Interdomain interactions regulate the activation of the heme-regulated eIF 2 alpha kinase

The heme-regulated inhibitor of protein synthesis (HRI) regulates translation through the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF 2). While HRI is best known for its activation in response to heme-deficiency, we recently showed that the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD) of HRI activated and suppressed its activity, respectively. Here, we examined the effect of hemin, NO, and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/Delta HBD). Hemin stabilized the interaction of NT-HBD with HRI/Delta HBD, and NO and CO disrupted and stabilized this interaction, respectively. Mutant HRI (Delta H-HRI), lacking amino acids 116-158 from the NT-HBD, was less sensitive to heme-induced inhibition, and mutant NT-HBD lacking these residues did not bind to HRI/Delta HBD. HRI/Delta HBD and Delta H-HRI also activated more readily than HRI in response to heme-deficiency. Thus, HRI's activity is regulated through the modulation of the interaction between its NT-HBD and catalytic domain.     

The N-terminal region of the heme-regulated eIF2alpha kinase is an autonomous heme binding domain.

The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation factor (eIF)2alpha kinase (HRI) was aligned to sequences in the NCBI data base using ENTREZ and a PAM250 matrix. Significant similarity was found between amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mammalian alpha-globins. Several conserved amino acid residues present in globins are conserved in the NTD of HRI. His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold. Recombinant NTD (residues 1-159) was expressed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate. These results indicate that the NTD of HRI is an autonomous heme-binding domain, with His83 possibly serving as the proximal heme binding ligand.     

Multiple autophosphorylation is essential for the formation of the active and stable homodimer of heme-regulated eIF2alpha kinase

In heme-deficient reticulocytes, protein synthesis is inhibited due to the activation of heme-regulated eIF2alpha kinase (HRI). Activation of HRI is accompanied by its phosphorylation. We have investigated the role of autophosphorylation in the formation of active and stable HRI. Two autophosphorylated species of recombinant HRI expressed in Escherichia coli were resolved by SDS-PAGE. Both species of HRI were multiply autophosphorylated on serine, threonine, and to a lesser degree also tyrosine residues. Species II HRI exhibited a much higher extent of autophosphorylation and thus migrates slower in SDS-PAGE than species I HRI. Similarly, HRI naturally present in reticulocytes also exhibited these species with different degrees of phosphorylation. Importantly, in heme-deficient intact reticulocytes, inactive species I HRI was converted completely into species II. We further separated and characterized these two species biochemically. We found that species I was inactive and had a tendency to aggregate while the more extensively autophosphorylated species II was an active heme-regulated eIF2alpha kinase and stable homodimer. Our results strongly suggest that autophosphorylation regulates HRI in a two-stage mechanism. In the first stage, autophosphorylation of newly synthesized HRI stabilizes species I HRI against aggregation. Although species I is an active autokinase, it is still without eIF2alpha kinase activity. Additional multiple autophosphorylation in the second stage is required for the formation of stable dimeric HRI (species II) with eIF2alpha kinase activity that is regulated by heme.     

Identification of crucial histidines for heme binding in the N-terminal domain of the heme-regulated eIF2alpha kinase

The heme-regulated eukaryotic initiation factor-2alpha (eIF2alpha) kinase (HRI) regulates the initiation of protein synthesis in reticulocytes. The binding of NO to the N-terminal heme-binding domain (NTD) of HRI positively modulates its kinase activity. By utilizing UV-visible absorption, resonance Raman, EPR and CD spectroscopies, two histidine residues have been identified that are crucial for the binding of heme to the NTD. The UV-visible absorption and resonance Raman spectra of all the histidine to alanine mutants constructed were similar to those of the unmutated NTD. However, the change in the CD spectra of the NTD construct containing mutation of His78 to Ala (H78A) indicated loss of the specific binding of heme. The EPR spectrum for the ferric H78A mutant was also substantially perturbed. Thus, His78 is one of the axial ligands for the NTD of HRI. Significant changes in the EPR spectrum of the H123A mutant were also observed, and heme readily dissociated from both the H123A and the H78A NTD mutants, suggesting that His123 was also an axial heme ligand. However, the CD spectrum for the Soret region of the H123A mutant indicated that this mutant still bound heme specifically. Thus, while both His78 and His123 are crucial for stable heme binding, the effects of their mutations on the structure of the NTD differed. His78 appears to play the primary role in the specific binding of heme to the NTD, acting analogously to the "proximal histidine" ligand of globins, while His123 appears to act as the "distal" heme ligand.     

Interdomain interactions regulate the activation of the heme-regulated eIF2α kinase

The heme-regulated inhibitor of protein synthesis (HRI) regulates translation through the phosphorylation of the α-subunit of eukaryotic initiation factor-2 (eIF2). While HRI is best known for its activation in response to heme-deficiency, we recently showed that the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD) of HRI activated and suppressed its activity, respectively. Here, we examined the effect of hemin, NO, and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/ΔHBD). Hemin stabilized the interaction of NT-HBD with HRI/ΔHBD, and NO and CO disrupted and stabilized this interaction, respectively. Mutant HRI (ΔH-HRI), lacking amino acids 116–158 from the NT-HBD, was less sensitive to heme-induced inhibition, and mutant NT-HBD lacking these residues did not bind to HRI/ΔHBD. HRI/ΔHBD and ΔH-HRI also activated more readily than HRI in response to heme-deficiency. Thus, HRI's activity is regulated through the modulation of the interaction between its NT-HBD and catalytic domain.     

