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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...

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Related Experiment Video

Updated: Jul 14, 2026

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells
06:44

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

A selective cellular screening assay for B-Raf and c-Raf kinases.

Tsuyoshi Ish1, Hiroshi Sootome, Yukiko Yagi

  • 1GlaxoSmithKline K.K., Ibaraki, Japan. tsu_ishii01@yahoo.co.jp

Journal of Biomolecular Screening
|May 23, 2007
PubMed
Summary

Researchers developed a B-Raf-specific cellular assay to profile inhibitors targeting the Ras/Raf pathway crucial in cancer. This assay enables quantitative determination of inhibitor selectivity for B-Raf and c-Raf kinases, aiding drug discovery.

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Identification of Kinase-substrate Pairs Using High Throughput Screening
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Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Related Experiment Videos

Last Updated: Jul 14, 2026

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells
06:44

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

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

Identification of Kinase-substrate Pairs Using High Throughput Screening
11:13

Identification of Kinase-substrate Pairs Using High Throughput Screening

Published on: August 29, 2015

Area of Science:

  • Oncology
  • Molecular Biology
  • Biochemistry

Background:

  • The Ras/Raf signaling pathway is integral to cancer biology.
  • Activating BRAF mutations are prevalent in melanoma (66%) and other cancers.

Purpose of the Study:

  • To develop a B-Raf-specific cellular assay for profiling cell-active B-Raf inhibitors.
  • To establish a platform for quantitatively determining inhibitor selectivity against B-Raf and c-Raf kinases.

Main Methods:

  • Developed a cellular assay using mifepristone-regulated expression of active B-Raf (V600E) and kinase-inactive MEK1.
  • Monitored B-Raf catalytic activity via MEK1 phosphorylation.
  • Established a parallel c-Raf assay to assess kinase selectivity.
  • Validated assay robustness with Z' factor values consistently above 0.50.

Main Results:

  • Demonstrated a B-Raf-specific cellular assay capable of profiling inhibitors.
  • Successfully established a c-Raf assay for selectivity profiling.
  • Quantitatively determined the selectivity profiles of compounds targeting B-Raf and c-Raf kinases.
  • Confirmed robust assay performance suitable for drug discovery.

Conclusions:

  • The developed platform provides a quantitative cellular readout for B-Raf and c-Raf kinase inhibitors.
  • This system is highly suitable for drug discovery efforts targeting the Ras/Raf pathway.