Elucidation of molecular mechanism of stability of the heme-regulated eIF2α kinase upon binding of its ligand, hemin in its catalytic kinase domain.

The eIF2α kinase activity of the heme-regulated inhibitor (HRI) is regulated by heme which makes it a unique member of the family of eIF2α kinases. Since heme concentrations create an equilibrium for the kinase to be active/inactive, it becomes important to study the heme binding effects upon the kinase and understanding its mechanism of functionality. In the present study, we report the thermostability achieved by the catalytic kinase domain of HRI (HRI.CKD) upon ligand (heme) binding. Our CD data demonstrates that the HRI.CKD retains its secondary structure at higher temperatures when it is in ligand bound state. HRI.CKD when incubated with hemin loses its monomeric state and attains a higher order oligomeric form resulting in its stability. The HRI.CKD fails to refold into its native conformation upon mutation of H377A/H381A, thereby confirming the necessity of these His residues for correct folding, stability, and activity of the kinase. Though our in silico study demonstrated these His being the ligand binding sites in the kinase insert region, the spectra-based study did not show significant difference in heme affinity for the wild type and His mutant HRI.CKD.

[Cloning and expression of PKR gene and affinity purification of PKR interacting proteins].

AIM: To clone and express protein kinase regulated by double-stranded RNA (PKR) gene, and to purify and isolate PKR interacting proteins. METHODS: By using specific primers, PKR gene fragments tagged with HA and FLAG (FLAG-PKR-HA, and HA-PKR-FLAG) were amplified by PCR, and cloned into pSG5 vector. The recombinant plasmids were transfected into PKR knockdown (PKR(kd);) HeLa cells by Lipfectamine(TM); 2000. The expression of tagged PKR was confirmed by Western blotting. Finally, the PKR interacting proteins were isolated by tandem affinity purification (TAP) system using HA and FLAG antibodies, and visualized by Western blotting and SDS-PAGE silver staining. RESULTS: PKR expression plasmids were constructed and TAP system was successfully established. Silver staining showed that PKR, and two potential PKR interacting protein fragments were isolated by SDS-PAGE. CONCLUSION: We have successfully purified and isolated PKR interacting proteins, thus providing a basis for future research.

Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.

In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

The Zalpha domain of PKZ from Carassius auratus can bind to d(GC)(n) in negative supercoils.

PKZ was the most recently discovered member of eIF2alpha kinase family in fish. CaPKZ, the first identified fish PKZ, possessed a conserved eIF2alpha kinase catalytic domain in C-terminal and two Z-DNA binding domains (Zalpha) in N-terminal. The Zalpha of CaPKZ closely resembled that of other Z-DNA binding proteins: ADAR1, DLM-1, and E3L. In order to understand more about the function of CaPKZ, we expressed and purified three constructed peptides of CaPKZ (P(Zalpha)): P(Zalpha1Zalpha2), P(Zalpha1Zalpha1) and P(Zalpha2)(Zalpha2). Moreover, most of the plasmids containing d(GC)(n) inserts were maintained in the Z-conformation, as confirmed by using inhibition of methylation experiments and anti-Z-DNA antibody. Gel mobility shift assays were then used to examine the affinity of these P(Zalpha) to the recombinant plasmids. Meanwhile, a competition experiment using P(Zalpha1Zalpha2) and anti-Z-DNA antibody was performed. The results revealed that P(Zalpha1Zalpha2) and P(Zalpha1Zalpha1) were able to bind to the recombinant plasmids with high affinity, whereas P(Zalpha2)(Zalpha2) could not bind to it. In addition, dimerization of P(Zalpha1Zalpha2) indicated the function unit of Zalpha of CaPKZ would be a dimer.

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.

Biochemical analyses of multiple fractions of PKR purified from Escherichia coli.

PKR is a cellular protein kinase activated by double-stranded RNA (dsRNA) that phosphorylates eukaryotic initiation factor alpha (eIF2alpha) and inhibits protein translation. Activation of PKR is accompanied by Ser/Thr autophosphorylation on multiple sites. Because PKR negatively regulates cell growth, overexpression and purification of PKR are difficult to achieve. Here, we describe overexpression and purification of recombinant PKR protein from Escherichia coli under native conditions at the milligram level. Affinity, ion exchange, and gel filtration chromatographies revealed multiple fractions of PKR with distinctive biochemical characteristics. During gel filtration, a small amount of PKR was found in a high molecular weight (>300 kDa) fraction that also contained endogenous bacterial RNA. The PKR in this fraction has a constitutive substrate phosphorylation activity. The majority of PKR is found in fractions of lower molecular weight and is free of RNA but is differentially phosphorylated as examined by isoelectric focusing electrophoresis and can be further separated by gradient anion exchange chromatography. PKR eluted with low salt has a lower level of basal autophosphorylation, and its kinase activity can be induced by dsRNA. With an increasing NaCl gradient, the purified PKR exhibits an increased level of autophosphorylation and constitutive kinase activity but reduced dsRNA inducibility. The highest salt eluent of PKR exhibits little dsRNA-induced activation. The inducible activation of high salt eluent PKR by dsRNA can be partially restored by treatment with protein phosphatase 1. The production of multiple fractions of PKR with different biochemical properties in E. coli suggests that the spectrum of PKR activity and regulation in mammalian cells is likely to be similarly complex.

