ABSTRACT
Inhibition of protein synthesis during heat shock limits accumulation of unfolded proteins that might damage eukaryotic cells. We demonstrate that chaperone Hsp27 is a heat shock-induced inhibitor of cellular protein synthesis. Translation of most mRNAs requires formation of a cap-binding initiation complex known as eIF4F, consisting of factors eIF4E, eIF4A, eIF4E kinase Mnk1, poly(A)-binding protein, and adaptor protein eIF4G. Hsp27 specifically bound eIF4G during heat shock, preventing assembly of the cap-initiation/eIF4F complex and trapping eIF4G in insoluble heat shock granules. eIF4G is a specific target of Hsp27, as eIF4E, eIF4A, Mnk1, poly(A)-binding protein, eIF4B, and eIF3 were not bound by Hsp27 and were not recruited into insoluble complexes. Dissociation of eIF4F was enhanced during heat shock by ectopic overexpression of Hsp25, the murine homolog of human Hsp27. Overexpression of Hsc70, a constitutive homolog of Hsp70, prevented loss of cap-initiation complexes and maintained eIF4G solubility. Purified Hsp27 specifically bound purified eIF4G in vitro, prevented in vitro translation, eliminated eIF4G interaction with protein binding factors, and promoted eIF4G insolubilization. These results therefore demonstrate that Hsp27 is a heat-induced inhibitor of eIF4F-dependent mRNA translation.
Subject(s)
HSP70 Heat-Shock Proteins , Heat-Shock Proteins , Molecular Chaperones/physiology , Neoplasm Proteins/metabolism , Neoplasm Proteins/physiology , Peptide Initiation Factors/metabolism , Protein Biosynthesis , Animals , Blotting, Western , Carrier Proteins/metabolism , Cell Line, Transformed , Dose-Response Relationship, Drug , Eukaryotic Initiation Factor-4F , Eukaryotic Initiation Factor-4G , Fluorescent Antibody Technique , Glutathione Transferase/metabolism , HSC70 Heat-Shock Proteins , HSP27 Heat-Shock Proteins , HeLa Cells , Hot Temperature , Humans , Intracellular Signaling Peptides and Proteins , Mice , Molecular Chaperones/metabolism , Phosphotransferases/metabolism , Plasmids , Precipitin Tests , Protein Binding , Protein Serine-Threonine Kinases/metabolism , RNA Caps , RNA, Messenger/metabolism , Temperature , TransfectionABSTRACT
Cytokine and proto-oncogene messenger RNAs (mRNAs) are rapidly degraded through AU-rich elements in the 3' untranslated region. Rapid decay involves AU-rich binding protein AUF1, which complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G, and poly(A) binding protein. AU-rich mRNA decay is associated with displacement of eIF4G from AUF1, ubiquitination of AUF1, and degradation of AUF1 by proteasomes. Induction of hsp70 by heat shock, down-regulation of the ubiquitin-proteasome network, or inactivation of ubiquitinating enzyme E1 all result in hsp70 sequestration of AUF1 in the perinucleus-nucleus, and all three processes block decay of AU-rich mRNAs and AUF1 protein. These results link the rapid degradation of cytokine mRNAs to the ubiquitin-proteasome pathway.
