Redox Regulated Proteins
The Implications of Akt2 Redox-regulation in Radiation-associated Diabetes and Muscle Function in Aging
Protein kinase B/Akt is a major signaling hub in cytokine, growth factor and integrin signaling pathways of consequence to many biological processes. In mammals, three isoforms of Akt, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, regulate protein metabolism, cell proliferation, energy storage, and apoptosis. Despite their high sequence identity, mouse knockout models lacking Akt isoforms have revealed extensive differences in the isoform phenotypes: Akt1 is involved in anabolic metabolism and regulation of apoptosis, Akt2 is instrumental in the signaling of glucose metabolism, while Akt3 is implicated in neurological function and development. In general, these functional differences are attributed to variations in their tissue expression, temporal activation, subcellular location and substrate-specific signaling. We discovered the first posttranslational modification that distinguishes between the Akt isoforms and demonstrated the isoform-specific oxidation of Akt2 at Cys124 in NIH 3T3 cells stimulated with platelet derived growth factor (PDGF). Cys124 is situated in the linker region connecting the pleckstrin homology (PH) domain to the catalytic kinase domain of Akt2 and is not conserved in the other Akt isoforms. The results are reported in two articles published in PNAS and Cell Cycle in 2011, which included the following findings:
- oxidation is inhibitory to Akt2 kinase activity;
- ~50% of phosphorylated Akt2 undergoes oxidation in cells stimulated with PDGF; this process is reversible - kinase activity can be restored by removal of PDGF or treatment of oxidized protein with reducing agents;
- the mechanism of inhibition of Akt2 kinase activity with oxidation involves formation of multiple disulfides involving cysteine residues 60 and 77 in PH domain, 124 in the linker region, and 297 and 311 in the kinase domain;
- redox regulation of Akt2 is linked to physiological outputs such as glucose uptake, cell migration, and cell cycle.
Our current research is aimed at determining structural changes induced by disulfide bond formation in Akt2, the mechanism that drives the formation and order of disulfides, and the consequence of oxidation on Akt2 trafficking within the cell. The working model is shown here. We are also investigating the contribution of oxidized Akt2 to pathological conditions such as radiation-associated diabetes and impaired muscle function associated with aging.