Related Experiment Video

Updated: Jun 6, 2026

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
07:49

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

Published on: July 17, 2019

Allosteric modulation of Ras-GTP is linked to signal transduction through RAF kinase

Greg Buhrman1, V S Senthil Kumar, Murat Cirit

  • 1Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.

The Journal of Biological Chemistry
|November 25, 2010
PubMed

Insights

Ras mutants G12V and Q61L impact GTPase activity, crucial in cancer. RasQ61L uniquely stabilizes an inactive conformation, leading to stronger MAPK pathway activation than RasG12V.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • Ras proteins are central to cellular signal transduction.
  • Mutations at Glycine 12 (G12) and Glutamine 61 (Q61) impair Ras GTPase activity and are common in various cancers.
  • Wild-type Ras-GTP utilizes an allosteric switch mechanism to regulate its activity.

Purpose of the Study:

  • To investigate the conformational dynamics of Ras mutants G12V and Q61L.
  • To determine how these mutations affect the allosteric switch and downstream signaling pathways.
  • To elucidate the mechanism controlling the duration of Ras-Raf complex formation.

Main Methods:

  • Structural analysis of Ras mutants.
  • Measurement of GTP hydrolysis rates.
  • Experiments in NIH-3T3 cells to assess pathway activation.

Main Results:

  • Both RasG12V and RasQ61L mutants can adopt 'on' and 'off' conformations of the allosteric switch.
  • RasQ61L stabilizes an anti-catalytic conformation of switch II when bound to Raf, unlike RasG12V.
  • This differential stabilization leads to more potent activation of the Ras/Raf/MEK/ERK pathway by RasQ61L compared to RasG12V.
  • No significant difference in activation was observed for the Raf-independent Ras/PI3K/Akt pathway.

Conclusions:

  • The allosteric switch plays a critical role in controlling Ras/Raf/MEK/ERK pathway signaling.
  • A GTPase-activating protein-independent model explains the duration of the Ras-Raf complex.
  • RasQ61L exhibits distinct signaling behavior compared to RasG12V due to differential allosteric switch stabilization.

More Related Videos

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

Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells10:27

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

Published on: March 9, 2012

Related Concept Videos

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 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...
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:
Interactions Between Signaling Pathways01:19

Interactions Between Signaling Pathways

Signaling cascades usually lack linearity. Multiple pathways interact and regulate one another, allowing cells to integrate and respond to diverse environmental stimuli.
Convergence and divergence, and cross-talk between signaling pathways
Two distinct signaling pathways can converge on a single functional unit, which may either be a single protein or a complex of proteins. The response is either functionally distinct or synergistic between the two pathways but different from the response...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...
GTPases and their Regulation02:14

GTPases and their Regulation

Guanine nucleotide-binding proteins (G-proteins), also known as GTPases, are a superfamily of proteins that regulate many cellular processes, such as cell signaling, vesicular transport, and the regulation of cell shape and motility. Mutation or dysfunction of these proteins can lead to disease. There are around 40,000 known G-proteins that can broadly be classified into two groups ‒  small G-proteins consisting of a single domain and large multi-domain G-proteins.
Large G-proteins, also known...