Fig. 4: GSK’157 activates GCN2 and induces eIF2α phosphorylation in vitro.
From: Activation of the integrated stress response by inhibitors of its kinases
a Immunoblots of indicated proteins from in vitro kinase reactions carried out with 7.5 nM GCN2, 2 μM eIF2α and indicated concentrations of ATP and incubated for 20 min at 30 °C. Active (A) and Inactive (I) GCN2 are indicated with arrows. Representative experiment from n = 3, biologically independent experiments. b Immunoblots of indicated proteins from reconstituted kinase reaction as in (a), with 6 μM ATP and indicated concentrations of GSK’157, without or with 10 μM of the GCN2 inhibitor A92 compound (last lane). Active (A) and Inactive (I) GCN2 are indicated with arrows. Representative experiment from n = 3, biologically independent experiments. c Ratio of P-eIF2α to eIF2α levels in immunoblots such as the ones shown in panel (b), normalised to 6 μM ATP conditions. Data are shown as mean ± SD (n = 3) with a Gaussian curve fitting, biologically independent experiments. ***p = 0.0009, ****p < 0.0001, as determined by one-way ANOVA with Dunnett’s multiple comparison test. d Structure of PERK kinase domain (KD) with GSK’157 bound to its ATP-binding pocket reported in ref. 11 compared with the structure of GCN2 KD from ref. 23 with GSK’157 fitted into the ATP-binding site. Yellow—PERK, blue—GCN2, green—GSK’157, red—residues in contact with GSK’157. e Melting temperature (Tm) of GCN2 kinase domain (KD) or pseudokinase domain (PKD) in the presence or absence of GSK’157 (100 μM) derived from Supplementary Fig. 9. f Immunoblots of indicated proteins from in vitro kinase reaction as in (a), (b), with 12 μM ATP and indicated concentrations of GCN2 ATP-competitive inhibitor A92. Active (A) and Inactive (I) GCN2 are indicated with arrows. Representative experiment from n = 2, biologically independent experiments. g Immunoblots of indicated proteins as in (a), (b), (f), from reconstituted in vitro kinase reaction with 12 μM ATP, with or without 0.04 μM A92 and with indicated concentrations of GSK’157. Active (A) and Inactive (I) GCN2 are indicated with arrows. Representative experiment from n = 2, biologically independent experiments. h Saturating concentrations of ATP-competitive PERK inhibitor (PERKi) occupy both ATP-pockets and inhibit GCN2 whilst sub-saturating concentrations of PERKi result in occupancy of one of the two ATP-binding sites of the GCN2 dimer and kinase activation. Source data are provided as a Source data file.
