Lipid-dependent Akt-ivity: where, when, and how

Abstract

Akt is an essential protein kinase activated downstream of phosphoinositide 3-kinase and frequently hyperactivated in cancer. Canonically, Akt is activated by phosphoinositide-dependent kinase 1 and mechanistic target of rapamycin complex 2, which phosphorylate it on two regulatory residues in its kinase domain upon targeting of Akt to the plasma membrane by PI(3,4,5)P₃ Recent evidence, however, has shown that, in addition to phosphorylation, Akt activity is allosterically coupled to the engagement of PI(3,4,5)P₃ or PI(3,4)P₂ in cellular membranes. Furthermore, the active membrane-bound conformation of Akt is protected from dephosphorylation, and Akt inactivation by phosphatases is rate-limited by its dissociation. Thus, Akt activity is restricted to membranes containing either PI(3,4,5)P₃ or PI(3,4)P₂ While PI(3,4,5)P₃ has long been associated with signaling at the plasma membrane, PI(3,4)P₂ is gaining increasing traction as a signaling lipid and has been implicated in controlling Akt activity throughout the endomembrane system. This has clear implications for the phosphorylation of both freely diffusible substrates and those localized to discrete subcellular compartments.

Keywords: 4; 4)P2; 5)P3; Akt; PI(3; kinase; lipids; phosphorylation/dephosphorylation.

Figure 1.. Akt at a glance – the primary effector of PI3K signaling.

PI3K can be activated by the engagement of either RTKs or GPCRs with growth factors or hormones, respectively. PI(3,4,5)P₃ production in the plasma membrane drives the translocation of PDK1 and Akt via their PH domains. PDK1 activates itself by homodimerization and trans-autophosphorylation, thereby permitting the activation of Akt by activation loop phosphorylation, aided by the regulatory phosphorylation of Akt by mTORC2. Akt is inactivated by PI(3,4,5)P₃ and PI(3,4)P₂ turnover, membrane dissociation, and dephosphorylation by the phosphatases PP2A and PHLPP.

Figure 2.. Switching Akt on and off.

(A) Domain schematic of human Akt1. Regulatory phosphorylation sites are highlighted in red. (B) Switching Akt on. Left panel – Akt activity in the cytosol is blocked by its PH domain. Docking of the PH domain to the kinase domain occludes substrate binding and sequesters the activation loop and probably also the hydrophobic motif in a conformation inaccessible to PDK1 and mTORC2. Middle panel – PI(3,4,5)P₃ generation in the plasma membrane leads to the binding of Akt and consequent displacement of its PH domain from its autoinhibited conformation, concomitantly exposing the activation loop and hydrophobic motif for phosphorylation. mTORC2 phosphorylation of the hydrophobic motif creates a docking site for PDK1, which subsequently mediates phosphorylation of the activation loop. Right panel – activation loop and hydrophobic motif phosphorylation drive a disorder-to-order transition of both segments, stabilized by ATP. The sequestration of both phosphorylated residues on the surface of the kinase domain protects them from dephosphorylation. (C) Switching Akt off. Left panel – Akt phosphorylated on both its activation loop and hydrophobic motif is primed for substrate binding and phosphorylation as long as Akt remains bound to PI(3,4,5)P₃ or PI(3,4)P₂. Middle panel – turnover of PI(3,4,5)P₃ and PI(3,4)P₂ by PTEN or PI(3,4)P₂ by INPP4 results in Akt dissociation and inactivation by its PH domain. Docking of the PH domain to the kinase domain displaces the phosphorylated activation loop and hydrophobic motif, rendering them accessible for dephosphorylation. Right panel – Akt is dephosphorylated in its activation loop and hydrophobic motif. PP2A and PHLPP have been identified as the respective phosphatases, but further work is required to determine precisely where and how Akt is dephosphorylated.

Figure 3.. Spatiotemporal dynamics of PI(3,4,5)P₃ and PI(3,4)P₂.

Left panel – Distribution of signaling phosphoinositides in the endomembranes of cells. PI(3,4,5)P₃- and PI(3,4)P₂-containing membranes are indicated in green and orange, respectively. Potential sites of Akt activity are expected to coincide with these lipids. Bona fide Akt substrates [50] are indicated in the black boxes at specific subcellular compartments. Substrates that exert their function in the nucleus, but are phosphorylated by Akt in the cytoplasm, are indicated with a dashed black box. Right panel – inter-conversion of phosphoinositides and the enzymes responsible.