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 heat ing, eukaryotic initiation factor 2-alpha (eIF-2alpha) becomes phospho rylated which prevents the binding of Met-tRNA to the 40s ribosomal su bunit inhibiting initiation of translation. Thermotolerant cells demon strate resistance to inhibition of translation by additional heating s uggesting 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-2alpha phosphorylation using rat fibroblasts stably transfected wi th a cloned human hsp70 gene. We observed a decrease in the rate of tr anslational inhibition following heat shock in both hsp70-transfected and thermotolerant cells. Upon recovery at 37-degrees-C, both hsp70-tr ansfected and thermotolerant cells exhibit a faster rate of translatio nal recovery. Utilizing slab gel isoelectric focusing coupled with imm unoblotting we demonstrate that 45-degrees-C heat shock leads to a rap id 4-5-fold increase in eIF-2alpha phosphorylation, with little differ ence seen between control cells and hsp70-transfected cells. However, dephosphorylation of eIF-2alpha occurs faster in the hsp70-transfected cells. These results suggest that hsp70 may play a role in facilitati ng the dephosphorylation of eIF-2alpha as well as reversing the inhibi tion of translation following heat shock.