Dual role for Hsc70 in the biogenesis and regulation of the heme-regulatedkinase of the a subunit of eukaryotic translation initiation factor 2

The heme-regulated kinase of the a subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a n umber of environmental conditions, including heme deficiency, heat shock, a nd oxidative stress. Activation of HRI causes an arrest of initiation of pr otein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in respons e to these environmental conditions. Hsc70 is also known to be a critical c omponent of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to a ctivate. Using de novo-synthesized HRI in synchronized pulse-chase translat ions, we have examined the role of Hsc70 in the regulation of HRI biogenesi s and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI tha t was matured to a state which was competent to undergo stimulus-induced ac tivation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhi bited the folding of HRI into a mature-competent conformation. Unlike Hsp90 , Hsc70 also interacted with transformed HRI, Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transfo rmed in the absence of the drug. Disruption of Hsc70 interaction with trans formed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermo re, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. T hese results indicate that Hsc70 is required for the folding and transforma tion of HRI into an active kinase but is subsequently required to negativel y attenuate the activation of transformed HRI.