Fig. 2.

Akt1 prepared by protein semisynthesis lacks a phosphorylated turn motif. (A) Phosphorylation state analysis of Akt1 after expressed protein ligation and in vitro phosphorylation with PDK1. Tandem mass spectrometry of pepsin digest. Missing turn motif phosphorylation highlighted in red. (B) Thermal stability analysis of Akt1 by differential scanning fluorimetry. Black curves, monophosphorylated Akt1^1P; red curves, diphosphorylated Akt1^2P. Solid lines, +1 mM ATP; dashed lines, no ATP. EPL, expressed protein ligation. (C) Liposome pelleting assay of Akt1^2P and Akt1^1P in the presence of 0% and 5% PIP₃-containing liposomes. (D) Akt1 kinase assay in the presence of liposomes containing 0 or 5 mol % PI(3,4,5)P₃, ± 10 μM MK-2206 (added postliposome binding). Diphosphorylated (T308/S473) Akt1^2P, red bars; Akt1^1P, black bars.