Metabolic stress regulates ERK activity by controlling KSR‐RAF heterodimerization

Abstract

Altered cell metabolism is a hallmark of cancer, and targeting specific metabolic nodes is considered an attractive strategy for cancer therapy. In this study, we evaluate the effects of metabolic stressors on the deregulated ERK pathway in melanoma cells bearing activating mutations of the or oncogenes. We report that metabolic stressors promote the dimerization of KSR proteins with CRAF in NRAS-mutant cells, and with oncogenic BRAF in BRAF-mutant cells, thereby enhancing ERK pathway activation. Despite this similarity, the two genomic subtypes react differently when a higher level of metabolic stress is induced. In NRAS-mutant cells, the ERK pathway is even more stimulated, while it is strongly downregulated in BRAF-mutant cells. We demonstrate that this is caused by the dissociation of mutant BRAF from KSR and is mediated by activated AMPK. Both types of ERK regulation nevertheless lead to cell cycle arrest. Besides studying the effects of the metabolic stressors on ERK pathway activity, we also present data suggesting that for efficient therapies of both genomic melanoma subtypes, specific metabolic targeting is necessary.

The MAPK and AMPK signalings: interplay and implication in targeted cancer therapy

“…It is well established that the hetero-/homo-dimerization of RAF/KSR family kinases plays a determinant role in the activation of MAPK signaling, which requires the association of 14-3-3, a dimeric scaffold protein with their carboxyl-terminus [ 228 , 229 ]. Mechanistic studies have revealed that a 14-3-3 dimer associates with the C-terminus of two individual RAF/KSR molecules and facilitates their dimerization and subsequent activation [ 25 , 230 , 231 ] (Fig. 4 a).…”

“…This molecular mechanism may also be responsible for the hyperactive MAPK signaling induced by metabolic stress in Ras-mutated melanoma. Upon metabolic perturbations, AMPK is activated in this type of melanoma cells and promotes KSR/CRAF heterodimerization likely through altering 14-3-3 binding manners, which leads to a highly activated MAPK signaling [ 230 ]. Besides CRAF and KSR, the association of BRAF with 14-3-3 is also regulated by AMPK-mediated phosphorylation.…”

Dual Targeting of BRAF and mTOR Signaling in Melanoma Cells with Pyridinyl Imidazole Compounds

“…Melanoma cells (A375) were seeded at 10 cm plate, washed once with ice-cold phosphate-buffered saline (PBS), scraped out, and lysed as previously described [ 82 , 83 ]. The lysate was centrifugated at 13,000× g for 25 min at 4 °C.…”

“…Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) were added and incubated for another 2–3 h at 4 °C on a slow rotator. Beads–immune complexes were washed as previously described [ 82 , 83 ]. The kinase assay was performed in the presence of 20 µM ATP and 500 ng of a recombinant human inactive MEK-1 as the substrate (Life Technologies, Prague, Czech Republic).…”

“…Acetonitrile extraction was employed to quench cell metabolism and to extract low molecular weight compounds from A375 melanoma cells quantitatively [ 83 , 88 ]. Following removal of acetonitrile via vacuum concentration, dried extracts were resuspended in 550 μL of D 2 O (Sigma-Aldrich) containing 0.005% sodium 3-(trimethylsilyl)-propionate-2,2,3,3-d4 (TSP) (Sigma-Aldrich) used as both chemical shift reference and internal standard for metabolite quantification.…”