Evidence for the Association of the Heme-regulated eIF-2α Kinase with the 90-kDa Heat Shock Protein in Rabbit Reticulocyte Lysate in Situ

Abstract

Abstract Inhibition of protein synthesis initiation in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation occurs due to the activation of a heme-regulated protein kinase (HRI), which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. How the activation of HRI in hemin-supplemented lysate occurs in response to oxidants and heat stress is not well understood. Recently, the 90-kDa heat shock protein (hsp 90) has been reported to co-purify with HRI activity. In this report, we have used monoclonal antibodies directed against hsp 90 to determine whether HRI and hsp 90 are functionally associated in the reticulocyte lysate in situ. The AC88 antibody recognizes only free hsp 90 and only bound significant amounts of hsp 90 upon prolonged incubation in the absence of heme or upon N-ethylmaleimide treatment of hemin-supplemented lysates. HRI activity is not absorbed by the AC88 antibody. The 8D3 monoclonal antibody, which binds to both free hsp 90 and hsp 90 complexed to steroid hormone receptors, absorbed the hsp 90 present in hemin-supplemented lysates and reduced the HRI activity by 70-95%. Progressively more HRI activity is not adsorbed by the 8D3 antibody the longer the reticulocyte lysate is incubated in the absence of hemin. The HRI that is adsorbed from heme-deficient lysates by the 8D3 antibody is also more active. The sedimentation rate of HRI was analyzed by glycerol gradient centrifugation. HRI present in hemin-supplemented lysate was found to have a sedimentation coefficient of approximately 7.5-8 S and was adsorbed from fractions by the 8D3 antibody in association with hsp 90. A second peak of HRI activity with a sedimentation coefficient of approximately 4.5-5 S was detected upon glycerol gradient centrifugation of heme-deficient lysates. Upon Western blot analysis, heme-deficient lysates were found to have less hsp 90 in the 7.5-8 S region of glycerol gradients than hemin-supplemented lysates. The data suggest that HRI is associated with hsp 90 in an inactive form in hemin-supplemented lysates and dissociates from hsp 90 upon activation. There also appears to be an intermediate of active HRI which is associated with hsp 90 or which can reversibly associate with hsp 90. Similarities between the stages of HRI activation and steroid hormone receptor activation and transformation are discussed.