Related Experiment Video

Updated: Jun 20, 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

Insights into the molecular function of the inactivating mutations of B-Raf involving the DFG motif

Sonia Moretti1, Valentina De Falco, Anna Tamburrino

  • 1Dipartimento di Medicina Interna, Università di Perugia, Perugia, Italy.

Biochimica Et Biophysica Acta
|September 9, 2009
PubMed

Insights

Inactivating BRAF mutations, like G596R, impair kinase function by altering protein structure. These BRAF mutants can form heterodimers with C-Raf, but their cancer-driving activity is minimal.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cancer Research

Background:

  • BRAF gene mutations are linked to various human cancers.
  • Specific mutations in the DFG motif of BRAF, such as G596R, are known to be inactivating.
  • Understanding the molecular basis of these inactivating mutations is crucial for cancer therapy.

Purpose of the Study:

  • To investigate the molecular mechanisms behind the loss of function in the inactivating B-Raf G596R mutation.
  • To examine the formation of heterodimers between B-Raf DFG motif mutants and C-Raf.
  • To assess the functional consequences of B-Raf/C-Raf heterodimer formation.

Main Methods:

  • Wet molecular experiments were conducted in HEK293T cells.
  • Molecular dynamics simulations were employed to analyze protein structures and interactions.
  • Heterodimerization assays were performed to study B-Raf and C-Raf interactions.

Main Results:

  • The B-Raf G596R mutant was confirmed to be kinase-impaired.
  • Molecular dynamics revealed that Arg596 restrains Asp594, disrupting ATP binding and leading to loss of function.
  • Functional B-Raf/C-Raf heterodimers formed with all DFG B-Raf mutants, despite conformational differences.
  • The transforming activity of these mutants was found to be negligible.

Conclusions:

  • Novel insights into the functional properties of inactivating B-Raf DFG motif mutants were provided.
  • Mechanisms governing B-Raf/C-Raf heterodimerization and C-Raf transactivation were elucidated.
  • The study highlights that while heterodimerization occurs, the oncogenic potential of these specific mutants is limited.

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

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation15:05

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation

Published on: May 20, 2020

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:
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 Retinoblastoma Gene01:20

The Retinoblastoma Gene

Tumor suppressor genes are normal genes that can slow down cell division, repair DNA mistakes, or program the cells for apoptosis in case of irreparable damage. Hence, they play an essential role in preventing the proliferation of damaged cells.
The first-ever tumor suppressor gene called Rb was identified in retinoblastoma - a rare eye tumor in children. In inherited forms of the disease, a child inherits one defective copy of the Rb gene, which predisposes them to retinoblastoma. However,...
mTOR Signaling and Cancer Progression03:03

mTOR Signaling and Cancer Progression

The mammalian target of rapamycin or mTOR protein was discovered in 1994 due to its direct interaction with rapamycin. The protein gets its name from a yeast homolog called TOR. The mTOR protein complex in mammalian cells plays a major role in balancing anabolic processes such as the synthesis of proteins, lipids, and nucleotides and catabolic processes, such as autophagy in response to environmental cues, such as availability of nutrients and growth factors.
The mTOR pathway or the...