Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases.
The phosphorylation of the alpha-subunit of initiation factor eIF-2 leads to an inhibition of protein synthesis in mammalian cells. We have performed site-directed mutagenesis on a cDNA encoding the alpha-subunit of human eIF-2 and have replaced the candidate sites of phosphorylation, Ser-48 and Ser-51, with alanines. The cDNAs were expressed in vitro by SP6 polymerase transcription and rabbit reticulocyte lysate translation, and the radiolabeled protein products were analyzed by high-resolution two-dimensional gel electrophoresis. The wild-type and Ser-48 mutant proteins became extensively phosphorylated by eIF-2 kinases present in the reticulocyte lysate, and when additional heme-controlled repressor or double-stranded RNA-activated kinase was present, phosphorylation of the proteins was enhanced. The Ser-51 mutant showed little covalent modification by the endogenous enzymes and showed no increase in the acidic variant with additional eIF-2 kinases, thereby suggesting that Ser-51 is the site of phosphorylation leading to repression of protein synthesis.
Pathak
V K
VK
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Schindler
D
D
Hershey
J W
JW
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D010448:Peptide Initiation Factors; D011506:Proteins; D004247:DNA; D011494:Protein Kinases; D019892:eIF-2 Kinase