Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells.
Protein synthesis was drastically inhibited in HeLa cells incubated for 5 min at 42.5 degrees C, but it resumed after 20 min at a rate about 50% that of control cells. After 10 min of heat shock, the binding of Met-tRNAf to 40 S ribosomal subunits was greatly reduced and a polypeptide identified by immunoprecipitation with the alpha subunit of eukaryotic initiation factor-2 (eIF-2) was phosphorylated. Extracts prepared from control and heat-shocked cells were assayed for in vitro protein synthesis. Both extracts were active when supplemented with hemin, but the extract from heat-shocked cells had little initiation activity without this addition. A Mr 90,000 polypeptide and eIF-2 alpha were phosphorylated in this extract, but hemin or an antibody which inhibits the protein kinase designated heme-controlled repressor reduced this phosphorylation. These findings implicated heme-controlled repressor as the kinase at least in part responsible for eIF-2 alpha phosphorylation. Furthermore, the initial inhibition of protein synthesis and eIF-2 alpha phosphorylation after heat shock were reduced by adding hemin to intact HeLa cells. These cells synthesized heat-shock proteins with some delay relative to cells without added hemin. The binding of Met-tRNAf to 40 S ribosomal subunits was inhibited by about 50% in extracts prepared from cells heat-shocked for 40 min, and eIF-2 alpha phosphorylation was increased in these cells. These results suggest that heme-controlled repressor is activated in heat-shocked cells and that eIF-2 alpha phosphorylation limits mRNA translation even after partial recovery of protein synthesis.
D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006367:HeLa Cells / Q000201:enzymology*; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D014176:Protein Biosynthesis / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D019892:eIF-2 Kinase
D006418:Heme; D006427:Hemin; D011494:Protein Kinases; D019892:eIF-2 Kinase