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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Published on: July 17, 2019

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction

J C Stone1, M Colleton, D Bottorff

  • 1Department of Biochemistry, University of Alberta, Edmonton, Canada.

Molecular and Cellular Biology
|December 1, 1993
PubMed

Insights

GTPase-activating proteins (GAPs) regulate p21ras function. A specific mutation (Pro-34-->Arg) in c-H-ras impairs GAP binding, suggesting distinct structural requirements for effector function and GAP interaction, and implicating other molecules as ras effectors.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Oncogenesis

Background:

  • GTPase-activating proteins (GAPs) like p120GAP and neurofibromatosis 1 gene product negatively regulate p21ras GTPase activity.
  • GAPs are hypothesized to be ras effectors, mediating downstream signaling pathways.

Purpose of the Study:

  • To identify activating substitutions in the c-H-ras effector domain that decouple effector function from GAP-mediated negative regulation.
  • To investigate the structural basis for ras effector function and GAP interaction.

Main Methods:

  • Site-directed mutagenesis to introduce the Pro-34-->Arg substitution in c-H-ras.
  • In vivo GTP-binding assays to assess GTP-bound p21ras levels.
  • In vitro assays to evaluate GAP stimulation and p120GAP binding.
  • Analysis of p21ras coupling to Raf-1 phosphoprotein.

Main Results:

  • The Pro-34-->Arg mutation resulted in a c-H-ras protein substantially bound to GTP in vivo.
  • This mutant protein showed impaired binding to p120GAP and was not stimulated by GAPs in vitro.
  • The Pro-34-->Arg p21 species effectively coupled to Raf-1, indicated by electrophoretic mobility shifts.

Conclusions:

  • The structural requirements for p21ras effector function and GAP interaction are distinct.
  • Molecules other than p120GAP likely serve as ras effectors.
  • The Pro-34-->Arg mutation provides a tool to study ras signaling independent of GAP regulation.

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