Related Experiment Video

Updated: Jun 3, 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

Single substitution within the RKTR motif impairs kinase activity but promotes dimerization of RAF kinase

Angela Baljuls1, Regina Mahr, Inge Schwarzenau

  • 1Theodor-Boveri Institute of Bioscience, Department of Microbiology, University of Wuerzburg, Wuerzburg, Germany. angela.baljuls@mail.uni-wuerzburg.de

The Journal of Biological Chemistry
|April 2, 2011
PubMed

Insights

The RKTR motif in RAF kinases is crucial for their function. Mutations in this motif inhibit activity and affect RAF protein behavior, highlighting its importance in the MAPK pathway.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Protein Kinase Research

Background:

  • RAF kinases are central to the MAPK cascade, a critical cell signaling pathway.
  • RAF activity regulation is complex, involving membrane recruitment, protein interactions, and phosphorylation.
  • Previous work identified a potential interaction between the RAF N-region and the RKTR motif.

Purpose of the Study:

  • To investigate the role of the RKTR motif in RAF kinase function.
  • To determine the impact of specific residue substitutions within the RKTR motif on RAF activity and behavior.
  • To differentiate the effects of RKTR motif mutations across RAF isoforms (A-RAF, B-RAF, C-RAF).

Main Methods:

  • Site-directed mutagenesis to substitute basic residues within the RKTR motif.
  • Assays to measure RAF kinase catalytic activity.
  • Analysis of protein phosphorylation patterns.
  • Cellular fractionation to assess protein localization (plasma membrane).
  • Dimerization studies of RAF isoforms.

Main Results:

  • Single substitutions of basic residues in the RKTR motif inhibited catalytic activity in all RAF isoforms.
  • C-RAF and A-RAF showed different inhibition and phosphorylation patterns compared to B-RAF.
  • Substitution of the first arginine residue caused hyperphosphorylation and plasma membrane accumulation of A-RAF and C-RAF, suggesting impaired recycling.
  • All RAF isoforms exhibited similar behavior regarding RKTR motif-dependent dimerization.
  • Substitution of the second arginine residue led to significantly increased dimerization, potentially due to structural rearrangements.

Conclusions:

  • Each basic residue within the RKTR motif is essential for proper RAF kinase function.
  • The RKTR motif plays distinct roles in RAF isoform regulation, particularly concerning membrane association and recycling.
  • Mutations in the RKTR motif can induce structural changes affecting RAF dimerization, offering insights into RAF inhibitor mechanisms.

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

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors10:33

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

Published on: October 26, 2015

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...
Receptor Tyrosine Kinases01:26

Receptor Tyrosine Kinases

Receptor tyrosine kinases or RTKs are membrane-bound receptors that phosphorylate specific tyrosine on protein substrates. RTKs regulate cellular growth, differentiation, survival, and migration. They contain an extracellular ligand binding domain, a transmembrane domain, and a cytosolic tail with intrinsic kinase activity. Several extracellular signaling molecules activate RTKs in one or more ways and relay the signal downstream. Ligands such as platelet-derived growth factor (PDGF) or...
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:
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 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...
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...