RNA-activated Protein Kinase PKR Research Articles

HCV‐Host Cell Interactions: Role of Ebp‐1 in Hepatitis C virus Replication

Persistent hepatitis C virus ( HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish chronic HCV infection are poorly understood. Also, the role of specific cellular factors in chronic HCV infection is not well established. We have recently identified a cellular factor ErbB3 binding protein‐1 (Ebp1) that specifically interacts with HCV RNA genome and is inhibitory to HCV replication. Ebp1 is a double stranded RNA binding protein that is a member of a family of proliferation‐regulating proteins. Ebp1 has two isoforms, p42 and p48, which are synthesized from two differently spliced mRNAs from the same gene. Our preliminary results show that overexpression of Ebp1 (the longer as well as the shorter isoform) has a strong negative impact on HCV replication. We also found that Ebp1 binds to the HCV 5′NTR. We established that Ebp1 is mainly localized in the cytosol, a site of HCV replication. Further experiments showed that Ebp1 interacts not only with HCV viral proteins NS5A and NS5B, but also with interferon‐inducible double stranded RNA activated protein kinase PKR, which is critical for cellular antiviral and anti proliferative responses induced by interferon. Taken together our results suggest the critical role of Ebp1 in negatively regulating Hepatitis C virus infection. We propose to further identify the role of PKR in Ebp1 mediated signaling in Hepatitis C virus infection. Elucidation of the role of Ebp1 in activating PKR or vice versa will provide valuable insight into the molecular mechanisms of these interactions and their implication on HCV‐replication and associated pathogenesis.

P5.03 - Control of cell fate by the mammalian target of rapamycin (mTOR) under stress depends on eIF2α serine 51 phosphorylation

ABSTRACT The mammalian/mechanist target of rapamycin (mTOR) plays an essential role in cell growth and metabolism by regulating protein and RNA stability, transcription as well as mRNA translation. Phosphorylation of the a subunit of translation initiation factor eIF2 at serine 51 (eIF2aS51P) is a key regulator of mRNA translation and an important determinant of cell fate under stress. Some studies have provided evidence for a cross-talk between mTOR and eIF2aS51P under stress through poorly defined mechanisms. Herein, we demonstrate that genetic as well as pharmacological inhibition of mTOR complex 2 (mTORC2) induces eIF2aS51P via the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK as a result of Akt inactivation. Analysis of the functional interplay between eIF2aS51P and mTOR in tuberous sclerosis complex (TSC)-deficient cells showed that mTORC2 inactivation induces the PERK-eIF2aS51P arm to substitute for the loss of Akt and promote survival in response to ER stress (see Figure above). On the other hand, TSC-deficient cells decrease PERK activity and increase the activity of the double-stranded RNA-activated protein kinase PKR to promote death via eIF2aS51P, JNK activation and increased autophagy in response to oxidative stress (see attached Figure). The balance between PERK inactivation and PKR activation in TSC-mutant cells under oxidative stress is controlled by mTORC1 and ribosomal S6 kinase 1, 2 (S6K1, 2) activity. Impaired eIF2aS51P accelerates TSC-mutant tumor initiation in mice, which is associated with an impaired production of reactive oxygen species (ROS) compared to eIF2aS51P-proficient TSC-mutant tumors. Our study reveals that eIF2aS51P acts as a double-edged sword downstream of mTOR to promote either survival or death in response to distinct forms of stress with implications in tumor development and treatment with chemotherapeutic drugs. Download : Download full-size image P5.03 FIGURES .

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.

A survey of host range genes in poxvirus genomes

Poxviruses are widespread pathogens, which display extremely different host ranges. Whereas some poxviruses, including variola virus, display narrow host ranges, others such as cowpox viruses naturally infect a wide range of mammals. The molecular basis for differences in host range are poorly understood but apparently depend on the successful manipulation of the host antiviral response. Some poxvirus genes have been shown to confer host tropism in experimental settings and are thus called host range factors. Identified host range genes include vaccinia virus K1L, K3L, E3L, B5R, C7L and SPI-1, cowpox virus CP77/CHOhr, ectromelia virus p28 and 022, and myxoma virus T2, T4, T5, 11L, 13L, 062R and 063R. These genes encode for ankyrin repeat-containing proteins, tumor necrosis factor receptor II homologs, apoptosis inhibitor T4-related proteins, Bcl-2-related proteins, pyrin domain-containing proteins, cellular serine protease inhibitors (serpins), short complement-like repeats containing proteins, KilA-N/RING domain-containing proteins, as well as inhibitors of the double-stranded RNA-activated protein kinase PKR. We conducted a systematic survey for the presence of known host range genes and closely related family members in poxvirus genomes, classified them into subgroups based on their phylogenetic relationship and correlated their presence with the poxvirus phylogeny. Common themes in the evolution of poxvirus host range genes are lineage-specific duplications and multiple independent inactivation events. Our analyses yield new insights into the evolution of poxvirus host range genes. Implications of our findings for poxvirus host range and virulence are discussed.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Specificity of the Double-Stranded RNA-Binding Domain from the RNA-Activated Protein Kinase PKR for Double-Stranded RNA: Insights from Thermodynamics and Small-Angle X-ray Scattering

