Ara G. Hovanessian

Molecular cloning and characterization of the human double-stranded RNA-activated protein kinase induced by interferon

The double-stranded (ds) RNA-activated protein kinase from human cells is a 68 kd protein (p68 kinase) induced by interferon. On activation by dsRNA in the presence of ATP, the kinase becomes autophosphorylated and can catalyze the phosphorylation of the alpha subunit of eIF2, which leads to an inhibition of the initiation of protein synthesis. Here we report the molecular cloning and characterization of several related cDNAs from which can be deduced the full-length p68 kinase sequence. All of the cDNAs identify a 2.5 kb RNA that is strongly induced by interferon. The deduced amino acid sequence of the p68 kinase predicts a protein of 550 amino acids containing all of the conserved domains specific for members of the protein kinase family, including the catalytic domain characteristic of serine/threonine kinases. In vitro translation of a reconstructed full-length p68 kinase cDNA yields a protein of 68 kd that binds dsRNA, is recognized by a monoclonal antibody raised against the native p68 kinase, and is autophosphorylated.

The cytopathic effect of HIV is associated with apoptosis.

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.

Human p68 kinase exhibits growth suppression in yeast and homology to the translational regulator GCN2.

The human p68 kinase is an interferon‐regulated enzyme that inhibits protein synthesis when activated by double‐stranded RNA. We show here that when expressed in Saccharomyces cerevisiae, the p68 kinase produced a growth suppressing phenotype resulting from an inhibition of polypeptide chain initiation consistent with functional protein kinase activity. This slow growth phenotype was reverted in yeast by two different mechanisms: expression of the p68 kinase N‐terminus, shown to bind double‐stranded RNA in vitro and expression of a mutant form of the alpha‐subunit of yeast initiation factor 2, altered at a single phosphorylatable site. These results provide the first direct in vivo evidence that the p68 kinase interacts with the alpha‐subunit of eukaryotic initiation factor 2. Sequence similarity with a yeast translational regulator, GCN2, further suggests that this enzyme may be a functional homolog in higher eukaryotes, where its normal function is to regulate protein synthesis through initiation factor 2 phosphorylation.

Functional expression and RNA binding analysis of the interferon-induced, double-stranded RNA-activated, 68,000-Mr protein kinase in a cell-free system.

Eukaryotic viruses have devised numerous strategies to downregulate activity of the interferon-induced, double-stranded (dsRNA)-activated protein kinase (referred to as p68 on the basis of its Mr of 68,000 in human cells). Viruses must exert this control to avoid extensive phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2) by p68 and the resultant negative effects on protein synthesis initiation. To begin to define the molecular mechanisms underlying this regulation, we optimized expression of p68 in an in vitro transcription-translation system utilizing the full-length cDNA clone. The in vitro-expressed kinase was autophosphorylated in response to dsRNAs and heparin in a manner similar to that for the native p68 provided that the kinase inhibitor, 2-aminopurine, was present during the in vitro translation reaction. Further, the activated kinase efficiently phosphorylated its natural substrate, the alpha subunit of eIF-2. Binding experiments revealed that the expressed kinase complexed with the dsRNA activator, reovirus dsRNA, as well as the adenovirus-encoded inhibitor, VAI RNA. Interestingly, both the reovirus RNAs and VAI RNA also complexed with protein kinase molecules that lacked the carboxyl terminus and all catalytic domains. Deletion analysis confirmed that the p68 amino terminus contained critical determinants for reovirus dsRNA and VAI RNA binding. Further, reovirus dsRNA efficiently bound to, but failed to activate, p68 kinase molecules containing a single amino acid substitution in the invariant lysine 295 present in catalytic domain II. Taken together, these data demonstrate that this expression system permits a detailed mutagenic analysis of the regions of p68 required for interaction with virus-encoded activators and repressors.

PKR Stimulates NF-κB Irrespective of Its Kinase Function by Interacting with the IκB Kinase Complex

ABSTRACT The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-κB. NF-κB is retained in the cytoplasm through binding to its inhibitor IκBα. The critical step in NF-κB activation is the phosphorylation of IκBα by the IκB kinase (IKK) complex. This activity releases NF-κB from IκBα and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-κB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2α-binding domain, can also activate NF-κB. This result indicated that NF-κB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2α relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR0/0 mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathioneS-transferase pull-down assay, we showed that PKR interacts with the IKKβ subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-κB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-κB signaling pathway without using its kinase activity.

Anticellular and antiviral effects of pppA(2′p5′A)n

Abstract pppA(2′p5′A)n, the 2′5′ linked oligoadenylic acid triphosphate commonly known as 2–5A, is synthesized by an enzyme fraction from interferon-treated cells and rabbit reticulocyte lysates. By the use of a calcium-phosphate coprecipitation technique, we show here that treatment of cells with 2–5A results in the inhibition of in vivo protein, RNA, and DNA synthesis. These inhibitory effects of 2–5A are transient at low concentrations of 2–5A ( M ). At higher concentrations, however, the inhibition of protein, RNA and DNA synthesis continues with an apparent effect on cell growth. Treatment of vesicular stomatitis virus (VSV)-infected cells with 2–5A results in the inhibition of virus RNA replication. In accord with this virus production is inhibited by 90–100% after treatment of infected cells with 10–100 n M of 2–5A. This inhibition seems to be mediated by the 2–5A-dependent nuclease which results in the degradation of viral RNA.

