Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo

Abstract

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention.

Akt2 interacts with Snail1 in the E-cadherin promoter

“…Binding of Snail1 requires the PH domain of Akt, similarly to other reported interactors (Sato et al, 2000;Jahn et al, 2002). It is possible that this interaction stabilizes the active Akt conformer, preventing the intramolecular association between the PH and the kinase domains (Calleja et al, 2007). Regarding Snaill1, and in contrast to its binding with other corepressors, the association with Akt is independent of the SNAG sequence but requires the C-terminal domain of Snail1 protein.…”

Analysis of 3-phosphoinositide-dependent kinase-1 signaling and function in ES cells

“…Fig. 2 shows that PDK1 LG is only slightly compromised compared to WT PDK1 in its to phosphorylate ΔPH-PKB/Akt, a PH domain deleted mutant of PKB/Akt that can be phosphorylated by PDK1 in the absence of PIP 3 . Importantly, all the analogues tested strongly inhibited the activity of PDK1 LG, whereas WT PDK1 was not inhibited, or only inhibited 50%.…”

“…Once activated, PI3K synthesizes the lipid second messenger phosphatidylinositol-3,4,5-triphosphate (PIP 3 ), which in turn acts as a docking site at the plasma membrane that recruits 3-phosphoinositide dependent kinase 1 (PDK1). PDK1 is a serine/threonine kinase that was originally identified as the kinase that phosphorylates the activation loop of protein kinase B (PKB)/Akt (T308) in the presence of PIP 3 [1,2].…”

“…PIP 3 recruits PKB/Akt and PDK1 to the membrane by binding their pleckstrin homology (PH) domains. This colocalizes the two enzymes and is thought to lead to a conformational change in PKB/Akt allowing PDK1 to phosphorylate the activation loop of PKB/Akt [3]. PDK1 has subsequently been shown to phosphorylate and activate a whole group of related protein kinases belonging to the AGC (cAMP-dependent, cGMP-dependent, and protein kinase C) kinase family at their activation, or T-loop site.…”

“…Other PDK1 substrates like S6K, SGK, and RSK do not have a PH-domain and do not bind PIP 3 , nor is their phosphorylation by PDK1 directly stimulated by PIP 3 . Instead, the phosphorylation of their T-loop by PDK1 seems to be dependent on the phosphorylation of these enzymes at a C-terminal Ser/Thr residue termed the hydrophobic motif (HM) site.…”