Molecular cloning and characterization of a mouse alpha 2C2 adrenoceptor subtype gene.

A clone MA2C2 encoding the murine homologue of adrenoceptor alpha 2C2 was isolated from a mouse genomic library using a subtype specific probe. The nucleotide and the deduced amino acid sequences derived from an ApaI fragment (2 kb) of the clone reveal a single open reading frame encoding a putative receptor protein of 455 amino acids. The 5' untranslated region (0.5 kb) sequenced is characterized by high GC content and CpG island count.     

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.     

Identification of Cys385 in the isolated kinase insertion domain of heme-regulated eIF2 alpha kinase (HRI) as the heme axial ligand by site-directed mutagenesis and spectral characterization

Heme-regulated eIF2alpha kinase (HRI) is an important enzyme that modulates protein synthesis during cellular emergency/stress conditions, such as heme deficiency in red cells. It is essential to identify the heme axial ligand(s) and/or binding sites to establish the heme regulation mechanism of HRI. Previous reports suggest that a His residue in the N-terminal region and a Cys residue in the C-terminal region trans to the His are axial ligands of the heme. Moreover, mutational analyses indicate that a residue located in the kinase insertion (KI) domain between Kinase I and Kinase II domains in the C-terminal region is an axial ligand. In the present study, we isolate the KI domain of mouse HRI and employ site-directed mutagenesis to identify the heme axial ligand. The optical absorption spectrum of the Fe(III) hemin-bound wild-type KI displays a broad Soret band at around 373nm, while that of the Fe(II) heme-bound protein contains a band at 422nm. Spectral titration studies conducted for both the Fe(III) hemin and Fe(II) heme complexes with KI support a 1:1 stoichiometry of heme iron to protein. Resonance Raman spectra of Fe(III) hemin-bound KI suggest that thiol is the axial ligand in a 5-coordinate high-spin heme complex as a major form. Electron spin resonance (ESR) spectra of Fe(III) hemin-bound KI indicate that the axial ligands are OH(-) and Cys. Since Cys385 is the only cysteine in KI, the residue was mutated to Ser, and its spectral characteristics were analyzed. The Soret band position, heme spectral titration behavior and ESR parameters of the Cys385Ser mutant were markedly different from those of wild-type KI. Based on these spectroscopic findings, we conclude that Cys385 is an axial ligand of isolated KI.     

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.     

Evidence that protein phosphatase 5 functions to negatively modulate the maturation of the Hsp90-dependent heme-regulated eIF2alpha kinase

The maturation and activation of newly synthesized molecules of the heme-regulated inhibitor of protein synthesis (HRI) in reticulocytes require their functional interaction with Hsp90. In this report, we demonstrate that protein phosphatase 5 (PP5), a previously documented component of the Hsp90 chaperone machine, is physically associated with HRI maturation intermediates. The interaction of PP5 with HRI is mediated through Hsp90, as mutants of PP5 that do not bind Hsp90 do not interact with HRI. PP5 was also present in Hsp90 heterocomplexes with another Hsp90 cohort, p50(cdc37), and expression of newly synthesized HRI enhanced the amount of p50(cdc37) associated with Hsp90/PP5-HRI heterocomplexes. The functional significance of the interaction of PP5 with Hsp90-HRI heterocomplexes was examined by characterizing the effects of compounds that impact PP5 activity in vitro. The protein phosphatase inhibitors okadaic acid and nodularin enhanced the kinase activity of HRI when applied during HRI maturation/activation, while the PP5 activators arachidonic and linoleic acid repressed HRI activity when applied during HRI maturation/activation. However, application of these compounds after HRI's "transformation" to an Hsp90-independent form did not similarly impact HRI's kinase activity. Furthermore, the Hsp90 inhibitor geldanamycin negated the effects of phosphatase inhibitors on HRI maturation/activation. The finding that PP5 downregulates an Hsp90-dependent process supports models for regulated Hsp90 function and describes a novel potential substrate for PP5 function in vivo.     

Hsp90 regulates p50(cdc37) function during the biogenesis of the activeconformation of the heme-regulated eIF2 alpha kinase

Recent studies indicate that p50(cdc37) 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 alpha 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.     

Functional characterization of Drosophila melanogaster PERK eukaryotic initiation factor 2alpha (eIF2alpha) kinase.

