Expression of the full-length neurofibromatosis type 1 gene product and localization of its microtubule binding domain.

Abstract

The gene for neurofibromatosis type 1 (NF1), a cancer predisposition disorder that primarily affects tissues derived from the neural crest, was cloned in 1990 and found to encode a 2818 amino acid protein with homology to the family of GTPase activating proteins (GAPs). This family, which includes the human protein p120-GAP and the yeast proteins IRA1 and IRA2, down-regulate the activity of p21-ras by catalyzing its intrinsic GTPase activity. The NF1 protein, called neurofibromin, is also able to stimulate the GTPase activity of ras and complement the temperature sensitive phenotype of IRA- yeast strains. An unexpected cellular localization of neurofibromin was observed by immunofluorescence. The immunostaining pattern appeared to coincide with that of microtubules. Neurofibromin also associated with the microtubule pellet in a microtubule cycling assay using rat brain homogenates, and a 55 kilodalton protein, identified as tubulin, co-sedimented when neurofibromin was purified from an overexpression system. The region of neurofibromin involved in the microtubule association was localized by expressing truncated forms of the protein in Sf9 insect cells and measuring microtubule cycling behavior. Results of these assays indicate that there are at least two independently acting regions capable of conferring binding. Both of these (amino acids 1125-1175 and 1300-1425) are located within the domain of the protein responsible for the GAP activity. As a negative control, the same assays were performed with the p120-GAP protein, which is known to be cytoplasmic. This protein did not associate with the microtubules and was located in the supernatant fraction after cycling. Although the evidence is strong that neurofibromin associates with microtubules, its cellular function is not known. There is evidence that its GAP activity is diminished when tubulin is bound, which suggests a regulatory function for the binding. Neurofibromin may serve as a connection between ras, which is involved in regulation of cell growth and differentiation through signal transduction pathways, and microtubules, which are important for cell division and cytoskeletal structure.