Regulation of Akt conformation and function

The kinase Akt is a central mediator of insulin signaling. In the inactive state, Akt exhibits a closed conformation whereby the kinase domain is inaccessible (Fig 1A). After insulin signaling, Akt is phosphorylated at two key sites, T308 in the kinase domain and S473 in the disordered C-terminal tail. This causes a conformational change that exposes the kinase domain and activates Akt. Using mice and primary murine hepatocytes, we have found unexpectedly that the essential trace element manganese (Mn) potently activates Akt activity without inducing Akt phosphorylation. Insulin causes an additive effect. Moreover, we have found that hepatocyte Mn efflux is regulated by fasting and feeding, suggesting that Mn is a physiologic modulator of insulin-Akt signaling. We have identified putative Mn-coordinating residues in Akt, and in this grant we propose to test the hypothesis that Mn activates Akt by causing a conformational change to a “primed-active” state that exposes the kinase domain and allows phosphorylation of Akt targets (Fig 1B). We will pursue this goal in two aims: Aim 1-Determine effects of Mn on Akt conformation. We will use hydrogen/deuterium exchange mass spectrometry, isothermal titration calorimetry, inductively coupled plasma mass spectrometry, and intramolecular fluorescence resonance energy transfer of Akt itself to determine the effects of Mn and the putative Mn-binding residues on Akt conformation. Aim 2-Determine effects of Mn-binding on Akt function. We will use fluorescence resonance energy transfer and western blot of Akt substrates in Akt-deficient cells where we’ve expressed wildtype versus Mn-binding-site-mutants of Akt. Success of this project would (i) reveal that Mn determines the conformation and activity of Akt, and (ii) suggest the possibility that micronutrient transport is an unexplored, physiologic mechanism to regulate signal transduction and metabolism.