 Akt1. Collectively, these results suggested that formation of the closed conformation of the Akt1 kinase domain is a prerequisite for ATP to exert allosteric protection of the pT308 site.
(A) Effects of T160A, F161A, E191A, H194R, E198A, R200A, K297A, and E298A mutants on phosphorylated Akt1. MyrAkt1 (myristoylated Akt1) and Akt1 mutants were transfected into HeLa cells. After 48 hr, cells were lysed for western blots. MyrAkt1, pT308-MyrAkt1, and GAPDH were analyzed by immunoblotting (upper panel). An unpaired (equal variance) t test was performed on the relative levels of pT308 in each MyrAkt1 mutant compared with that of MyrAkt1 wild-type (WT) (lower panel) (n = 3; ∗∗p < 0.01).
(B) The signal propagation pathway from ATP to pT308 in the ATP-bound Akt1.
(C) Schematic representation of the interactions of residue 191 with F161 and H194 in the WT, E191A, E191G, E191L, E191H, and E191W mutants. The generated steric hindrances in the E191L, E191H, and E191W mutants are colored red.
(D) Effects of E191G, E191A, E191L, E191H, and E191W mutants on phosphorylated Akt1. MyrAkt1 and mutants in both (A) and (D) were transfected into HeLa cells for 48 hr followed by immunoblot analysis of Akt1 T308 phosphorylation and HA-tagged MyrAkt1 expression.
See also Figures S3–S5.
NetworkView: 3D display and analysis of protein·RNA interaction networks.
) is supported by the energy change of H194⋯pT308 calculated by the two-layer ONIOM-based quantum mechanical/molecular mechanical method, with the interaction energy in the E191A mutant approximately −3.2 kcal/mol lower than in the WT (Text S2). Taken together, these data suggest that phosphatase resistance can be sufficient in the E191A mutant, more so than in the WT Akt1.
To further investigate the role of residue 191 side chain in the allosteric pathway from ATP to pT308, four additional mutants, E191G, E191L, E191H, and E191W, were constructed and tested. In the allosteric modeling, the lack of side chain in the E191G mutant abrogates the allosteric signal propagation from F161 to H194 (Figure 3C); larger volumes of side chains such as L191, H191, and W191 are not compatible with the cavity formed by F161 and H194 due to steric hindrance (Figure 3C). As expected, the E191G mutant showed a significant decrease in the pT308 level compared with WT Akt1 in our experimental test (Figure 3D), which is in good agreement with the theoretical model of allosteric communication from F161 to H194. In addition, protein expression of E191L, E191H, and E191W mutants was significantly reduced, and these effects are likely derived from structural instability due to steric conflicts. Thus, the combination of theoretical and experimental data lends support to the allosteric pathway from ATP to pT308, linking ATP binding and pT308 protection.
). We explored how the four mutations affected the dephosphorylation of pT308 by PP2A. The results showed that in the presence of ATP-γ-S, T160A/F161A/H194R mutants reduced the pT308 level compared with WT Akt1(144–443) while the E191A mutant effectively increased the resistance to T308 dephosphorylation (Figure S4), which is in good agreement with our full-length Akt1 results in HeLa cells (Figure 3A), suggesting that the identified allosteric pathway plays a protective role in the independent kinase domain.
), similar to the Akt1 R273H mutant observed here (Figure 5A). Conformational analysis of the MD trajectories indicated that the pT308 phosphate moiety lost salt bridges/H bonds with residues 194 and 273 (from 100% to 0%) when R273 was replaced by H273, and was exposed from the catalytic cleft throughout the MD trajectory (Figure 4A). This persistent exposure of pT308 may cause its enhanced dephosphorylation by the phosphatase in the pathology-related mutant.
(A) Details of the intramolecular interactions of pT308 with three residues in the catalytic cleft (H194, H273, and K297) of the Akt1 R273H mutant from 200 ns of MD simulation. The surface of pT308 is depicted by dots, and green dotted lines represent salt bridges or H bonds.