CO binding study of mouse heme-regulated eIF-2alpha kinase: kinetics and resonance Raman spectra.

Heme-regulated eukaryotic initiation factor (eIF)-2alpha kinase (HRI) regulates the synthesis of globin chains in reticulocytes with heme availability. In the present study, CO binding kinetics to the 6-coordinated Fe(II) heme of the amino-terminal domain of mouse HRI and resonance Raman spectra of the Fe(II)-CO complex are examined to probe the character of the heme environment. The CO association rate constant, k(on)', and CO dissociation rate constant, k(off), were 0.0029 microM(-1)s(-1) and 0.003 s(-1), respectively. These values are very slow compared with those of mouse neuroglobin and sperm whale myoglobin, while the k(off) value of HRI was close to those of the 6-coordinated hemoglobins from Chlamydomonas and barley (0.0022 and 0.0011 s(-1)). The dissociation rate constant of an endogenous ligand, which occurs prior to CO association, was 18.3 s(-1), which was lower than those (197 and 47 s(-1)) of the same 6-coordinated hemoglobins. Resonance Raman spectra suggest that the Fe-C-O adopts an almost linear and upright structure and that the bound CO interacts only weakly with nearby amino acid residues.

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA.

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.

Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.

PERK and IRE1 are type-I transmembrane protein kinases that reside in the endoplasmic reticulum (ER) and transmit stress signals in response to perturbation of protein folding. Here we show that the lumenal domains of these two proteins are functionally interchangeable in mediating an ER stress response and that, in unstressed cells, both lumenal domains form a stable complex with the ER chaperone BiP. Perturbation of protein folding promotes reversible dissociation of BiP from the lumenal domains of PERK and IRE1. Loss of BiP correlates with the formation of high-molecular-mass complexes of activated PERK or IRE1, and overexpression of BiP attenuates their activation. These findings are consistent with a model in which BiP represses signalling through PERK and IRE1 and protein misfolding relieves this repression by effecting the release of BiP from the PERK and IRE1 lumenal domains.

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.

RNA binding and modulation of PKR activity.

PKR is an RNA-dependent protein kinase that is induced in mammalian cells by interferon treatment. It is present in a latent or inactive form in mammalian cells and is activated by very low concentrations of double-stranded (ds) RNA. Activated PKR phosphorylates eIF2, an essential initiation factor of protein synthesis, as well as other substrates including histone IIA, a 90-kDa protein from rabbit reticulocytes, the inhibitor, IkappaB, of the transcription factor, NF-kappaB, and the HIV-1 Tat protein. PKR interacts with several cellular and viral products and these interactions modulate its activation by dsRNA. Here we describe methods that are used to study the activation or inhibition of PKR by RNA modulators. Specifically, we detail (1) the purification of PKR from interferon-treated mammalian cells, (2) functional assays for PKR activation and inhibition in vitro, using purified enzyme or crude cell lysates, and (3) assays allowing evaluation of the binding of dsRNA and single-stranded RNA to PKR.

The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon-inducible, double-stranded RNA-dependent kinase.

The parapoxvirus orf virus was resistant to type 1 (IFN-alpha) and type 2 (IFN-gamma) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon-resistance gene, and is referred to as the (putative) orf virus interferon-resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR-Tx) inhibited the activation (by autophosphorylation) of an interferon-inducible, double-stranded (ds) RNA-dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus-infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon-treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon-resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.

Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.

We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and online capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with > 60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and minimizes sample handling. The use of MALDI/TOF and LCQ, two types of mass spectrometers that are widely available to the biological community, will make this procedure readily accessible to biologists. We applied this technique to identify two autophosphorylation sites and to assign at least another 12 phosphorylation sites to two tryptic peptides in a series of experiments using a gel slice containing only 200 ng (3 pmol) of human double-stranded RNA-activated protein kinase expressed in a mutant strain of the yeast Saccharomyces cerevisiae.

The molecular basis of viral oncolysis: usurpation of the Ras signaling pathway by reovirus.

NIH-3T3 cells, which are resistant to reovirus infection, became susceptible when transformed with activated Sos or Ras. Restriction of reovirus proliferation in untransformed NIH-3T3 cells was not at the level of viral gene transcription, but rather at the level of viral protein synthesis. An analysis of cell lysates revealed that a 65 kDa protein was phosphorylated in untransformed NIH-3T3 cells, but only after infection with reovirus. This protein was not phosphorylated in infected or uninfected transformed cells. The 65 kDa protein was determined to be the double-stranded RNA-activated protein kinase (PKR), whose phosphorylation leads to translation inhibition. Inhibition of PKR phosphorylation by 2-aminopurine, or deletion of the Pkr gene, led to drastic enhancement of reovirus protein synthesis in untransformed cells. The emerging picture is one in which early viral transcripts trigger PKR phosphorylation in untransformed cells, which in turn leads to inhibition of translation of viral genes; this phosphorylation event is blocked by an element(s) in the Ras pathway in the transformed cells, allowing viral protein synthesis to ensue. The usurpation of the Ras signaling pathway therefore constitutes the basis of reovirus oncolysis.

Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum.

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.