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).

Mutating Akt1 H194 and R273 abolishes ATP-mediated dephosporylation resistance. (A) Schematic view of the Akt1 catalytic cleft showing the nucleotide-binding pocket with bound ATP and Mg²⁺ and phosphorylated T308 interacting with H194 and R273 (19). The structure shown is modeled on active human Akt2 crystal structures bound to ATP analog AMP-PNP and Mn²⁺ (PDB 1o6k). However, we denoted ATP and Mg²⁺ and corresponding Akt1 amino acid residues for clarity. (B) MyrAkt1 H194A and R273A mutants were transfected into Hela cells. After 48 h, cells were propagated for four additional hours in exogenous growth factor-free medium followed by adding calyculin phosphatase inhibitor for 15 min. Akt T308 and S473 phosphorylation and HA-tag was analyzed by immunoblotting. (C) R273A mutation in WT Akt1 and in MyrAkt1 abolished ATP-mediated dephosphorylation resistance. Prephosphorylated MyrAkt1, MyrAkt1-R273A, or WT-Akt1-R273A were immunoprecipitated and incubated with recombinant PP2A in the presence of 100 uM ATP or 100 uM ATPγS for 30 min, 30 °C. Akt expression, T308 phosphorylation, and PP2A-C expression were analyzed by immunoblotting. (D) Quantification of T308 phosphorylation in MyrAkt1 mutants after PP2A treatment. Graph shows the percentage pT308 dephosphorylation by PP2A normalized to total Akt expression (Dephos. %) for MyrAkt (n = 5–7), H194A mutant (n = 3), R273A mutant (n = 6), and H194A/R273A mutant (n = 2–3). *P < 0.001 vs. PP2A for MyrAkt group. Not significant differences for H194A, R273A and H194A/R273A groups.

The “Phosphatase Shielding Cage” induced by ATP binding of Akt. (A) Structural representation of the closed Akt activation loop conformation in the presence of substrate peptide and ATP. Under these conditions phosphorylated T308 is stabilized by H194 and R273 interactions. The structure is modeled on active human Akt2 crystal structures bound to ATP analog AMP-PNP and Mn²⁺ (PDB 1o6k). However, we indicated ATP and Mg²⁺ and corresponding Akt1 amino acid residues for clarity. (B) Stable interactions with H194 and R273 prevent access of phosphatases to phosphorylated T308. (C) Abolishing either pT308 interactions (H194A and R273A) or ATP binding (K179M) renders pT308 sensitive to phosphatase attack.

ATP binding site occupancy regulates Akt1 dephosphorylation. (A) H9C2 cells transfected with HA-tagged MyrAkt1 were incubated for 4 h in medium free of exogenous growth factors before adding 1 ug/mL insulin for 30 min. Protein extracts were subjected to immunoblot analysis using antibodies to phospho-Akt T308 and GAPDH. (B) MyrAkt1 expressing H9C2 cells were incubated in growth factor-free medium with 10 uM VIII (allosteric Akt inhibitor) in the presence or absence of 10 uM A-443654, 100 nM calyculin or 2.5 uM okadaic acid for 30 min. Protein extracts were subjected to immunoblot analysis using phospho-Akt T308 antibody. (C) H9C2 cells grown in exogenous growth factor-free medium were stimulated with 1 ug/mL insulin for 20 min followed by 10 min in growth factor-free media supplemented with 10 uM A-443654, 200 nM wortmannin (PI-3 kinase inhibitor) or 0.5 uM UCN01 (PDK1 inhibitor) as indicated. Levels of Akt phosphorylation on T308 and S473, total Akt and GAPDH were determined by immunoblotting. Quantification shows percentage of Akt dephosphorylation (Dephos. %). (D) MyrAkt1 was mutated to ablate either ATP binding (K179M) or T308 phosphorylation (T308A). MyrAkt1 and mutants were transiently expressed in H9C2 cells for 48 h followed by immunoblot analysis of Akt T308 phosphorylation and HA-tag expression. (E) K179M mutation did not alter intracellular MyrAkt1 localization. H9C2 cells expressing MyrAkt1-HA and K179M mutant were fixed and stained. Immunofluorescence staining of cells with anti-HA-tag antibody (green). 60X whole cell images (small) and enlarged portion of the yellow square image are shown.