The Anti-HIV Cytokine Midkine Binds the Cell Surface-expressed Nucleolin as a Low Affinity Receptor

The growth factor midkine (MK) is a cytokine that inhibits the attachment of human immunodeficiency virus particles by a mechanism similar to the nucleolin binding HB-19 pseudopeptide. Here we show that the binding of MK to cells occurs specifically at a high and a low affinity binding site. HB-19 prevents the binding of MK to the low affinity binding site only. Confocal immunofluorescence laser microscopy revealed the colocalization of MK and the cell-surface-expressed nucleolin at distinct spots. The use of various deletion constructs of nucleolin then indicated that the extreme C-terminal end of nucleolin, containing repeats of the amino acid motif RGG, is the domain that binds MK. The specific binding of MK to cells is independent of heparan sulfate and chondroitin sulfate expression. After binding to cells, MK enters cells by an active process. Interestingly, the cross-linking of surface-bound MK with a specific antibody results in the clustering of surface nucleolin along with glycosylphosphatidylinositol-linked proteins CD90 and CD59, thus, pointing out that MK binding induces lateral assemblies of nucleolin with specific membrane components of lipid rafts. Our results suggest that the cell surface-expressed nucleolin serves as a low affinity receptor for MK and could be implicated in its entry process.

Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity

We have investigated the interaction of VAI RNA with the interferon‐induced, double‐stranded (ds) RNA‐activated protein kinase, P68, both of which regulate protein synthesis in adenovirus‐infected cells. Previous work has shown that during infection by the VAI RNA‐negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha‐subunit of the eukaryotic initiation factor, eIF‐2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331‐infected, wild‐type Ad2‐infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected‐cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild‐type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF‐1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon‐treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.

Surface Expressed Nucleolin Is Constantly Induced in Tumor Cells to Mediate Calcium-Dependent Ligand Internalization

Background Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in tumorigenesis and angiogenesis. Emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy. Methodology/Principal Findings By monitoring the expression of nucleolin mRNA, and by measuring the level of nucleolin protein recovered from the surface and nucleus of cells, here we show that the presence of nucleolin at the cell surface is dependent on the constant induction of nucleolin mRNA. Indeed, inhibitors of RNA transcription or translation block expression of surface nucleolin while no apparent effect is observed on the level of nucleolin in the nucleus. The estimated half-life of surface nucleolin is less than one hour, whereas that of nuclear nucleolin is more than 8 hours. Nucleolin mRNA induction is reduced markedly in normal fibroblasts that reach confluence, while it occurs continuously even in post-confluent epithelial tumor cells consistent with their capacity to proliferate without contact inhibition. Interestingly, cold and heat shock induce nucleolin mRNA concomitantly to enhanced mRNA expression of the heat shock protein 70, thus suggesting that surface nucleolin induction also occurs in response to an environmental insult. At the cell surface, one of the main functions of nucleolin is to shuttle specific extracellular ligands by an active transport mechanism, which we show here to be calcium dependent. Conclusion/Significance Our results demonstrate that the expression of surface nucleolin is an early metabolic event coupled with tumor cell proliferation and stress response. The fact that surface nucleolin is constantly and abundantly expressed on the surface of tumor cells, makes them a preferential target for the inhibitory action of anticancer agents that target surface nucleolin.

Suppression of Tumor Growth and Angiogenesis by a Specific Antagonist of the Cell-Surface Expressed Nucleolin

Background Emerging evidences suggest that nucleolin expressed on the cell surface is implicated in growth of tumor cells and angiogenesis. Nucleolin is one of the major proteins of the nucleolus, but it is also expressed on the cell surface where is serves as a binding protein for variety of ligands implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Methodology/Principal Findings By using a specific antagonist that binds the C-terminal tail of nucleolin, the HB-19 pseudopeptide, here we show that the growth of tumor cells and angiogenesis are suppressed in various in vitro and in vivo experimental models. HB-19 inhibited colony formation in soft agar of tumor cell lines, impaired migration of endothelial cells and formation of capillary-like structures in collagen gel, and reduced blood vessel branching in the chick embryo chorioallantoic membrane. In athymic nude mice, HB-19 treatment markedly suppressed the progression of established human breast tumor cell xenografts in nude mice, and in some cases eliminated measurable tumors while displaying no toxicity to normal tissue. This potent antitumoral effect is attributed to the direct inhibitory action of HB-19 on both tumor and endothelial cells by blocking and down regulating surface nucleolin, but without any apparent effect on nucleolar nucleolin. Conclusion/Significance Our results illustrate the dual inhibitory action of HB-19 on the tumor development and the neovascularization process, thus validating the cell-surface expressed nucleolin as a strategic target for an effective cancer drug. Consequently, the HB-19 pseudopeptide provides a unique candidate to consider for innovative cancer therapy.