Phosphomimetic Constructs of Akt/PKB do not Mimic Activated Akt in Cells

Akt/protein kinase B is a serine/threonine kinase of the AGC family of kinases which plays a key role in signaling downstream of phosphatidylinositol 3‐kinase. Akt is activated following localization to PIP 3 via its PH domain, and once recruited to the plasma membrane, Akt is phosphorylated at Thr308 within its activation loop by its upstream kinase PDK1 followed by phosphorylation at the C‐terminal hydrophobic site, Ser473. Here we show that phosphomimetic substitutions at these positions do not mimic the global signaling output of phosphorylated Akt. Specifically, we use live cell imaging with a FRET‐based Akt activity reporter, B Kinase Activity Reporter (BKAR), to show that constructs of Akt with Asp at position 308, or Asp at both Thr308 and Ser473, are neither basally active nor activatable following stimulation of COS cells with agonists such as EGF. In contrast, wild‐type Akt is robustly activated following EGF stimulation. Consistent with the lack of activity of Akt‐T308D revealed by live cell imaging, Western blot analysis using a phospho‐specific antibody to Akt consensus substrate phosphorylation sites also reveals no basal or EGF‐stimulated phosphorylation by the phosphomimetic construct. Although some previous reports indicate that substitution with Asp at both sites yields an active kinase, our data reveal that Asp is a poor phosphomimetic at the key regulatory sites of Akt, thus one should exercise caution in the use of such constructs to examine the biology of Akt signaling. Support or Funding Information This work was supported by NIH P01 DK5444.