillary freckling, optic glioma, specific bone lesions and an increased risk of malignant tumours. It is caused by a wide spectrum of mutations affecting the NF1 gene. Most mutations result in the loss of one allele at the DNA, mRNA or protein level and thus in the loss of any function of the gene product neurofibromin. The idea of the simultaneous loss of several different neurofibromin functions has been postulated to explain the pleiotropic effects of its loss. However, we have identified a novel missense mutation in a family with a classical multi-symptomatic NF1 phenotype, including a malignant schwannoma, that specifically abolishes the Ras-GTPase-activating function of neurofibromin. In this family, Arg1276 had mutated into proline. Based on complex biochemical studies as well as the analysis of the crystal structure of the GTPase-activating protein (GAP) domain of p120GAP in the presence of Ras, we unequivocally identified this amino acid as the arginine finger of the neurofibromin GAP-related domain (GRD)-the most essential catalytic element for RasGAP activity. Here, we present data demonstrating that the mutation R1276P, unlike previously reported missense mutations of the GRD region, does not impair the secondary and tertiary protein structure. It neither reduces the level of cellular neurofibromin nor influences its binding to Ras substantially, but it does completely disable GAP activity. Our findings provide direct evidence that failure of neurofibromin GAP activity is the critical element of NF1 pathogenesis. Thus, therapeutic approaches aimed at the reduction of Ras.GTP levels in neural crest-derived cells can be expected to relieve most of the NF1 symptoms.

Next-generation sequencing identifies rare variants associated with Noonan syndrome

“…NF1 encodes neurofibromin, a RAS-GTPase-activating protein (RAS-GAP). Although most causative NF1 point mutations result in premature translation termination (18), some disease-associated missense mutations encode unstable NF1 proteins without affecting NF1 mRNA levels (19), and others impair RAS-GAP activity (20)(21)(22). The leucine at position 1361 of NF1 is located in the GAP-related domain (NF1-GRD) and is highly conserved (Fig.…”

Structural and biochemical consequences of NF1 associated nontruncating mutations in the Sec14-PH module of neurofibromin

“…Reduced neurofibromin levels were detected in NF1 patients with truncating mutations (NF71, NF10 and NF106 (Kaufmann, et al, 1999b)). Neurofibromin content was measured in peripheral blood cells by immunoprecipitation and Western blotting as described (Griesser, et al, 1997;Klose, et al, 1998) . Lipid profiles of wild-type NF1 Sec14-PH and selected representative mutants.…”

“…Reduced neurofibromin levels were detected in NF1 patients with truncating mutations (NF71, NF10 and NF106 (Kaufmann, et al, 1999b)). Neurofibromin content was measured in peripheral blood cells by immunoprecipitation and Western blotting as described (Griesser, et al, 1997;Klose, et al, 1998) . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

STAT3 and HIF1α Signaling Drives Oncogenic Cellular Phenotypes in Malignant Peripheral Nerve Sheath Tumors

“…Neurofibromin functions as a tumor suppressor by acting as a GTPase-activating protein (GAP) toward the small G-protein Ras (3,4). Consequently, inactivating mutations to NF1 lead to increased signal transduction through Ras to promote uncontrolled cell growth and tumorigenesis (5,6).…”