Figure 1. Constitutive dephosphorylation resistance of phospho-PKA, but not phospho-Akt, in a cell-free assay.

(A) Superimposition of Akt kinase domain (Akt2–Mn–AMP-PNP–G–SK3, green, PDB code 1o6k [28]) and PKA kinase domain (PKA–Mn–ATP–PKI, brown, PDB code 1cdk [88]). In the presence of bound ATP analogues, the phosphorylated activation loops (Thr³⁰⁸ in Akt1, Thr¹⁹⁷ in PKA) shown are stabilized by invariable histidine (His¹⁹⁴ in Akt1, His⁸⁷ in PKA) and arginine (Arg²⁷³ in Akt1, Arg¹⁶⁵ in PKA) residues. The structure was modelled on active human Akt2 crystal structures bound to the ATP analogue AMP-PNP and Mn^{2 +} (PDB code 1o6k). Since most of our biochemical studies were performed with Akt1, we indicate the corresponding Akt1 amino acid residues in the structure for clarity. (B) Cell extract dephosphorylation of Akt and PKA. H9C2 cells expressing constitutively phosphorylated Myr-Akt1-HA and phosphorylated endogenous PKA were flash-frozen and extracted on ice. Aliquots of cell extracts without phosphatase inhibitors were incubated at 30°C for 90 min. After incubation, protein extracts were subjected to immunoblot analysis using antibodies detecting phosphorylated Akt (Thr³⁰⁸), HA-tagged Akt, phosphorylated PKA (Thr¹⁹⁷) and total PKA. (C) Recombinant phospho-PKA catalytic subunit, but not that of recombinant phospho-Akt, resists dephosphorylation. Recombinant Akt1 and PKA (50 ng) were incubated with PP2A (70 ng of PP2A-C) or pan-substrate bacteriophage λ phosphatase (40 units) for 60 min at 30°C. Images show Akt1 Thr³⁰⁸ phosphorylation, total Akt, PKA Thr¹⁹⁷ phosphorylation and total PKA. The histograms show the percentage of pThr³⁰⁸ or pThr¹⁹⁷ dephosphorylation. n=4/group. *P < 0.05 compared with PKA dephosphorylation by PP2A.