FUNCTIONAL-SIGNIFICANCE OF LYSINE-1423 OF NEUROFIBROMIN AND CHARACTERIZATION OF A 2ND SITE SUPPRESSOR WHICH RESCUES MUTATIONS AT THIS RESIDUE AND SUPPRESSES RAS2(VAL-19)-ACTIVATED PHENOTYPES

Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [N F1]) plays a crucial role in the function of NF1. Mutations of this ly sine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of th is residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is t he only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activat ing protein (GAP) activity is drastically reduced as a result of a dec rease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GA P-related domain. We also report that another residue, phenylalanine 1 434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 142 3 to other amino acids can be rescued by a second site intragenic muta tion at residue 1434. The mutation partially restored GAP activity in the lysine mutant. When the mutation phenylalanine 1434 to serine was introduced into a wild-type NF1 protein, the resulting protein acquire d the ability to suppress activated phenotypes of RA4S2Val-19 cells. T his suppression, however, does not involve Ras interaction, since the phenylalanine mutant does not stimulate the intrinsic GTPase activity of RAS2Val-19 protein and does not have an increased affinity for Ras proteins.