Differential regulation of Akt dephosphorylation by different nucleotides. (A) Effects of ATP and ADP analogs on MyrAkt1 dephoshorylation. Immuno-purified MyrAkt1 was incubated with recombinant PP2A, 25 uM I-T15A inhibitor peptide and as indicated, 100 uM ATP, 20 uM A-443654, 100 uM ATPγS, 100 uM ADP or 100 uM ADPβS. Akt expression and phosphorylation and PP2A-C expression were analyzed by immunoblotting. Quantification shows the percentage of decrease in pT308 phosphorylation (Dephos. %) relative to untreated control after 30 min at 30 °C. (B) ATP or A443654 do not inhibit phospho-PRAS40 dephosphorylation. Hela cells expressing HA-tagged PRAS40 were stimulated with 100 uM Per-VO4 for 15 min to induce phosphorylation. Immunopreciptated phospho-PRAS40 was incubated with recombinant PP2A with 100 uM ATP/25 uM I-T15A peptide or 20 uM A-443654 as indicated. PRAS40 phosphorylation (Thr246), total PRAS40, and PP2A-C expression were analyzed by immunoblotting. (C) ATP and A443654 inhibit WT Akt1 dephosphorylation. HA-tagged WT-Akt1 or Flag-Tagged WT-Akt were prephosphorylated with 100 uM Per-VO4 stimulation and immunoprecipitated with anti-Flag beads or ant-HA beads. Phospho-WT-Akt was incubated with recombinant PP2A in the presence of 100 uM ATP or 10 uM A-443654 as indicated. T308 phosphorylation, total Akt1 and PP2A-C expression were analyzed by immunoblotting.

(A) MD simulations compared the differences in the dynamics of the ATP and ADP bound Akt conformations and followed changes in H194 and pT308 interactions. The ATP and ADP complexes with Mg²⁺ ions were modeled into the crystal structure of AKT kinase bound to AMP-PNP (1o6 k). H194 and pT308 interactions were measured by the distance of a hydrogen bond from the E2 hydrogen to the phosphate oxygen. The first 1 ps of the simulation was for equilibration and the ensemble average distance was calculated over 1–10 ps. Images show modeled H194 to pT308 distance at 10 ps (green lines). (B) An Akt2 mutation (R274H) associated with human diabetes type II abolishes ATP-mediated dephosporylation resistance. MyrAkt2-R274H was transfected into Hela cells. After 48 h, cells were propagated for four additional hours in growth factor-free medium followed by adding 50 nM calyculin to inhibit cellular phosphatases for 15 min. Cell extracts were analyzed for Akt T309 phosphorylation and GAPDH by immunoblotting. (C) MyrAkt2 R274H mutant fails to resist dephosphorylation in the presence of ATP or ATPγS. Prephosphorylated MyrAkt2 or R274H mutant proteins were incubated with recombinant PP2A in the presence of 100 uM ATP or 100 uM ATPγS for 30 min, 30 °C. Akt expression and phosphorylation and PP2A-C expression were analyzed by immunoblotting.

The “Phosphatase Shielding Cage” induced by occupancy of the ATP-binding site of Akt kinases. (A) Structural representation of the “closed” Akt1 conformation in which phosphorylated threonine 308 is stabilized by intramolecular interactions with histidine 194 and arginine 273 in the presence of substrate peptide and ATP. Immunoblots showed that MyrAkt1, but not the arginine 273A mutant, resisted PP2A phosphatases in an ATP or ATPγS-dependent manner, suggesting arginine 273A mutation abolishes ATP-mediated dephosphorylation resistance. (B) Schematic model of sensitization of phosphorylated threonine 308 to phosphatase attack by disrupting either pthreonine 308 interactions (Akt1 mutants histidine 194A and arginine 273A) or ATP binding (Akt1 mutant K179M).