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Title: The interaction of p21Ras with neurofibromin
Author: Sermon, Beth A.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1997
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rasgenes encode guanine nucleotide binding proteins that act as molecular switches for signal transduction pathways controlling cell growth and differentiation. In the GTP- bound form the Ras protein is active and interacts with effector proteins to propagate a signal from the outside of the cell to the nucleus or cytoskeleton. Ras has a low intrinsic GTPase activity which is accelerated by the GTPase-activating proteins (GAPs), p120-GAP and neurofibromin. Two aspects of the interaction between Ras and the catalytic domain of neurofibromin (NF1334) have been studied. (1) The GTPase-activating activity of both p120-GAP and neurofibromin are inhibited by mitogenic lipids such as phosphatidic acid and arachidonic acid. Previous data on the differential inhibition of the two GAPs led to the hypothesis that both were effectors in a Ras-controlled mitogenic pathway. The mechanism of inhibition of NF1334 by arachidonic acid was studied by measuring the catalytic activity under multiple turnover conditions, using p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid as a fluorescent probe for ligands binding to GAPs and using a scintillation proximity assay to measure direct binding of Ras to NF1334. The inhibition by arachidonic acid included a major component that is competitive with Ras.GTP and an additional non-competitive type effect consistent with protein denaturing activity. This suggested that insomuch as the mitogenic effects of lipids are mediated via inhibition of GAPs, GAPs are not Ras effector proteins. (2) Basic residues within the catalytic domains of p120-GAP and neurofibromin have been suggested to play an important role in GAP-stimulated catalysis and Ras binding. Two invariant arginine residues within NF1334, R1276 and R1391, were mutated to alanine and their effects on maximal catalysis (kcat) affinity (Kd) for Ras measured under single turnover conditions. Both R1276 and R1391 are required for efficient catalysis by NF1334 as removal of either results in a 1000-fold loss of activity. R1276 is not thought to be directly involved in Ras binding as the affinity of R1276A for H-Ras or [Leu61]H-Ras is only moderately reduced (ca. 3-fold). The reduction in affinity of R1391A for H-Ras is more marked (ca. 10-20 fold) but most notable for [Leu61]H-Ras (ca. 100-fold) suggesting a role for this residue in Ras binding. The high affinity of [Leu61]H-Ras for NF1334 over H-Ras is completely lost with the R1391 NF1334, suggesting that the high affinity is related to an interaction with R1391 of NF1334.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available