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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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Assaying Protein Kinase Activity with Radiolabeled ATP
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MAPK Pathway Inhibition Reshapes Kinase Chemical Probe Reactivity Reflecting Cellular Activation States.

Andrew F Jarvis1, Mohd Younis Bhat1,2, Timothé Maujean1,2

  • 1Department of Cancer Biology, Perelman School, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.

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Summary
This summary is machine-generated.

Activity-based protein profiling (ABPP) reveals changes in amino acid reactivity in kinases, offering insights into cancer drug response and resistance. This chemical biology approach tracks kinase conformational dynamics and pathway adaptation in live cells.

Keywords:
ABPPBRAFMAPK pathwayMEKchemoproteomicskinases

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Area of Science:

  • Chemical Biology
  • Molecular Oncology
  • Proteomics

Background:

  • Oncogenic kinases are crucial in cancer, but profiling their activity and dynamics in live cells is difficult.
  • Current methods struggle to capture inhibitor-bound kinase states, limiting understanding of treatment response and resistance.
  • Genomic data alone is insufficient to fully understand kinase behavior in cancer.

Purpose of the Study:

  • To utilize activity-based protein profiling (ABPP) for monitoring kinase activity and conformational changes in live cancer cells.
  • To investigate how kinase inhibitor binding affects amino acid reactivity and cellular signaling.
  • To explore ABPP's potential in understanding cancer therapeutic resistance mechanisms.

Main Methods:

  • Employed activity-based protein profiling (ABPP) using electrophilic probes to assess amino acid reactivity (cysteine, lysine, carboxylic acid residues).
  • Applied ABPP to BRAFV600E mutant melanoma cells treated with kinase inhibitors (vemurafenib, trametinib, dabrafenib).
  • Analyzed changes in residue reactivity in response to inhibitor treatment, order of addition, and in resistant cell models.

Main Results:

  • Kinase inhibitor treatment decreased cysteine and lysine reactivity in BRAFV600E and MEK1/2, indicating inhibitor binding and conformational changes.
  • Varying probe and inhibitor addition order affected labeling, supporting competitive engagement and stabilization.
  • ABPP detected distinct labeling patterns with different BRAFV600E inhibitors, suggesting detection of inhibitor-specific conformations.
  • ABPP identified altered residue reactivity in resistant melanoma models, correlating with known resistance features.
  • Global proteome analysis revealed changes in KSR2 cysteine labeling, suggesting MAPK pathway remodeling.

Conclusions:

  • ABPP is a valuable chemical biology tool for studying inhibitor-dependent kinase residue reactivity in live cells.
  • ABPP provides a framework to investigate kinase conformational dynamics and signaling pathway adaptation in cancer therapy.
  • This approach can offer insights into how these factors influence therapeutic response and resistance.