Heat shock effects on phosphorylation of protein synthesis initiation factor proteins eIF-4E and eIF-2 alpha in Drosophila.
Heat shock of mammalian cells causes changes in initiation factor phosphorylation that likely contribute to or cause the translation reprogramming characteristic of heat shock. In these investigations we have carried out a parallel analysis of Drosophila, focusing on eIF-4E and eIF-2 alpha. 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-2 alpha has been identified by in vitro translation of T7 RNA polymerase-transcribed mRNA, and immunoblotting with anti-Drosophila eIF-2 alpha antiserum. 32P-labeling analysis (unfractionated cell lysates and immunoprecipitates) detects phosphorylated eIF-2 alpha, 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-2 alpha phosphorylated detects a single eIF-2 alpha 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.
Duncan
R F
RF
Department of Molecular Pharmacology and Toxicology, University of Southern California School of Pharmacy, Los Angeles.
Cavener
D R
DR
Qu
S
S
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004330:Drosophila; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E; D006358:Hot Temperature; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis
D018076:DNA, Complementary; D015852:Eukaryotic Initiation Factor-2; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D010710:Phosphates; D010750:Phosphoproteins