Related Experiment Video

Updated: Jul 7, 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

The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits

Oliver Rath1, Sungdae Park, Hui-hui Tang

  • 1The Beatson institute for Cancer Research, Cancer Research UK Garscube Estate, Bearsden, Glasgow G61 1BD, UK.

Cellular Signalling
|February 26, 2008
PubMed

Insights

The Raf-MEK-ERK pathway

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • The Raf-MEK-ERK pathway is crucial for cellular processes.
  • Raf kinase inhibitory protein (RKIP) negatively regulates this pathway.
  • RKIP inhibits MEK phosphorylation by Raf-1 via complex formation.

Purpose of the Study:

  • To investigate the role of RKIP's conserved ligand-binding pocket in its inhibitory function.
  • To determine if specific residues within the pocket are essential for RKIP activity.

Main Methods:

  • Site-directed mutagenesis to create RKIP loss-of-function mutants.
  • Analysis of RKIP-Raf-1 complex formation and stability.
  • Assessment of RKIP's inhibitory effect on Raf-1 mediated MEK activation.

Main Results:

  • The conserved ligand-binding pocket of RKIP is essential for inhibiting Raf-1 kinase activity.
  • Mutations in key pocket residues result in loss-of-function RKIP.
  • Mutants bind Raf-1 but show reduced complex stability with a Raf-1 phosphopeptide.

Conclusions:

  • RKIP's conserved pocket is critical for its function in the Raf-MEK-ERK pathway.
  • The pocket may act as a novel phosphoamino-acid binding motif.
  • This binding is necessary for RKIP's inhibitory mechanism.

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

Assaying Protein Kinase Activity with Radiolabeled ATP08:05

Assaying Protein Kinase Activity with Radiolabeled ATP

Published on: May 26, 2017

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...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
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...
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...
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...