HSP90 IS OBLIGATORY FOR THE HEME-REGULATED EIF-2-ALPHA KINASE TO ACQUIRE AND MAINTAIN AN ACTIVABLE CONFORMATION

The heme regulated eukaryotic initiation factor 2 alpha (eIF-2 alpha) kinase (HRI) interacts with hsp90 in situ in rabbit reticulocyte lysat e (RRL). In this report, we have examined the role of hsp90 in the mat uration of newly synthesized HRI in both hemin-supplemented and heme-d eficient RRL. Analysis of translating polyribosomes indicated that hsp 90 interacts with nascent HRI cotranslationally. Coimmunoadsorption of HRI with hsp90 by the 8D3 anti-hsp90 antibody indicated that this int eraction persisted after release of newly synthesized HRI from ribosom es. Incubation of HRI in heme-deficient lysate resulted in the transfo rmation of a portion of the HRI polypeptides into an active heme-regul atable eIF-2 alpha kinase that exhibited slower electrophoretic mobili ty. Transformation of HRI was dependent on autophosphorylation, and tr ansformed HRI was resistant to aggregation induced by treatment of RRL with N-ethylmaleimide. Transformed HRI did not coimmunoadsorb with hs p90, and regulation of the activity of transformed HRI by hemin was no t hsp90-dependent. The hsp90 binding drug geldanamycin disrupted the i nteraction of hsp90 with HRI and inhibited the maturation of HRI into a form that was competent to undergo autophosphorylation. Additionally geldanamycin inhibited the transformation of HRI into a stable heme-r egulatable kinase. These results indicate that hsp90 plays an obligato ry role in HRI acquiring and maintaining a conformation that is compet ent to become transformed into an aggregation-resistant activable kina se.