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.

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the a-subunit of eukaryotic initiation factor 2 (eIF2a). To identify kinases responsible for eIF2a phosphorylation [eIF2a(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2a(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2a kinase (HRI), or the amino acidregulated eIF2a kinase (GCN2). A 10-fold increase in eIF2a(P) was observed in reperfused wild-type mice and in the HRI±/± or GCN2±/± mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2a kinase (PERK) exhibited an isoform mobility shift on SDS±PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2a(P), and in conjunction with our previous report that eIF2a(P) is produced in the brain of reperfused PKR±/± mice, provides evidence that PERK is the kinase responsible for eIF2a phosphorylation in the early post-ischemic brain.

Recent studies indicate that p50cdc37 facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50 cdc37 is required for the biogenesis of the heme-regulated eIF2␣ kinase (HRI) in reticulocyte lysate. p50 cdc37 interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50 cdc37 stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50 cdc37 function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50 cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50 cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50 cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50 cdc37 with Hsp90. A C-terminal truncated mutant of p50 cdc37 inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50 cdc37 were detected in cultured K562 erythroleukemia cells. These results suggest that p50 cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.The heme-regulated inhibitor (HRI) 1 of protein synthesis is a protein-serine kinase which coordinates the synthesis of globin chains with the availability of heme in reticulocytes (reviewed in Refs. 1 and 2). Under heme-deficient conditions, HRI phosphorylates the ␣-subunit of eukaryotic translational initiation factor eIF2. Phosphorylation of eIF2␣ causes an inhibition of polypeptide chain initiation and the arrest of protein synthesis, preventing the synthesis of apo-globin chains in the absence of heme. HRI is also activated under heme-replete conditions in response to a host of other adverse environmental stimuli, such as heat shock, agents that generate oxidative stress, and the presence of denatured proteins (1, 2).The biogenesis and activation of HRI into an active hemeregulatable eIF2␣ kinase requires its functional interaction with the chaperone machinery containing the 90-kDa heat shock protein (Hsp90) and the 70-kDa heat shock cognate protein (Hsc70) (3, 4). During HRI biogenesis and its subsequent transformation and activation, several discrete HRI intermediates are generated; these intermediates can be distinguished on the basis of their competence to become an active kinase in response to heme deficiency or upon treatment with sulfhydryl reactive compounds such as N-ethylmaleimide. Immediately after their synthesis, HRI molecules are not active in hemereplete or heme-deficient rabbit reticulocyte lysate (RRL) and cannot be activated by N-ethylmaleimide treatment. This immature population interacts with Hsp90 and Hsc70 (3-7). Subsequent to this immature phase, a "mature-competent" HRI population appear...

In heme deficiency, protein synthesis in reticulocytes is inhibited by activation of heme-regulated ␣-subunit of eukaryotic initiation factor-2␣ (eIF-2␣) kinase (HRI). Previous studies indicate that HRI contains two distinct heme-binding sites per HRI monomer. To study the role of the N terminus in the heme regulation of HRI, two N-terminally truncated mutants, Met2 and Met3 (deletion of the first 103 and 130 amino acids, respectively), were prepared. Met2 and Met3 underwent autophosphorylation and phosphorylated eIF-2␣ with a specific activity of approximately 50% of that of the wild type HRI. These mutants were significantly less sensitive to heme regulation both in vivo and in vitro. In addition, the heme contents of purified Met2 and Met3 HRI were less than 5% of that of the wild type HRI. These results indicated that the N terminus was important but was not the only domain involved in the heme-binding and heme regulation of HRI. Heme binding of the individual HRI domains showed that both N terminus and kinase insertion were able to bind hemin, whereas the C terminus and the catalytic domains were not. Thus, both the N terminus and the kinase insertion, which are unique to HRI, are involved in the heme binding and the heme regulation of HRI.

The role of the heat-shock cognate protein, Hsc70, in regulating the activity of the heme-regulated eIF-2A kinase (HRI) in hemin-supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF-2A phosphorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42°C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg 2ϩ . The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein-synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg 2ϩ , Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF-4 E from heat-induced dephosphorylation, and its inability to protect translational elongation from GSSG-induced inhibition, respectively. Activation of HRI in heat-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss of 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 inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.