Related Experiment Video

Updated: Aug 17, 2026

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
10:27

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells

Published on: March 9, 2012

A single amino acid change in Raf-1 inhibits Ras binding and alters Raf-1 function

J R Fabian1, A B Vojtek, J A Cooper

  • 1Molecular Mechanisms of Carcinogenesis Laboratory, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702.

Proceedings of the National Academy of Sciences of the United States of America
|June 21, 1994
PubMed

Insights

The Ras/Raf-1 interaction is crucial for cell signaling. A specific mutation (Arg89Leu) in Raf-1 disrupts this binding, impacting Raf-1 activity and blocking Ras-mediated pathways.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Ras and Raf-1 proteins are central to transmitting signals for cell development and proliferation.
  • Raf-1 acts downstream of Ras in numerous signaling pathways, with direct association observed between GTP-bound Ras and Raf-1.
  • The in vivo consequences of the Ras/Raf-1 interaction on Raf-1 activity remain largely unestablished.

Purpose of the Study:

  • To investigate the biological impact of the Ras/Raf-1 interaction in vivo.
  • To identify critical Raf-1 residues essential for binding to Ras.

Main Methods:

  • Utilized site-directed mutagenesis to create an Arg89Leu mutation in Raf-1.
  • Assessed Ras-Raf-1 interaction using in vitro binding assays and the yeast two-hybrid system.
  • Evaluated Raf-1 enzymatic activation in the baculovirus/Sf9 expression system.
  • Examined the effect of the mutation on Ras-mediated signal transduction in Xenopus laevis oocytes.

Main Results:

  • A single amino acid mutation (Arg89Leu) in Raf-1 abolished Ras binding in vitro and in yeast.
  • This mutation abrogated Ras-mediated Raf-1 activation but not tyrosine kinase-mediated activation.
  • Kinase-defective Raf-1 mutants with the Arg89Leu mutation lost their dominant-inhibitory capacity and failed to block Ras signaling in oocytes.

Conclusions:

  • The physical association between Ras and Raf-1 directly modulates Raf-1's kinase activity and biological function.
  • Residue Arg89 of Raf-1 is critical for the interaction with Ras.
  • Raf-1 can be activated through Ras-independent pathways, as indicated by tyrosine kinase stimulation of the mutated Raf-1.

More Related Videos

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods07:49

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

Published on: July 17, 2019

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells06:44

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

Published on: March 1, 2024

Related Concept Videos

Rab Proteins01:14

Rab Proteins

Rab proteins constitute the largest family of monomeric GTPases, of which 70 members are present in humans. Rab proteins and their effectors regulate consecutive stages of vesicle transport such as vesicle transport, docking, and fusion to the correct recipient membrane.
Rab proteins switch between a cytosolic, GDP-bound inactive state and a membrane-anchored, GTP-bound active state. By themselves, Rabs show slow rates of GDP/GTP exchange and GTP hydrolysis. Thus, Rab proteins are considered...
The Ras Gene02:38

The Ras Gene

The Ras-gene-encoded proteins are regulators of signaling pathways controlling cell proliferation, differentiation, or cell survival. The Ras-gene family in humans constitutes three primary members—the HRas, NRas, and KRas. These genes code for four functionally distinct yet closely related proteins—the HRas, NRas, KRas4A, and KRas4B. The involvement of mutant Ras genes in human cancer was first discovered in 1982 and is among the most common causes of human tumorigenesis.
Ras is a superfamily...
Rab Cascades01:25

Rab Cascades

Rab GTPases act in a regulated cascade during membrane fusion, helping the lipid bilayers mix. The Rab family of proteins are active when bound to GTP, and inactive when bound to GDP. Hence, they act as guanine nucleotide-dependent molecular switches. Rab-GTP recognizes and binds to long or short-range tethering proteins to capture the target vesicle. These tethers coordinate with SNAREs on the vesicle and the target membrane to assemble the trans SNARE complex that locks the mixing bilayers.
Small GTPases - Ras and Rho01:24

Small GTPases - Ras and Rho

Ras and Rho are small monomeric GTPases that act downstream of receptor tyrosine kinase (RTK) and regulate various cellular processes. These GTPases switch between active and inactive states by binding to guanine nucleotides.
Three regulatory proteins control their activity:
MAPK Signaling Cascades01:07

MAPK Signaling Cascades

Mitogen-activated protein kinase, or MAPK pathway, activates three sequential kinases to regulate cellular responses such as proliferation, differentiation, survival, and apoptosis. The canonical MAPK pathway starts with a mitogen or growth factor binding to an RTK. The activated RTKs stimulate Ras, which recruits Raf or MAP3 Kinase (MAPKKK), the first kinase of the MAPK signaling cascade. Raf further phosphorylates and activates MEK or MAP2 Kinases (MAPKK), which in turn phosphorylates MAP...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...