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. 1988 Feb;8(2):993–995. doi: 10.1128/mcb.8.2.993

Generation of a mutant form of protein synthesis initiation factor eIF-2 lacking the site of phosphorylation by eIF-2 kinases.

V K Pathak 1, D Schindler 1, J W Hershey 1
PMCID: PMC363234  PMID: 3352609

Abstract

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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