The dynamic role of GRP78/BiP in the coordination of mRNA translation with protein processing.
The role of GRP78/BiP in coordinating endoplasmic reticular (ER) protein processing with mRNA translation was examined in GH3 pituitary cells. ADP-ribosylation of GRP78 and eukaryotic initiation factor (eIF)-2alpha phosphorylation were assessed, respectively, as indices of chaperone inactivation and the inhibition of translational initiation. Inhibition of protein processing by ER stress (ionomycin and dithiothreitol) resulted in GRP78 deribosylation and eIF-2 phosphorylation. Suppression of translation relative to ER protein processing (cycloheximide) produced approximately 50% ADP-ribosylation of GRP78 within 90 min without eIF-2 phosphorylation. ADP-ribosylation was reversed in 90 min by cycloheximide removal in a manner accelerated by ER stressors. Cycloheximide sharply reduced eIF-2 phosphorylation in response to ER stressors for about 30 min; sensitivity returned as GRP78 became increasingly ADP-ribosylated. Reduced sensitivity of eIF-2 to phosphorylation appeared to derive from the accumulation of free, unmodified chaperone as proteins completed processing without replacements. Prolonged (24 h) incubations with cycloheximide resulted in the selective loss of the ADP-ribosylated form of GRP78 and increased sensitivity of eIF-2 phosphorylation in response to ER stressors. Brefeldin A decreased ADP-ribosylation of GRP78 in parallel with increased eIF-2 phosphorylation. The cytoplasmic stressor, arsenite, which inhibits translational initiation through eIF-2 phosphorylation without affecting the ER, also produced ADP-ribosylation of GRP78.
Laitusis
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Department of Pharmacology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Brostrom
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MA
Brostrom
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CO
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones / Q000378:metabolism*; D010766:Phosphorylation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D015852:Eukaryotic Initiation Factor-2; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D020126:Brefeldin A; D000246:Adenosine Diphosphate Ribose; D003513:Cycloheximide