Global view of the RAF-MEK-ERK module and its immediate downstream effectors

Sci Rep. 2019 Jul 26;9(1):10865. doi: 10.1038/s41598-019-47245-x.

Abstract

Small molecule inhibitors of BRAF and MEK have proven effective at inhibiting tumor growth in melanoma patients, however this efficacy is limited due to the almost universal development of drug resistance. To provide advanced insight into the signaling responses that occur following kinase inhibition we have performed quantitative (phospho)-proteomics of human melanoma cells treated with either dabrafenib, a BRAF inhibitor; trametinib, a MEK inhibitor or SCH772984, an ERK inhibitor. Over nine experiments we identified 7827 class I phosphorylation sites on 4960 proteins. This included 54 phosphorylation sites that were significantly down-modulated after exposure to all three inhibitors, 34 of which have not been previously reported. Functional analysis of these novel ERK targets identified roles for them in GTPase activity and regulation, apoptosis and cell-cell adhesion. Comparison of the results presented here with previously reported phosphorylation sites downstream of ERK showed a limited degree of overlap suggesting that ERK signaling responses may be highly cell line and cue specific. In addition we identified 26 phosphorylation sites that were only responsive to dabrafenib. We provide further orthogonal experimental evidence for 3 of these sites in human embryonic kidney cells over-expressing BRAF as well as further computational insights using KinomeXplorer. The validated phosphorylation sites were found to be involved in actin regulation, which has been proposed as a novel mechanism for inhibiting resistance development. These results would suggest that the linearity of the BRAF-MEK-ERK module is at least context dependent.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Cell Line, Tumor
  • Drug Resistance, Neoplasm / drug effects
  • Extracellular Signal-Regulated MAP Kinases / antagonists & inhibitors*
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Humans
  • Imidazoles / pharmacology*
  • Indazoles / pharmacology*
  • MAP Kinase Signaling System / drug effects
  • Melanoma / metabolism*
  • Melanoma / pathology
  • Mitogen-Activated Protein Kinase Kinases / antagonists & inhibitors*
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Oximes / pharmacology*
  • Phosphorylation / drug effects
  • Piperazines / pharmacology*
  • Proteome
  • Proteomics / methods
  • Proto-Oncogene Proteins B-raf / antagonists & inhibitors*
  • Proto-Oncogene Proteins B-raf / metabolism
  • Pyridones / pharmacology*
  • Pyrimidinones / pharmacology*
  • Skin Neoplasms / metabolism*
  • Skin Neoplasms / pathology

Substances

  • Imidazoles
  • Indazoles
  • Oximes
  • Piperazines
  • Proteome
  • Pyridones
  • Pyrimidinones
  • SCH772984
  • trametinib
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Extracellular Signal-Regulated MAP Kinases
  • Mitogen-Activated Protein Kinase Kinases
  • dabrafenib