Activation of hemin-regulated initiation factor-2 kinase in heat-shocked HeLa cells

• Published in: The Journal of biological chemistry 1986-01, Vol.261 (1), p.338-342
• Main Authors: De Benedetti, A, Baglioni, C
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; eIF-2 Kinase; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Fundamental and applied biological sciences. Psychology; HeLa Cells - enzymology; Heme - analogs & derivatives; Hemin - metabolism; Hot Temperature; Humans; Molecular and cellular biology; Molecular genetics; Molecular Weight; Phosphorylation; Protein Biosynthesis - drug effects; Protein Kinases - metabolism; Translation. Translation factors. Protein processing
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)42476-8

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.