N the cell lysates are also shown. B HA-epitope-tagged BRAFV600E was transfected into HEK293 cells. Right after 24 h, cells have been treated with 2DG (11 mM) and/or metformin (Met; ten mM) for 3 h. HA-tagged BRAFV600E was immunoprecipitated (IP) with HA antibody, and the immunocomplexes had been Western-blotted for HA, endogenous KSR1, and endogenous AMPKa. HA, endogenous KSR1, phospho-AMPKa T172 (pAMPKa), and total AMPKa levels inside the cell lysates are also shown. C HA-epitope-tagged BRAFV600E and V5-epitope-tagged KSR1WT were transfected into HEK293 cells; 6 h later, one of the transfected dishes was transfected with an AMPKa siRNA. After 48 h, cells were treated with 2DG (11 mM) and rotenone (Rot; five lM) for 2 h. HA-tagged BRAFV600E was immunoprecipitated (IP) with HA antibody, as well as the immunocomplexes were Western-blotted for HA and V5. HA, V5, phospho-ERK1/2 (pERK1/2), and total ERK1/2 levels within the cell lysates are also shown. The efficiency in the AMPKa knockdown is shown within the reduce panel. D Among the dishes of A375 cells was transfected with an AMPKa siRNA. Soon after 48 h, cells have been treated with 2DG (5.5 mM) and rotenone (Rot; five lM) for 2 h. Endogenous BRAFV600E was immunoprecipitated (IP), along with the immunocomplexes have been Western-blotted for endogenous BRAFV600E and endogenous KSR2. Endogenous BRAFV600E and KSR2 levels inside the cell lysates are also shown. The efficiency from the AMPKa knockdown is presented inside the reduced panel. E MelJuso cells had been treated with 2DG (11 mM) and/or rotenone (Rot; 5 lM) and/or metformin (Met; ten mM) for two h. Endogenous CRAF was immunoprecipitated (IP), plus the immunocomplexes have been Western-blotted for endogenous CRAF, KSR2, and AMPKa. Endogenous CRAF, KSR2, phospho-AMPKa T172 (pAMPKa), total AMPKa, phospho-ERK1/2 (pERK1/2), and total ERK1/2 levels within the cell lysates are also shown. Source data are accessible on the net for this figure.EMBO reports Vol 19 | No two |2017 The AuthorsAmandine Verlande et alMetabolic stress controls KSR-RAF dimersEMBO reportshas been demonstrated that a sustained activation of your ERK pathway is required for G1 to S phase progression [35,36]. On the other hand, the duration along with the magnitude from the activation both have to be controlled as not merely the inhibition but additionally as well powerful activation of this pathway can bring about a reversible or permanent cell cycle arrest [37,38]. In this study, we showed that 2DG promoted a strongeractivation in the ERK pathway in MelJuso than in A375 cells (Figs 1A and 4A, and EV3A) and the difference became even more prominent when the level of metabolic tension was greater (Fig 4F). These data suggested that in MelJuso cells, the enhanced ERK pathway activation after 2DG may be higher enough to induce cell cycle arrest in G0/G1 phase, but not in A375 where the activation wasAHA-BRAFV600E2DG 2DG 2DG Rot MetBHA-BRAFV600E2DG 2DG MetKSR1 AMPK HA IP HA-BRAFV600EKSR1 AMPK HA IP HA-BRAFV600EKSR1 HA Lysates pAMPKKSR1 HA Lysates pAMPKAMPKAMPKCHA-BRAFV600E V5-KSR1WTD2DG Rot 2DG + 2DG Rot AMPK siRNAE2DG 2DG 2DG Rot MetKSR2 KSR2 AMPK IP BRAFV600E CRAF IP CRAFV5 IP HA KSR2 V5 BRAFV600E HA HA-BRAFV600E BRAFV600EKSR2 CRAF Lysates pERK1/2 Lysates ERK1/2 LysatespERK1/pAMPK AMPK -tubulin AMPKERK1/AMPK-tubulinFigure 5.2-Chloro-5-fluoro-6-methylpyridine custom synthesis 2017 The AuthorsEMBO reports Vol 19 | No 2 |EMBO reportsMetabolic tension controls KSR-RAF dimersAmandine Verlande et alABCDMelJuso 2DG p21Cip1 -tubulinEA375 – 2DGF2DG Rot 2DG RotGHCleaved PARP PARP Cleaved Caspase 3 Caspase 3 Cleaved Caspase 9 CaspaseCytosolic fractionCytochrome c -tubuli.2-Chloro-5,7-difluorobenzo[d]thiazole Data Sheet PMID:23710097
