Mechanisms regulating phosphorylation of the alpha-subunit of eukaryotic intitiation factor-2 during post-ischemic brain reperfusion

Protein synthesis is inhibited at the initiation step in neurons following brain ischemia and reperfusion due to the phosphorylation of the eukaryotic initiation factor 2α (eIF2α). In order to understand the causal mechanism of phosphorylation of this initiation factor, I looked at the following possibilities: (1) deglycosylation (inactivation) of the glycoprotein p67; (2) inactivation of a specific phosphatase; or (3) activation of the eIF2α kinase, PERK.; Using immunocytochemistry and Western Blot analysis, I discovered that the glycosylation state, and total levels of p67 do not change following brain ischemia and reperfusion. Previous work in our lab utilizing individual transgenic knockout mice for three of the four kinases (PKR, HRI and GCN2), had shown that these kinases were not responsible for the phosphorylation of eIF2α. In addition, we found no change in phosphatase activity at 5 minutes reperfusion when eIF2α(P) levels are ∼18-fold greater than normal. So this left activation of PERK as the probably cause of eIF2α phosphorylation. We had recently discovered that PERK undergoes a mobility shift by 10 minutes of reperfusion, as determined by Western Blot analysis, indicating that it was undergoing phosphorylation and thus activation. Indeed, in my study, using three different transgenic PERK knockout mouse models (EMX, CamKII, and RIP), eIF2α phosphorylation is substantially decreased by 85% in EMX, 80% in CamKII, and 99% in RIP mice during reperfusion compared to wild type mice. This confirmed that PERK is responsible for phosphorylation of eIF2α following global brain ischemia and reperfusion. However, recent studies in our lab have shown that by 90 minutes of reperfusion, PERK was degraded ∼80% in the cortex, and ∼50% in the hippocampus and brainstem. Several lines of evidence suggested that this may be due to activation of μ-calpain. We have previously shown that calpain is activated as a consequence of ischemia and reperfusion, thus these experiments were undertaken to determine if PERK is a substrate for μ-calpain. In vitro protease reactions containing PERK together with μ-calpain showed that it is indeed degraded by this enzyme.; These results demonstrate that PERK is the kinase responsible for the phosphorylation of eIF2α(P) following ischemia and reperfusion. They also suggest a possible mechanism for the degradation of PERK by 90 minutes of reperfusion.