cJun N-terminal kinase (JNK) phosphorylation of serine 36 is critical for p66Shc activation

Sci Rep. 2016 Feb 12;6:20930. doi: 10.1038/srep20930.

Abstract

p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Death / drug effects
  • Cell Hypoxia / drug effects
  • DNA Damage
  • Enzyme Activation / drug effects
  • Gene Knockout Techniques
  • JNK Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • JNK Mitogen-Activated Protein Kinases / metabolism*
  • Mice
  • Mitochondria / drug effects
  • Mitochondria / metabolism
  • Mutant Proteins / metabolism
  • Oxidants / toxicity
  • Oxidative Stress / drug effects
  • Oxygen / pharmacology
  • Phenotype
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism*
  • Protein Binding / drug effects
  • Protein Kinase C beta / metabolism
  • Protein Kinase Inhibitors / pharmacology
  • Reactive Oxygen Species / metabolism
  • Recombinant Proteins / metabolism
  • Src Homology 2 Domain-Containing, Transforming Protein 1 / metabolism*

Substances

  • Mutant Proteins
  • Oxidants
  • Protein Kinase Inhibitors
  • Reactive Oxygen Species
  • Recombinant Proteins
  • Shc1 protein, mouse
  • Src Homology 2 Domain-Containing, Transforming Protein 1
  • Phosphoserine
  • Protein Kinase C beta
  • JNK Mitogen-Activated Protein Kinases
  • Oxygen