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Ischemia‐Induced Phosphorylation of Initiation Factor 2 in Differentiated PC12 Cells
Author(s) -
Muñoz F.,
Martín M. E.,
Tomico J. Manso,
Berlanga J.,
Salinas M.,
Fando J. L.
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0752335.x
Subject(s) - phosphatase , protein phosphatase 1 , dephosphorylation , kinase , phosphorylation , biology , eukaryotic initiation factor , eif2 , protein kinase a , protein biosynthesis , microbiology and biotechnology , biochemistry , messenger rna , translation (biology) , gene
An in vitro model of ischemia was obtained by subjectingPC12 cells differentiated with nerve growth factor to a combination of glucosedeprivation plus anoxia. Immediately after the ischemic period, the proteinsynthesis rate was significantly inhibited (80%) and western blots of cellextracts revealed a significant accumulation of phosphorylated eukaryoticinitiation factor 2, α subunit, eIF2(αP) (42%). Upon recovery,eIF2(αP) levels returned to control values after 30 min, whereas proteinsynthesis was still partially inhibited (33%) and reached almost controlvalues within 2 h. The activities of the mammalian eIF2α kinases,double‐stranded RNA‐activated protein kinase, mammalian GCN2 homologue, andendoplasmic reticulum‐resident kinase, were determined. None of theeIF2α kinases studied showed increased activity in ischemic cells ascompared with controls. Exposure of cells to cell‐permeable inhibitors ofprotein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose‐ andtime‐dependent accumulation of eIF2(αP), mimicking an ischemic effect.Protein phosphatase activity, as measured with [ 32 P]phosphorylase a as a substrate, diminished during ischemia and returned to controllevels upon 30‐min recovery. In addition, the rate of eIF2(αP)dephosphorylation was significantly lower in ischemic cells, paralleling boththe greatest translational inhibition and the highest eIF2(αP) levels.The endogenous phosphatase activity from control and ischemic extracts showeddifferent sensitivity to inhibitor 2 and fostriecin in in vitro assays,inhibitor‐2 effect in ischemic cells being lower than in control cells.Together these results indicate that an eIF2α phosphatase, probablyprotein phosphatase 1, is implicated in the ischemia‐induced eIF2(αP)accumulation in PC12 cells.