(B) Nearby residues including H194, K297, M306, K307, C310, and Y326 within a radius of 6 Å around pT308 are displayed in the Akt1 R273H mutant.
(C) In silico mutagenesis analysis of pT308-adjoining residues based on the Akt1 R273H mutant. For each mutant, MD simulation was carried out for 100 ns. The distances between the two Cα atoms of residues in positions 194 and 308 were measured in each mutant.
The results reported above revealed that exposed pT308 failed to resist dephosphorylation by phosphatase in the pathology-related mutant, unlike in the embedded protective state. To confirm the hypothesis, we designed a rescue strategy for pT308 in the Akt1 R273H mutant by enhancing the interactions between other residues and pT308. First, most of the residues in the vicinity of pT308 including H194, K297, M306, K307, C310, and Y326 (Figure 4B) were respectively changed to H, K, Q, R, W, and Y based on the R273H mutant, yielding a total of 32 double mutants in silico (Table S1). Then 100-ns MD simulations were performed on each to investigate the effect of these substitutions on the protective ability of pT308. Among these putative double mutants, only the H194R mutation could decrease the distance between the N and C lobes (Figure 4C) and the SASA of pT308 from 63.8% to 24.1%, providing a significant potential recovery of the phosphatase resistance of pT308 of the double mutant. Finally, the pT308 level of H194R/R273H double mutant was obtained and compared with the WT Akt1. The results indicated that the double mutant restored the WT pT308 level (Figure 5A). To avoid some non-specific function by the additional H194R mutation, single H194R and double H194R/R273S mutants were tested. Both mutants were incapable of increasing the pT308 level in Akt1 on their own (Figure 5A), revealing the restoration by the additional H194R mutation in the R273H mutant.
(A) Effects of H194R and R273H single mutants as well as H194R/R273H and H194R/R273S double mutants on phosphorylated Akt1. MyrAkt1 and mutants were transfected into HeLa cells. After 48 hr, cells were lysed for western blots. MyrAkt1, pT308-MyrAkt1, and GAPDH were analyzed by immunoblotting (upper panel). An unpaired (equal variance) t test was performed on the relative levels of pT308 in each MyrAkt1 mutant compared with that of MyrAkt1-WT (lower panel) (n = 3; ∗p < 0.05, ∗∗p < 0.01).
(B) Details of the intramolecular interactions of pT308 with three residues in the catalytic cleft (R194, H273, and K297) of the Akt1 H194R/R273H double mutant from 200 ns of MD simulation. The surface of pT308 is depicted by dots, and green dotted lines represent salt bridges or H bonds.
(C) The signal propagation pathway from ATP to pT308 in the Akt1 H194R/R273H double mutant.
To elucidate the reconstructed protection of pT308 in the H194R/R273H double mutant, the H194R/R273H MD simulation was extended to 200 ns and analyzed. As shown in Figure 5B, the phosphate moiety of pT308 formed two salt bridges with R194 and K297, and H273 formed an H bond with the backbone carbonyl group of pT308. These intramolecular interactions shielded the phosphate moiety of pT308 in the catalytic cleft, stabilized thereby the closed conformation of the kinase domain in the H194R/R273H double mutant, and rebuilt the allosteric communication from ATP to pT308 in the H194R/R273H double mutant (Figure 5C).
). At the membrane, T308 of Akt1 undergoes phosphorylation by PDK1. To test whether the mutations in the allosteric pathway impair the recruitment of PDK1 or phosphatase PP2A, the consequences of Akt1 mutations (T160A, F161A, E191A, H194R, and H194R/R273H) on pT308 levels were measured in the presence of the PP2A inhibitor Calyculin A and PDK1 inhibitor BX-795. The results showed that similar to changes in WT Akt1 (Figure 6A), pT308 levels in all mutants were markedly increased in the presence of Calyculin A, which was counteracted by co-treatment with BX-795 (Figure 6B). This result suggested