The interferon-inducible, double-stranded (ds) RNA-activated protein kinase (PKR) contains a dsRNA-binding domain (dsRBD) and plays key roles in viral pathogenesis and innate immunity. Activation of PKR is typically mediated by long dsRNA, and regulation of PKR is disfavored by most RNA imperfections, including bulges and internal loops. Herein, we combine isothermal titration calorimetry (ITC), electrophoretic mobility shift assays, and small-angle X-ray scattering (SAXS) to dissect the thermodynamic basis for the specificity of the dsRBD termed "p20" for various RNAs and to detect any RNA conformational changes induced upon protein binding. We monitor binding of p20 to chimeric duplexes containing terminal RNA-DNA hybrid segments and a central dsRNA segment, which was either unbulged ("perfect") or bulged. The ITC data reveal strong binding of p20 to the perfect duplex (K(d) ~ 30 nM) and weaker binding to the bulged duplex (K(d) ~ 2-5 μM). SAXS reconstructions and p(r) distance distribution functions further uncover that p20 induces no significant conformational change in perfect dsRNA but largely straightens bulged dsRNA. Together, these observations support the dsRBD's ability to tightly bind to only A-form RNA and suggest that in a noninfected cell, PKR may be buffered via weak interactions with various bulged and looped RNAs, which it may straighten. This work suggests that PKR-regulating RNAs with complex secondary and tertiary structures likely mimic dsRNA and/or engage portions of PKR outside of the dsRBD.

Abstract 1350: Transgenic expression of PKR induces a myelodysplastic syndrome/pre-leukemia-like condition in mice

Abstract Double stranded (ds) RNA activated protein kinase PKR was discovered over 30 years ago as a downstream mediator of the interferon signal pathway. Initial studies focused on PKR's anti-virus and pro-inflammatory effects. More recently, PKR has been found to be involved in signal transduction pathways critical for the negative regulation of cell growth and initiation of apoptosis. Significantly, loss of PKR expression/activity has been associated with increased growth of human breast carcinoma, B-cell CLL, and T-cell ALL using patient samples. Furthermore, PKR activity is increased in bone marrow progenitor cells isolated from patients with myelodysplastic syndrome (MDS) which undergo significant apoptosis compared to normal marrow progenitors not observed when evolved to AML. Thus, in the process of MDS evolution to leukemia, PKR expression/activity may be downregulated by a process that is currently unclear. To explore the role of PKR in MDS/AML we have constructed a transgenic mouse model expressing human PKR (TgPKR) under control of vav regulatory elements to drive expression specifically in hematopoietic-tissue. We hypothesized transgenic expression of human PKR would exert an inhibitory effect on mouse hematopoiesis, cause ineffective blood cell production, and thus simulate a disease process similar to human MDS. In our preliminary data, quantitative PCR and immunoblotting results confirm that human PKR is specifically expressed in spleen, thymus and bone marrow (BM) but not in liver, kidney, intestine, muscle and other tissues outside hematopoiesis in TgPKR mice. Aberrant activation of transcription factor STAT1 and MAPKinases ERK1/2 occurs in hematopoietic cells from the TgPKR mice. In addition, inhibitory cytokines including interferon gamma, CXCL9 and 10 are overexpressed. Initial characterization of BM cells from TgPKR mice indicates a higher level of uninduced/basal apoptosis in growth medium and increased apoptosis upon withdrawal of growth factors compared to cells from wild type (WT) littermates. Furthermore, BM cells from TgPKR mice display >20% reduced colonies (CFU-GM and CFU-GEMM) which contain 40% less cells than those from WT littermates. Significantly, histopathological analysis of BM and spleen from TgPKR mice reveals a ∼20% increase in bone marrow cellularity with a striking increase in dysplastic megakaryocytes that are either small without nuclear lobation or of normal size but with hypolobation or separated nuclear lobes. Taken together our results demonstrate that transgenic expression of hPKR induces an MDS-like condition in mice characterized by reduced BM cell survival, increased apoptosis and reduced proliferation and differentiation of BM progenitor/stem cells. Thus, increased PKR expression in patients may, at least in part, lead to a BM failure state similar to MDS. Thus, future anti-MDS therapies that inhibit PKR may be clinically useful. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1350. doi:1538-7445.AM2012-1350

