Rat fibroblasts transfected with the human 70-kDa heat shock gene exhibit altered translation and eukaryotic initiation factor 2 alpha phosphorylation following heat shock.
Heat shock inhibits translation in a wide variety of cells. After heating, eukaryotic initiation factor 2-alpha (eIF-2 alpha) becomes phosphorylated which prevents the binding of Met-tRNA to the 40s ribosomal subunit inhibiting initiation of translation. Thermotolerant cells demonstrate resistance to inhibition of translation by additional heating suggesting that heat shock proteins may help to maintain translational integrity following thermal stress. Here we have examined the effects of increased intracellular levels of hsp70 protein on translation and eIF-2 alpha phosphorylation using rat fibroblasts stably transfected with a cloned human hsp70 gene. We observed a decrease in the rate of translational inhibition following heat shock in both hsp70-transfected and thermotolerant cells. Upon recovery at 37 degrees C, both hsp70-transfected and thermotolerant cells exhibit a faster rate of translational recovery. Utilizing slab gel isoelectric focusing coupled with immunoblotting we demonstrate that 45 degrees C heat shock leads to a rapid 4-5-fold increase in eIF-2 alpha phosphorylation, with little difference seen between control cells and hsp70-transfected cells. However, dephosphorylation of eIF-2 alpha occurs faster in the hsp70-transfected cells. These results suggest that hsp70 may play a role in facilitating the dephosphorylation of eIF-2 alpha as well as reversing the inhibition of translation following heat shock.
Chang
G C
GC
Department of Radiation Oncology, University of California, San Francisco 94143.
Liu
R
R
Panniers
R
R
Li
G C
GC
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature / Q000009:adverse effects*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D014176:Protein Biosynthesis; D051381:Rats; D014162:Transfection
D015852:Eukaryotic Initiation Factor-2; D018840:HSP70 Heat-Shock Proteins; D003513:Cycloheximide