Heat Shock Effects on Phosphorylation of Protein Synthesis Initiation Factor Proteins eIF-4E and eIF-2α in Drosophila

Roger F. Duncan, Douglas R. Cavener, Shimian Qu

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

Heat shock of mammalian cells causes changes in initiation factor phosphorylation that likely contribute to or cause the translational reprogramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eIF-4E and eIF-2α. eIF-4E plus associated proteins was purified from lysates by m7GTP-Sepharose chromatography. A minor fraction (<10%) of eIF-4E is phosphorylated under normal growth conditions, and phosphorylation decreases during heat shock. Drosophila eIF-2α has been identified by in vitro translation of T7 RNA polymerasetranscribed mRNA, and immunoblotting with mti-Drosophila eIF-2α antiserum. 32P-labeling analysis (unfractionated cell lysates and immunoprecipitates) detects phosphorylated eIF-2α, whose amount increases approximately 2-3-fold upon heat shock. Immunoblotting analysis of two-dimensional gel-resolved proteins to determine the mass fraction of eIF-2α phosphorylated detects a single eIF-2α spot in both normal temperature and heat shocked cells, indicating less than 5% phosphorylation after and before heat shock. Staining quantification is consistent with this low prevalence. A major phosphoprotein which copurifies with eIF-4E on m7GTP-Sepharose shows decreased overall phosphorylation and decreased association with eIF-4E following heat shock. Several distinctive characteristics of this phosphoprotein suggest it is Drosophila eIF-4B.

Original languageEnglish (US)
Pages (from-to)2985-2997
Number of pages13
JournalBiochemistry
Volume34
Issue number9
DOIs
StatePublished - Mar 1995

All Science Journal Classification (ASJC) codes

  • Biochemistry

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