title: In situ phosphorylation of the alpha subunit of eukaryotic initiation factor 2 in reticulocyte lysates inhibited by heme deficiency, double-stranded RNA, oxidized glutathione, or the heme-regulated protein kinase.
abstract: Protein synthesis initiation in reticulocyte lysates is inhibited by heme deficiency, low levels of double-stranded RNA (dsRNA), oxidized glutathione (GSSG), or the purified kinase (HRI) that acts on the alpha polypeptide of eukaryotic initiation factor 2 (eIF-2alpha). The phosphoprotein profiles produced in lysates in response to these various conditions have been monitored directly in lysates after labeling for brief periods with pulses of [gamma-(32)P]ATP. The [(32)P]phosphoprotein profiles were analyzed by electrophoresis in sodium dodecyl sulfate/polyacrylamide slab gels under conditions in which the HRI and eIF-2alpha polypeptides were clearly distinguished. All four modes of inhibition produced a rapid phosphorylation of eIF-2alpha compared to control lysates, which displayed little or no phosphorylation of eIF-2alpha. In heme-deficient lysates, phosphorylation of eIF-2alpha occurred rapidly both before and after the shut-off of protein synthesis; the delayed addition of hemin to these lysates resulted in a decrease in the phosphorylation of eIF-2alpha and the subsequent restoration of protein synthesis. These data suggest that rapid turnover of phosphate occurs at the site(s) of eIF-2alpha phosphorylation. In lysates inhibited by heme deficiency, GSSG, or added HRI, the phosphorylation of eIF-2alpha was accompanied by the rapid in situ phosphorylation of HRI. The inhibition of initiation induced by dsRNA was accompanied by the phosphorylation of eIF-2alpha and a 67,000-dalton polypeptide but not HRI. These observations in situ indicate that (i) the phosphorylation of eIF-2alpha is the critical event in these inhibitions of protein chain initiation, and (ii) the phosphorylation of HRI is associated with its activation in heme deficiency.
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005978:Glutathione / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006418:Heme / Q000172:deficiency; D006427:Hemin / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012313:RNA / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D010448:Peptide Initiation Factors; D006418:Heme; D012313:RNA; D006427:Hemin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D005978:Glutathione