Cytochrome c Is Tyrosine 97 Phosphorylated by Neuroprotective Insulin Treatment

Thomas H Sanderson,Qinqin Ji,Kebing Yu,Renee N Tousignant,Petr Pecina,Melissa J Bukowski,Christopher Sinkler,Arthur R Salomon,Gargi Mahapatra,Rita Kumar,Maik Hüttemann,Ashwathy Varughese,Icksoo Lee,Claudia Miele

doi:10.1371/journal.pone.0078627

Abstract

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Highlights

Brain ischemia, caused by stroke or cardiac arrest, results in extensive brain damage and is a major cause of death and disability [1]
We first investigated the effect of insulin treatment on release of cytochrome c (Cytc) from the mitochondria into the cytosol and subsequent cell death in a rat model of global brain ischemia/reperfusion injury
We previously demonstrated that Cytc release following global brain ischemia reaches a maximum level by 24 hours of reperfusion [4]

Summary

Introduction

Brain ischemia, caused by stroke or cardiac arrest, results in extensive brain damage and is a major cause of death and disability [1]. We have previously shown that administration of insulin immediately following an ischemic insult activates the PI3K-Akt cell survival pathway, inhibits Bax translocation to the mitochondria, promotes favourable Bcl-2 family protein interactions on the mitochondria, inhibits Cytc release, and importantly, protects hippocampal structure and function [4,5]. While these studies revealed insulin induced cell signaling events associated with inhibition of Cytc release and attenuation of cell death, the specific cell signaling events responsible for this effect remain to be elucidated

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Conclusion