Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance.

Phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post-ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1alpha and PP1gamma immunoprecipitates. The highest levels of phosphorylated eIF2alpha found after 30 min reperfusion in rats without IT, correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and HSC70 proteins. After 4 h reperfusion, the levels of these proteins in PP1c complexes had returned to control values, in parallel to a significant decrease in eIF2alpha phosphorylated levels. IT that promoted a decrease in eIF2alpha phosphorylated levels after 30 min reperfusion induced the association of GADD34 with PP1c, while prevented that of DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1alpha and PP1gamma isoforms, whereas GRP78 was only detected in PP1gamma immunoprecipitates. Here we suggest that PP1, through different signaling complexes with their interacting proteins, may modulate the eIF2alpha phosphorylation/dephosphorylation during reperfusion after a transient global ischemia in the rat brain. Of particular interest is the potential role of GADD34/PP1c complexes after tolerance acquisition.