Four distinct eukaryotic initiation factor 2alpha (eIF2alpha) kinases phosphorylate eIF2alpha at S51 and regulate protein synthesis in response to various environmental stresses. These are the hemin-regulated inhibitor (HRI), the interferon-inducible dsRNA-dependent kinase (PKR), the endoplasmic reticulum (ER)-resident kinase (PERK) and the GCN2 protein kinase. Whereas HRI and PKR appear to be restricted to mammalian cells, GCN2 and PERK seem to be widely distributed in eukaryotes. In this study, we have characterized the second eIF2alpha kinase found in Drosophila, a PERK homologue (DPERK). Expression of DPERK is developmentally regulated. During embryogenesis, DPERK expression becomes concentrated in the endodermal cells of the gut and in the germ line precursor cells. Recombinant wild-type DPERK, but not the inactive DPERK-K671R mutant, exhibited an autokinase activity, specifically phosphorylated Drosophila eIF2alpha at S50, and functionally replaced the endogenous Saccharomyces cerevisiae GCN2. The full length protein, when expressed in 293T cells, located in the ER-enriched fraction, and its subcellular localization changed with deletion of different N-terminal fragments. Kinase activity assays with these DPERK deletion mutants suggested that DPERK localization facilitates its in vivo function. Similar to mammalian PERK, DPERK forms oligomers in vivo and DPERK activity appears to be regulated by ER stress. Furthermore, the stable complexes between wild-type DPERK and DPERK-K671R mutant were mediated through the N terminus of the proteins and exhibited an in vitro eIF2alpha kinase activity.     

Molecular cloning and characterization of mouse aquaporin 6

In the rat kidney, aquaporin (AQP) 6 is localized in the intracellular vesicle membranes of type-A intercalated cells of the collecting duct; mouse AQP6 (mAQP6) has not been characterized. Although mAQP6 was originally cloned from cDNA in a mouse cerebellum library (GenBank NM 175087), we have independently cloned a cDNA encoding mAQP6 from an adult kidney cDNA library (C57BL/6J strain). We identified two different spliced variants of mAQP6: mAQP6a and mAQP6b. The mAQP6a isoform is almost identical to that of rat AQP6, whereas mAQP6b is identical to that reported in the mouse cerebellum library mentioned above. We found that the mRNA expression of these two spliced variants is regulated in a tissue-specific and age-dependent manner. Functional analyses of water and ion permeation revealed that mAQP6a functions like rat AQP6 and that mAQP6b does not function as either a water channel or an ion channel under our experimental conditions.     

Molecular cloning and functional characterization of mouse chitotriosidase

Mammalian chitinase and chitinase-like proteins are members of a recently discovered gene family. Thus far, neither chitin nor chitin synthase has been found in mammals. The existence of chitinase genes in mammals is intriguing and the physiologic functions of chitinases are not clear. Human chitotriosidase, also called chitinase 1 (chit1), has been cloned. It has been found that high levels of serum chitotriosidase are associated with several diseases, but the physiologic functions of this enzyme are still unclear. To facilitate the studies in animal models we cloned and characterized a cDNA that encodes the mouse chitotriosidase. The open reading frame of this cDNA predicts a protein of 464 amino acids with a typical chitinase structure, including a signal peptide, a highly conserved catalytic domain and a chitin-binding domain. The predicted amino acid sequence is highly homologous to that of human chitotriosidase and to that of mouse acidic mammalian chitinase. Sequence analysis indicates that the mouse chitotriosidase gene has 12 exons, spanning a 40-kb region in mouse chromosome 1. The constitutive expression of mouse chitotriosidase is restricted to brain, skin, bone marrow, kidney, tongue, stomach and testis. Recombinant expression of the cloned cDNA demonstrated that the encoded protein is secreted and has chitinolytic activity that is sensitive to the specific chitinase inhibitor allosamidin and has the ability to bind to chitin particles. Substitution mutations at the conserved catalytic site completely abolished the enzymatic activity of the recombinant protein. These studies illustrate that mouse chitotriosidase is a typical chitinase that belongs to the mammalian chitinase gene family.     

Molecular cloning of the human casein kinase II alpha subunit

A human cDNA encoding the alpha subunit of casein kinase II and a partial cDNA encoding the rat homologue were isolated by using a Drosophila casein kinase II cDNA probe. The 2.2-kb human cDNA contains a 1.2-kb open reading frame, 150 nucleotides of 5' leader, and 850 nucleotides of 3' noncoding region. Except for the first 7 deduced amino acids that are missing in the rat cDNA, the 328 amino acids beginning with the amino terminus are identical between human and rat. The Drosophila enzyme sequence is 90% identical with the human casein kinase II sequence, and there is only a single amino acid difference between the published partial bovine sequence and the human sequence. In addition, the C-terminus of the human cDNA has an extra 53 amino acids not present in Drosophila. Northern analysis of rat and human RNA showed predominant bands of 5.5, 3.1, and 1.8 kb. In rat tissues, brain and spleen had the highest levels of casein kinase II alpha subunit specific RNA, while skeletal muscle showed the lowest. Southern analysis of human cultured cell and tissue genomic DNA using the full-length cDNA probe revealed two bands with restriction enzymes that have no recognition sites within the cDNA and three to six bands with enzymes having single internal sites. These results are consistent with the possibility that two genes encode the alpha subunits.