Fig. 7: GSK’157 activates GCN2 by increasing its affinity for ATP.
From: Activation of the integrated stress response by inhibitors of its kinases
a Size-exclusion chromatography–multiangle light scattering (SEC-MALS) profile of in-house recombinant GST-GCN2 fragment (MW ~ 135 kDa) in presence of 50 μM GSK’157 or DMSO. The molecular weight measured by light scattering was 244–245 kDa in both conditions, consistent with a dimeric state of the recombinant protein. Representative experiment from n = 2, biologically independent experiments. b Immunoblots of GCN2 from in vitro kinase reactions with 7.5 nM GCN2 and indicated concentrations of ATP in presence of DMSO or 5 μM GSK’157. Active (A) and Inactive (I) GCN2 are indicated with arrows. Representative experiment from n = 4, biologically independent experiments. c Ratio of active to total (active + inactive) GCN2 in the reactions fit to Michaelis-Menten nonlinear model. Data are shown as mean ± SEM (n = 4, except 12 μM ATP n = 3 for GSK’157), biologically independent experiments. d Velocity (Vmax) and apparent affinity (Kobs) for GCN2 and ATP in presence of DMSO or 5 μM GSK’157 derived from panel (c). Data are shown as mean ± SEM (n = 4), biologically independent experiments. *p = 0.0385, **p = 0.0046, as determined by two-sided, unpaired t-test. Source data are provided as a Source data file.
