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Phillips2003 - The Mechanism of Ras GTPase Activation by Neurofibromin

ABSTRACT: Phillips2003 - The Mechanism of Ras GTPase Activation by Neurofibromin A mathematical model for Ras-GTP activation by neurofibromin and the kinetic rates of the relevant reactions. This model is described in the article: The mechanism of Ras GTPase activation by neurofibromin. Phillips RA, Hunter JL, Eccleston JF, Webb MR. Biochemistry 2003 Apr; 42(13): 3956-3965 Abstract: Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly. This model is hosted on BioModels Database and identified by: BIOMD0000000692. To cite BioModels Database, please use: Chelliah V et al. BioModels: ten-year anniversary. Nucl. Acids Res. 2015, 43(Database issue):D542-8. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

SUBMITTER: Sharat Vayttaden

PROVIDER: BIOMD0000000692 | BioModels | 2018-03-19

REPOSITORIES: BioModels

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The mechanism of Ras GTPase activation by neurofibromin.

Phillips Robert A RA Hunter Jackie L JL Eccleston John F JF Webb Martin R MR

Biochemistry 20030401 13

Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate rele ...[more]

PMID: 12667087

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Project description:Smith2009 - RGS mediated GTP hydrolysis This model is described in the article: Dual positive and negative regulation of GPCR signaling by GTP hydrolysis. Smith B, Hill C, Godfrey EL, Rand D, van den Berg H, Thornton S, Hodgkin M, Davey J, Ladds G. Cell Signal. 2009 Jul;21(7):1151-60. Abstract: G protein-coupled receptors (GPCRs) regulate a variety of intracellular pathways through their ability to promote the binding of GTP to heterotrimeric G proteins. Regulator of G protein signaling (RGS) proteins increases the intrinsic GTPase activity of Galpha-subunits and are widely regarded as negative regulators of G protein signaling. Using yeast we demonstrate that GTP hydrolysis is not only required for desensitization, but is essential for achieving a high maximal (saturated level) response. Thus RGS-mediated GTP hydrolysis acts as both a negative (low stimulation) and positive (high stimulation) regulator of signaling. To account for this we generated a new kinetic model of the G protein cycle where Galpha(GTP) enters an inactive GTP-bound state following effector activation. Furthermore, in vivo and in silico experimentation demonstrates that maximum signaling output first increases and then decreases with RGS concentration. This unimodal, non-monotone dependence on RGS concentration is novel. Analysis of the kinetic model has revealed a dynamic network motif that shows precisely how inclusion of the inactive GTP-bound state for the Galpha produces this unimodal relationship. To reproduce dose-response plots in the publication, the model is simulated with 12 different concentrations (see parameter Ligand_conc). For each concentration, a single value must be obtained from the integral of the trajectory of species z3 from time=0 to time=30. These values are then used to build a dose-response plot (authors used GraphPad Prism). Mutant strains are simulated with alternative parameter values or initial conditions in Table S3. This model is hosted on BioModels Database and identified by: MODEL1212040001 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. PMID: 20587024 . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3’5’cyclic AMP (cAMP)-dependent Protein Kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in Acrodysostosis. How mutations on PDEs and RIα interfere with regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit. PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα:PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: Translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis Step 2) Processivity: Binding of free cAMP from the cytosol at both CNBs of RIα and Step 3) Product (5’AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that trigger subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, (PDE8 T690P) and RIα (T207A) that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and Fluorescence Polarization (FP) reveal that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5’AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5’AMP release from the PDE, T207A greatly slowed release of substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα:PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.

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Phillips2003 - The Mechanism of Ras GTPase Activation by Neurofibromin

Publications

The mechanism of Ras GTPase activation by neurofibromin.

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