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 differentiated with nerve growth factor to a combination of glucose deprivation plus anoxia. Immediately after the ischemic period, the protein synthesis rate was significantly inhibited (80%) and western blots of cell extracts revealed a significant accumulation of phosphorylated eukaryotic initiation factor 2, alpha subunit, eIF2(alphaP) (42%). Upon recovery, eIF2(alphaP) levels returned to control values after 30 min, whereas protein synthesis was still partially inhibited (33%) and reached almost control values within 2 h. The activities of the mammalian eIF2alpha kinases, double-stranded RNA-activated protein kinase, mammalian GCN2 homologue, and endoplasmic reticulum-resident kinase, were determined. None of the eIF2alpha kinases studied showed increased activity in ischemic cells as compared with controls. Exposure of cells to cell-permeable inhibitors of protein phosphatases 1 and 2A, calyculin A or tautomycin, induced dose- and time-dependent accumulation of eIF2(alphaP), mimicking an ischemic effect. Protein phosphatase activity, as measured with [(32)P]phosphorylase a as a substrate, diminished during ischemia and returned to control levels upon 30-min recovery. In addition, the rate of eIF2(alphaP) dephosphorylation was significantly lower in ischemic cells, paralleling both the greatest translational inhibition and the highest eIF2(alphaP) levels. The endogenous phosphatase activity from control and ischemic extracts showed different 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 eIF2alpha phosphatase, probably protein phosphatase 1, is implicated in the ischemia-induced eIF2(alphaP) accumulation in PC12 cells.
Muñoz
F
F
Department of Biochemistry and Molecular Biology, Alcalá University, Madrid, Spain.
Martín
M E
ME
Manso-Tomico
J
J
Berlanga
J
J
Salinas
M
M
Fando
J L
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D020925:Hypoxia-Ischemia, Brain / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016716:PC12 Cells; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D054645:Protein Phosphatase 1; D011506:Proteins / Q000378:metabolism; D051381:Rats
D004791:Enzyme Inhibitors; D015852:Eukaryotic Initiation Factor-2; D011506:Proteins; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1