Fig 1: W→A mutation in both isoforms resists inactivation by MK-2206 in HeLa cells cultured under steady state conditions.A. HeLa cells were treated with 5μM MK-2206 for 3h before lysis. Cell lysates were analyzed for pAkt (S473), pan-Akt, and total and phosphoproteins for PRAS40, GSK3β, AS160, and FoxO3a by immunoblotting. GAPDH was blotted as a loading control. B and C. HeLa cells were either mock transfected (lanes 1, 2) or transfected with the WT (lanes 3, 4) or W→A (lanes 5, 6) constructs of HA-Akt1 (B), HA-Akt2 (C). Twenty-four hours post-transfection, cells were treated with DMSO or MK-2206 for 3h, lysed and the cell lysates were analyzed for pAkt (S473), HA and GAPDH (loading control) by immunoblotting.
Fig 2: Dose response of MK-2206 based inhibition of Akt in HEK293T cells.A. Structure of MK-2206. B. Schematic of the domain architecture of Akt isoforms. The N-terminal PH domain, central kinase domain and the C-terminal hydrophobic tail are shown. The residues important for MK-2206 binding (W80 in Akt1 and Akt2, W79 in Akt3) and for activation of the kinase (T308 and S473 in Akt1, T309 and S474 in Akt2 and T305 and S472 in Akt3) are indicated. The domains are not drawn to scale. Total number of residues in the three isoforms is indicated. C. HEK293T cells were treated with the indicated concentrations of MK-2206 for 3h before lysis. Cell lysates were analyzed for pAkt (T308 and S473), pan-Akt, and total and phosphoproteins for PRAS40, GSK3β, AS160, and FoxO3a by immunoblotting. GAPDH was blotted as a loading control.
Fig 3: In vitro phosphorylation of recombinant, purified PRAS40 by the W→A Akt isoforms.A and B. Lysates of HEK293T (A) or HeLa (B) cells transfected with the indicated Akt constructs were immunoprecipitated with the mouse monoclonal anti-HA antibody overnight. The immunoprecipitates were incubated with 0.5 μg of purified recombinant PRAS40 C-terminal fragment in the kinase assay buffer at 30°C for the indicated timepoints. A parallel reaction was set up in the kinase buffer without ATP and incubated for 90 minutes. The reactions were analyzed by immunoblotting for pPRAS40, PRAS40 and HA. C and D. The phospho-PRAS40 band was quantified and normalized to the corresponding total PRAS40 protein band in that reaction. The 90-minute timepoint was set at 1 and the fractional phosphorylation at the earlier timepoints was calculated and graphed. An ordinary one-way analysis of variance was performed to determine the statistical significance between the differences of means. Error bars represent SEM, n = 3, *p<0.05, **p<0.01.
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