The Heme-regulated Eukaryotic Initiation Factor 2α Kinase

Abstract

Nitric oxide (NO) has been reported to inhibit protein synthesis in eukaryotic cells by increasing the phosphorylation of the ␣-subunit of eukaryotic initiation factor (eIF) 2. However, the mechanism through which this increase occurs has not been characterized. In this report, we examined the effect of the diffusible gases nitric oxide (NO) and carbon monoxide (CO) on the activation of the heme-regulated eIF2␣ kinase (HRI) in rabbit reticulocyte lysate. Spectral analysis indicated that both NO and CO bind to the N-terminal heme-binding domain of HRI. Although NO was a very potent activator of HRI, CO markedly suppressed NO-induced HRI activation. The NO-induced activation of HRI was transduced through the interaction of NO with the N-terminal heme-binding domain of HRI and not through S-nitrosylation of HRI. We postulate that the regulation of HRI activity by diffusible gases may be of wider physiological significance, as we further demonstrate that NO generators increase eIF2␣ phosphorylation levels in NT2 neuroepithelial and C2C12 myoblast cells and activate HRI immunoadsorbed from extracts of these non-erythroid cell lines.Over 2 decades ago, the heme-regulated inhibitor (HRI) 1 of protein chain initiation in rabbit reticulocyte lysate (RRL) was identified as a heme-regulated protein kinase that phosphorylated the ␣-subunit of eukaryotic initiation factor eIF2 (reviewed in Refs. 1-3). eIF2 delivers Met-tRNA i in a complex with GTP to 43 S ribosomal initiation complexes and is released as a complex with GDP at the completion of the initiation cycle. Recycling of eIF2⅐GDP and the formation of eIF2⅐GTP⅐Met-tRNA i complexes requires the action of the guanine nucleotide exchange factor, eIF2B. Under heme-deficient conditions, HRI is activated and phosphorylates eIF2. Phosphorylated eIF2 avidly binds eIF2B, sequestering eIF2B in a poorly dissociable complex, which subsequently leads to the inhibition of the initiation of translation, as eIF2⅐GDP complexes that are present in excess of eIF2B fail to recycle. Thus, HRI functions to coordinate globin synthesis with heme availability in RRL.The amino acid sequence deduced from HRI cDNA indicates that it is composed of at least five distinct domains (4). The unique N-terminal domain of HRI contains ϳ165 amino acids. The second and fourth domains contain conserved sequence motifs I-V and motifs VI-XI, which comprise the N-terminal and C-terminal catalytic lobes of protein kinases, respectively. The third and fifth domains are also unique, consisting of ϳ140 amino acids that are inserted between the two conserved kinase lobes and ϳ50 amino acids at the C terminus, respectively.HRI is a hemoprotein that contains two distinct of hemebinding sites (5-7). Heme binding to the first site is stable, and remains associated with purified HRI, whereas heme-binding to the second site is reversible and appears to be responsible for the rapid heme-induced down-regulation of HRI activity . We have recently demonstrated that the N-terminal domain of HRI contains the stable heme-binding...

Activation of Heme-regulated Eukaryotic Initiation Factor 2α Kinase by Nitric Oxide Is Induced by the Formation of a Five-coordinate NO-Heme Complex

“…Previous analyses reveal that NO-induced activation of HRI in rabbit reticulocyte lysates requires high concentrations of NOC9 (0.25-0.5 mM) (7). In the present investigation, HRI was activated at a range of physiological NO concentrations (ϳ20 M).…”

“…In Vitro eIF2␣ Kinase Assay-A previous study on HRI in rabbit reticulocyte lysates showed that NO binds to the heme iron and stimulates kinase activity, whereas binding of CO to the heme iron suppresses kinase activity (7). It is possible that in this system, regulation of HRI by NO or CO is mediated through interaction with HSP90 and its partner protein or modification of cysteine by NO.…”

“…Notably, HRI is expressed not only in reticulocytes but also in numerous tissues and cultured cells (7,16). Although only a limited quantity of HRI is present in the brain, reperfusion after brain ischemia results in the suppression of protein synthesis.…”

“…Specifically, NO affects reticulocyte HRI in situ and in vitro complexed with heat shock protein (HSP90) and its partner proteins (7,8). To determine the mechanism of NOinduced regulation of HRI, the heme environment of the isolated N-terminal heme-binding domain (HRI-NTD) was investigated using physicochemical methods and site-directed mutagenesis (9 -11).…”

Identification of Crucial Histidines for Heme Binding in the N-terminal Domain of the Heme-regulated eIF2α Kinase

“…The other heme-binding site is located in the N-terminal domain (NTD) of HRI, which consists of ϳ165 amino acids (4). This domain is responsible for the stable "constitutive" binding of heme to HRI (4) and appears to be the active center for nitric oxide (NO)-induced activation of HRI (5). The isolated NTD stably binds heme but shows no kinase activity (4).…”