Global redox proteome and phosphoproteome analysis reveals redox switch in Akt

Zhiduan Su,Lan K Nguyen,Guang Yang,Qiao‐Ping Wang ,Sean J Humphrey,Serdar Kuyucak,Dorthe Francis ,Benjamin L Parker,Joyce Chiu,Sung-Young Shin,Daniel J Fazakerley,Philip J Hogg,Jonathan Scavuzzo,Alison L Kearney ,Dougall M Norris,Reichelle X Yeo ,Sabina Yasmin,Kelsey H Fisher‐Wellman ,James G Burchfield,Fatemeh Vafaee,Miro A Astore,Pengyi Yang,David E James

doi:10.1038/s41467-019-13114-4

Abstract

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Here, we delineate global changes in adipocyte signalling networks following acute oxidative stress and reveal considerable crosstalk between cysteine oxidation and phosphorylation-based signalling. Oxidation of key regulatory kinases, including Akt, mTOR and AMPK influences the fidelity rather than their absolute activation state, highlighting an unappreciated interplay between these modifications. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized. Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. Our data provide insights into the interplay between oxidative stress-derived redox signalling and protein phosphorylation networks and serve as a resource for understanding the contribution of cellular oxidation to a range of diseases.

Highlights

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear
We combine a model of oxidative stress with a multi-omics approach to explore the interaction between Reactive oxygen species (ROS)-dependent protein oxidation and phospho-signalling
ROS has a profound influence on the adipocyte phosphorylation signalling network principally by influencing the fidelity of signalling at key regulatory nodes including Akt, mammalian target of rapamycin (mTOR) and AMPK

Summary

Introduction

Protein oxidation sits at the intersection of multiple signalling pathways, yet the magnitude and extent of crosstalk between oxidation and other post-translational modifications remains unclear. Mechanistic analysis of the redox regulation of Akt identified two cysteine residues in the pleckstrin homology domain (C60 and C77) to be reversibly oxidized Oxidation at these sites affected Akt recruitment to the plasma membrane by stabilizing the PIP3 binding pocket. The activity of kinases and phosphatases can be altered by redox-mediated post-translational modifications (PTMs), providing a link between ROS, phosphorylation-based signalling (phospho-signalling)[1,5,6,7,8] and physiological processes including cell growth and proliferation[4]. We have developed a simple model for studying widespread cellular oxidation based on inhibition of the two major thiolbased antioxidant systems (glutathione and thioredoxin)[26] This model allows ROS effects to be studied without the complex phenotypes that may occur in disease-specific models[27]. We identify oxidation of critical cysteine residues in the Ser/Thr kinase Akt that are essential for its plasma membrane (PM)recruitment and activation

Methods

Results

Conclusion