Functional significance of lysine 1423 of neurofibromin and characterization of a second site suppressor which rescues mutations at this residue and suppresses RAS2Val-19-activated phenotypes.
Lysine 1423 of neurofibromin (neurofibromatosis type I gene product [NF1]) plays a crucial role in the function of NF1. Mutations of this lysine were detected in samples from a neurofibromatosis patient as well as from cancer patients. To further understand the significance of this residue, we have mutated it to all possible amino acids. Functional assays using yeast ira complementation have revealed that lysine is the only amino acid that produced functional NF1. Quantitative analyses of different mutant proteins have suggested that their GTPase-activating protein (GAP) activity is drastically reduced as a result of a decrease in their Ras affinity. Such a requirement for a specific residue is not observed in the case of other conserved residues within the GAP-related domain. We also report that another residue, phenylalanine 1434, plays an important role in NF1 function. This was first indicated by the finding that defective NF1s due to an alteration of lysine 1423 to other amino acids can be rescued by a second site intragenic mutation 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 acquired the ability to suppress activated phenotypes of RAS2Val-19 cells. This 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.
Poullet
P
P
Department of Biochemistry and Molecular Biology, University of Chicago, Illinois 60637.
Lin
B
B
Esson
K
K
Tamanoi
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D020690:GTPase-Activating Proteins; D016514:Genes, Neurofibromatosis 1; D016153:Genes, Suppressor; D011905:Genes, ras; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D025542:Neurofibromin 1; D010641:Phenotype; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020703:ras GTPase-Activating Proteins
D017931:DNA Primers; D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; C065725:IRA1 protein, S cerevisiae; D025542:Neurofibromin 1; D011506:Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D020703:ras GTPase-Activating Proteins