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. 2011 Oct 26;108(46):E1120-E1127. doi: 10.1073/pnas.1109879108

Fig. 2.

Fig. 2.

A443654 or ATP binding inhibits Akt dephosphorylation. (A) A443654 inhibits MyrAkt1 dephosphorylation in cell extracts. MyrAkt1 expressing Hela cells were flash-frozen and extracted in ice. Aliquots of cell extracts were treated with A443654 or calyculin on ice before incubation at 30 °C for 15 min. After incubation, protein extracts were subjected to immunoblot analysis using antibodies detecting phosphorylated Akt (T308), GSK3β (S9), and Pras40 (T246) and GAPDH. (B) A443654 inhibits recombinant PP2A-mediated Akt dephosphorylation. Immunoprecipitated MyrAkt1 was incubated with recombinant PP2A (40 ng PP2A-C) for 30 min, 30 °C in the presence of A443654 (2–20 uM). Images show MyrAkt1 T308 phosphorylation, HA-tagged MyrAkt1 and PP2A-C expression. Graph shows the percentage pT308 dephosphorylation. *P < 0.001 for PP2A vs. PP2A+A44 at 30 min. (C) ATP inhibits recombinant PP2A-mediated Akt dephosphorylation in the presence of Akt-specific pseudosubstrate peptide (I-T15A). Immunoprecipitated MyrAkt1 was incubated with recombinant PP2A for 30 min, 30 °C in a buffer containing 5 mM MgCl2, 400 uM sonicated lipid micelles, 25 uM high affinity Akt pseudosubstrate inhibitor (I-T15A) and increasing concentrations of ATP (0, 20 uM or 100 uM) as indicated. Immunoblot images show MyrAkt1 T308 and S473 phosphorylation, HA-tagged MyrAkt1 and PP2A-C expression. Graph shows the percentage pT308 dephosphorylation in the presence PP2A alone (n = 11), PP2A with 100 uM ATP (n = 8), with 25 uM I-T15A peptide (n = 3) or both (n = 7). *P < 0.001 vs. PP2A, #P < 0.05 vs. PP2A/ATP, ^P < 0.05 vs. PP2A/I-15A (Nonparametric ANOVA, Kruskal-Wallis).