title: Hsp90 is obligatory for the heme-regulated eIF-2alpha kinase to acquire and maintain an activable conformation.
abstract: The heme-regulated eukaryotic initiation factor 2alpha (eIF-2alpha) kinase (HRI) interacts with hsp90 in situ in rabbit reticulocyte lysate (RRL). In this report, we have examined the role of hsp90 in the maturation of newly synthesized HRI in both hemin-supplemented and heme-deficient RRL. Analysis of translating polyribosomes indicated that hsp90 interacts with nascent HRI cotranslationally. Coimmunoadsorption of HRI with hsp90 by the 8D3 anti-hsp90 antibody indicated that this interaction persisted after release of newly synthesized HRI from ribosomes. Incubation of HRI in heme-deficient lysate resulted in the transformation of a portion of the HRI polypeptides into an active heme-regulatable eIF-2alpha kinase that exhibited slower electrophoretic mobility. Transformation of HRI was dependent on autophosphorylation, and transformed HRI was resistant to aggregation induced by treatment of RRL with N-ethylmaleimide. Transformed HRI did not coimmunoadsorb with hsp90, and regulation of the activity of transformed HRI by hemin was not hsp90-dependent. The hsp90 binding drug geldanamycin disrupted the interaction 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-regulatable kinase. These results indicate that hsp90 plays an obligatory role in HRI acquiring and maintaining a conformation that is competent to become transformed into an aggregation-resistant activable kinase.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: S
authors.initials: S
authors.lastname: Uma
authors.forename: S D
authors.initials: SD
authors.lastname: Hartson
authors.forename: J J
authors.initials: JJ
authors.lastname: Chen
authors.forename: R L
authors.initials: RL
authors.lastname: Matts
mesh_terms: D000818:Animals; D016227:Benzoquinones; D002474:Cell-Free System; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006418:Heme / Q000494:pharmacology*; D047029:Lactams, Macrocyclic; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes; D019892:eIF-2 Kinase
chemical_list: D016227:Benzoquinones; D004791:Enzyme Inhibitors; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D047428:Protein Kinase Inhibitors; D011809:Quinones; D006418:Heme; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D005033:Ethylmaleimide; C001277:geldanamycin