EVIDENCE THAT HSC70 NEGATIVELY MODULATES THE ACTIVATION OF THE HEME-REGULATED EIF-2-ALPHA KINASE IN RABBIT RETICULOCYTE LYSATE

The role of the heat-shock cognate protein, Hsc70, in regulating the a ctivity of the heme-regulated eIF-2 alpha kinase (HRI) in hemin-supple mented rabbit reticulocyte lysate (RRL) in response to heat and oxidat ive stress was examined and compared with the effect of Hsc70 on HRI a ctivation in response to heme deficiency. Hsc70 suppressed eIF-2a phos phorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42 degrees C), denatured protein (reduced carb oxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg2+. The ability of Hsc70 to inhibit HRI activation was mediated thro ugh its ability to inhibit the hyper-autophosphorylation of transforme d HRI, which causes the hyperactivation of HRI, Maintenance of protein -synthesis rate was observed to be an unreliable indicator of the abil ity of Hsc70 to suppress HRI activation in response to stress. While H sc70 completely reversed protein synthesis inhibition caused by Hg2+, Hsc70 only partially reversed translational inhibition caused by oxidi zed glutathione (GSSG) or heat shock, The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSS G was due to inability of Hsc70 to protect eIF-4 E from heat-induced d ephosphorylation, and its inability to protect translational elongatio n from GSSG-induced inhibition, respectively. Activation of HRI in hea t-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss o f the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and in hibit the ability of Hsc70 to suppress HRI activation. Our results ind icate that Hsc70 not only negatively modulates the activation of HRI i n heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.