Related Experiment Video
Updated: Aug 1, 2026
12:42
Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
Published on: December 22, 2015
Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf
C Gorman1, R H Skinner, J V Skelly
1Wellcome Research Laboratories, Langley Court, South Eden Park Road, Beckenham, Kent BR3 3BS, United Kingdom.
The Journal of Biological Chemistry
|March 22, 1996
Summary
This study characterizes Ras-Raf interactions using equilibrium binding assays, revealing that Raf and neurofibromin compete for Ras binding. These findings suggest a rapid equilibrium between Ras, Raf, and neurofibromin within cells.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Raf is a serine/threonine kinase crucial for activating the mitogen-activated protein kinase pathway.
- Raf interacts with the GTP-bound form of Ras via its amino-terminal regulatory domain.
Purpose of the Study:
- To characterize the binding interaction between the Ras-binding domain of Raf and Ras.
- To compare equilibrium binding methods with non-equilibrium methods for studying Ras-Raf interactions.
- To investigate the competitive binding of neurofibromin and Raf to Ras.
Main Methods:
- Utilized equilibrium binding methods: scintillation proximity assay and fluorescence anisotropy.
- Employed glutathione S-transferase (GST) fusion proteins of Raf (residues 1-257, 1-190, and 51-131) and Ras.
- Assessed inhibition of binding by the catalytic domain of neurofibromin.
Main Results:
- Equilibrium binding assays revealed challenges with full-length Raf due to inactive protein percentages.
- A stable GST-Raf fusion protein (residues 51-131) enabled reliable measurement of Ras binding under equilibrium conditions.
- Raf and neurofibromin were found to bind to Ras competitively, with similar affinities for Harvey-RasLeu-61.
- The dissociation rate constant for Raf from Ras was estimated to be >1 min-1.
Conclusions:
- Equilibrium binding methods are essential for accurate characterization of Ras-Raf interactions, overcoming limitations of non-equilibrium assays.
- Raf and neurofibromin bind to Ras in a mutually exclusive manner, indicating a direct competition for the binding site.
- The Ras-Raf and Ras-neurofibromin interactions are likely in rapid equilibrium within the cellular environment.
- Residues 132-147 of Raf do not appear to be critical binding determinants for Ras interaction.
Related Concept Videos
02:18The Equilibrium Binding Constant and Binding Strength
The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
01:14Rab 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...
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...
02:38The 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...
Ras is a superfamily...
01:25Rab 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.
01:24Small 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:
Three regulatory proteins control their activity:
01:07MAPK 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...