Ischemia-induced phosphorylation of initiation factor 2 in differentiated PC12 cells: Role for initiation factor 2 phosphatase

An in vitro model of ischemia was obtained by subjecting PC12 cells differe ntiated with nerve growth factor to a combination of glucose deprivation pl us anoxia, Immediately after the ischemic period, the protein synthesis rat e was significantly inhibited (80%) and western blots of cell extracts reve aled a significant accumulation of phosphorylated eukaryotic initiation fac tor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) level s returned to control values after 30 min, whereas protein synthesis was st ill partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2 alpha kinases, double-stranded RNA-ac tivated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum -resident kinase, were determined. None of the eIF2 alpha kinases studied s howed increased activity in ischemic cells as compared with controls. Expos ure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation o f eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [P-32]phosphorylase a as a substrate, diminished during i schemia and returned to control levels upon 30-min recovery. In addition, t he rate of eIF2(alphaP) dephosphorylation was significantly lower in ischem ic cells, paralleling both the greatest translational inhibition and the hi ghest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different sensitivity to inhibitor 2 and fost riecin in in vitro assays, inhibitor-2 effect in ischemic cells being lower than in control cells. Together these results indicate that an eIF2 alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia- induced eIF2(alphaP) accumulation in PC12 cells.