Purification and characterization of eukaryotic initiation factor (eIF)-2 alpha kinases from Ehrlich ascites tumor cells.
A major mechanism of regulation of mammalian protein synthesis initiation is accomplished by the phosphorylation of the alpha subunit of eukaryotic initiation factor (eIF) 2. This modification inhibits the activity of another initiation factor, guanine nucleotide exchange factor, preventing conversion of eIF-2.GDP to eIF-2.GTP and hence binding of initiator tRNA and formation of ternary complex (eIF-2.GTP.Met-tRNAf). Inhibition of protein synthesis and phosphorylation of eIF-2 occurs in Ehrlich cells when they are amino acid- or serum-deprived or heat-shocked as well as in other nucleated cells under similar conditions. This paper describes the purification of two eIF-2 alpha kinases from Ehrlich cells. Unlike the two well characterized eIF-2 alpha kinases, HRI (heme-regulated inhibitor from reticulocytes) and P68 (double-stranded RNA-dependent kinase found in interferon-treated cells), the Ehrlich cell kinases do not appear to autophosphorylate. The two kinases chromatograph differently on DEAE-cellulose and Mono Q. Furthermore, their Michaelis constants (Km values) for ATP are different. Both kinases can inhibit purified guanine nucleotide exchange factor (GEF) from stimulating ternary complex formation. However, only one kinase inhibits reticulocyte lysate cell free protein synthesis. The other kinase co-purifies with a factor that suppresses inhibition of protein synthesis in reticulocyte lysates by eIF-2 alpha kinases. This suppressing activity is probably guanine nucleotide exchange factor.
Olmsted
E A
EA
Department of Biochemistry, University of Rochester, New York 14642.
O'Brien
L
L
Henshaw
E C
EC
Panniers
R
R
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D005033:Ethylmaleimide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors; D007525:Isoelectric Focusing; D007700:Kinetics; D051379:Mice; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D012358:RNA, Transfer, Met; D011494:Protein Kinases; D019892:eIF-2 Kinase; D005033:Ethylmaleimide