Fig. 5.

PIP₃ binding promotes Akt hydrophobic motif exposure. (A) SAXS scattering curve for Akt1^ΔC. (B) Pair-distribution function (PDF) for Akt1^ΔC indicating the radius of gyration (R_g) and maximum dimension of the particle (D_max). (C) HDX-MS of Akt1^1P ± PIP₃ liposomes. Exposure (deprotection) of N-lobe peptide 218 to 225 indicated in red on structure of kinase domain. Plot: deuterium incorporation as a function of time for Akt1^1P ± PIP₃ liposomes. Deuterium incorporation plots were reproduced with permission. Adapted from ref. 20, which is licensed under CC BY-NC-ND 4.0. (D) HDX-MS of Akt1^1P versus Akt1^ΔC. Two regions showed significant increases in exchange (meeting the three criteria: ≥6% change in exchange, ≥0.4 Da difference in exchange, and a P value <0.01 using a two-tailed Student's t test). Regions 171 to 183 (YAMKILKKEVIVA) in the N-lobe and 260 to 274 (HSEKNVVYRDLKLEN) in the C-lobe are indicated in red on the structure of the kinase domain. Deuterium incorporation plots for these peptides as a function of time for Akt1^1P and Akt1^ΔC are shown to the right. (E) Plot of changes in deuterium incorporation between Akt1^1P and Akt1^ΔC. Changes in deuterium incorporation are plotted against the center of each peptide. Regions of protection and deprotection are indicated above the plot and correspond to those mapped in D. Error bars indicate the SD of three independent replicates. Red data points indicate increases or decreases in exchange that passed the three significance criteria. (F) Fluorescence anisotropy binding assay for C-terminal tail peptide (FITC-SMEAVDSERRPHFPQFSYSASGTA) to Akt1^ΔC. The K_D was estimated from three independent titrations. Each data point is the mean of 50 technical replicates with an integration time of 1 s. Error bars indicate the SD from the mean. Data were fit to a one-site binding model. (G) Composite model of full-length Akt1. The model comprises autoinhibited Akt1 (PH domain, PH-kinase linker, and kinase domain C-lobe), the N-lobe of active Akt1 (4EKK), and the phosphorylated C-terminal regulatory tail of PKCι (4DC2). The inactive conformation of the activation loop (unknown) is indicated with dashed magenta lines. (H) Stepwise activation of Akt by PIP₃ and phosphorylation. Activating steps are indicated with green arrows. Inactivating steps are indicated with red arrows. Phosphorylation state of each species in the activation and inactivation cycle is indicated in the blue boxes for each of the three regulatory residues: T308, T450, and S473.