Redox Regulation of Protein Kinase B/Akt Function by an Allosteric Disulphide Bond

Abstract

Most proteins in nature are chemically modified after they are made to control how, when and where they function. One type of chemical modification is the cleavage of disulphide bonds that link pairs of cysteine residues in the polypeptide chain. These cleavable bonds are known as allosteric disulphides. From an analysis of labile disulphide bonds in all protein structures from the Protein Data Bank (PDB), my colleagues and I identified a potential allosteric disulphide in the serine/threonine protein kinase B/Akt; linking cysteine residues 60 and 77 in the N-terminus pleckstrin homology (PH) domain. Akt plays a central role in glucose metabolism, cell survival and angiogenesis and is often hyper-activated in cancer cells. Akt is activated at the plasma membrane via binding to phosphatidylinositol-3,4,5-trisphosphate (PIP3) through its PH domain. Dissociation of Akt from the plasma membrane leads to PH domain-mediated autoinhibition of the kinase by a mechanism that is currently unknown. I hypothesised that the PH domain Cys60–Cys77 disulphide is an allosteric bond that regulates autoinhibition and inactivation of the kinase. To elucidate the role of the Cys60–Cys77 disulphide bond in Akt function, wild-type and reduced (Cys60 and/or Cys77 substituted with Ser) PH domain or full-length Akt mutants were analysed for PIP3 plasma membrane binding, Akt phosphorylation and Akt downstream substrate activation, transformation of fibroblasts, and angiogenesis, survival and development of zebrafish. Ablation of the Cys60–Cys77 disulphide bond did not appreciably affect binding of recombinant PH domain to PIP3, but markedly impaired insulin-stimulated binding of full-length Akt to the plasma membrane of adipocytes. Ablation of the Cys60–Cys77 disulphide bond had mixed effects on insulin-stimulated phosphorylation of Akt in fibroblasts. The Cys60Ser mutant was phosphorylated to the same extent as the wild-type, while the Cys77Ser mutant was poorly phosphorylated. Wild-type but not disulphide mutant Akt induced transformation of fibroblasts, indicating an oncogenic role for oxidised but not reduced Akt. Expression of disulphide mutant Akt in zebrafish increased the induction of angiogenesis and development of embryos but did not affect zebrafish survival. My findings imply that the Cys60–Cys77 disulphide bond in the PH domain of Akt is an allosteric disulphide involved in autoinhibition and functioning of the kinase