Fig. 6.

C60 and C77 are required for Akt activation. a In vitro protein-lipid overlay assay. Aliquots of purified Akt2 PH domain (N-terminal 111 residues) was reduced or oxidized by DTT or H₂O₂, respectively, and then incubated with membranes spotted with serial dilutions of PIP3. Binding affinity was assessed by immunoblotting. b Quantification of in vitro protein-lipid overlay assay. Data are shown as mean ± SEM from n = 3. For oxidized Akt2 PH domain, Hillslope = 2.407, EC50 = 28.84; for reduced Akt2 PH domain, Hillslope = 1.908, EC50 = 52.30. p-value from two-way ANOVA. c Assessment of Akt translocation to the PM by TIRF. Adipocytes overexpressing TagRFPt-Akt2-WT or TagRFPt-Akt2-C60/77S were serum starved, followed by insulin treatments (1 nM and 100 nM). Data are shown as mean ± SEM. d Representative immunoblots of Akt2 phosphorylation at T309. 3T3-L1 fibroblasts overexpressing TagRFPt tagged Akt2 -WT, C60S, C77S or C60/77S were serum starved and then treated with MK-2206 (10 μM, 30 min) prior to stimulation with insulin. e Quantification of T309 phosphorylation. Data is mean ± SEM from n = 3. f Translocation of tagRFPt tagged Akt1 WT, C77F or C60/C77S to the plasma membrane in response to 1 nM or 100 nM insulin, measured by TIRFm. g, f Translocation of tagRFPt tagged Akt1 WT, C77F or C60/C77S PH domains to the plasma membrane in response to 1 nM or 100 nM insulin, measured by TIRFm. h Time series of the E17(O)-R86(N) distance in Akt1-C77F. Only the distance between the closest O–N atoms are shown. i Time series of the F55(CZ)-F77(CD1) distance in the C77F mutant Akt1. Equilibrium configuration is reached after 60 ns. j C77F hydrophobic interactions. The F77 side chain strongly interacts with the F55, I75 and I84 residues which form a hydrophobic pocket. k Residue specific RMSDs of the loop 1 residues 12-26 for the WT and C60/77S and protonated C60/77 mutant Akts.