Abstract 2042: The RAX/PACT-PKR stress-signaling pathway promotes p53 sumoylation and stability to induce G1 arrest

Abstract Cellular stresses including IL-3 withdrawal from factor-dependent hematopoietic cells, inflammatory cytokine treatment or viral infection promote RAX/PACT-dependent activation of the interferon-induced, double-stranded RNA activated protein kinase PKR. Previously, we and others demonstrated that activated PKR phosphorylates the alpha subunit of eIF2 to inhibit protein synthesis and initiate apoptosis. In addition, recent reports indicate that PKR may regulate the transcription factors p53, STAT1 and NF-κB. Now we find that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, stable or transient expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while expression of RAX/PACT, PKR or p53 alone has little effect on the cell cycle of p53-null H1299 cells, co-expression of p53 with either RAX/PACT or PKR promotes a 25 - 35% increase of cells in G1. In contrast, co-expression of RAX/PACT with the sumoylation-deficient p53 (K386R) mutant or with p53 and the desumoylase SENP1 fails to induce such a G1 arrest. Furthermore, RAX/PACT-induced, p53-dependent G1 growth arrest is inhibited by expressing the dominant-negative PKR (K296R) mutant, and expression of RAX/PACT in pkr+/+ but not pkr-/- MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53 (K386R) on serine 392, and p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment. Collectively, results indicate that in response to stress the RAX/PACT-PKR signaling pathway may promote p53 translational activation leading to G1 cell cycle arrest by a mechanism dependent on p53 sumoylation and phosphorylation that regulates protein turnover. Importantly, these novel findings may indicate a wider role for the RAX/PACT-PKR signaling pathway to direct sumoylation-dependent subcellular localization, protein stability or protein-protein interactions of downstream targets in response to cellular stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2042. doi:1538-7445.AM2012-2042

Plasmacytoid dendritic cells as guardians in hepatitis C virus–infected liver

The innate immune response to viruses involves type I IFN (i.e., IFN-α and -β)–modulated up-regulation of cellular genes that are key mediators of antiviral defenses. Cellular proteins such as RNA helicase retinoic acid–inducible gene I (RIG-I) and Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, which are the hallmark of viral infections (1, 2). Consequently, many viruses have developed strategies to evade the IFN system. Hepatitis C virus (HCV), a liver-tropic virus that chronically infects approximately 170 million people worldwide (3), is able to attenuate the IFN response at multiple levels within infected hepatocytes. For example, viral NS3/4A protease is able to cleave key intermediate proteins that are involved in TLR3- and RIG-I–mediated antiviral signaling (Fig. 1) in cultured cells (reviewed in ref. 4). Furthermore, viral NS5A protein is able to block activation of the double-stranded RNA–activated protein kinase PKR (reviewed in ref 4). Curiously, HCV infection induces a robust production of IFN-stimulated genes in the liver (5, 6). These observations present a conundrum: HCV inhibits production of IFN and IFN-stimulated genes (ISGs) in the cultured liver, yet induces IFN production in the infected organ. Either the virus cannot block IFN-mediated antiviral responses in the highly differentiated liver organ, or IFN is produced in the liver by cells other than HCV-infected hepatocytes.

Modulation of innate immunity system by Epstein–Barr virus‐encoded non‐coding RNA and oncogenesis

Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are polyA-, non-coding RNAs that are expressed abundantly in all forms of cells latently infected with EBV. EBERs (EBER1 and EBER2) contribute to the clonal proliferation of EBV-negative Burkitt's lymphoma (BL) cells in soft agar, tumorigenicity in SCID mice, up-regulation of the bcl-2 oncoprotein, resistance to apoptosis, and maintenance of malignant phenotypes in BL cells. EBERs induce the expression of interleukin (IL)-10 in BL cells, insulin-like growth factor 1 (IGF-I) in gastric and nasopharyngeal carcinoma cells, IL-9 in T cells, and IL-6 in lymphoblastoid cell lines. Additionally, each of these cytokines acts as an autocrine growth factor. In BL cells, EBERs bind the double-stranded RNA-activated protein kinase PKR, inhibit its phosphorylation, and thereby prevent IFN-alpha-mediated apoptosis. In epithelial cells, EBERs confer resistance to Fas-mediated apoptosis by blocking PKR activity. EBERs form complexes with PKR, ribosomal protein L22, lupus erythematosis-associated antigen (La), and retinoic acid-inducible gene I (RIG-I). In BL cells, EBERs activate RIG-I signaling and induce the expression of type-I IFNs and interferon stimulated genes (ISGs) through the activation of RIG-I substrates, nuclear factor-kappa B (NF-kappaB), and IFN regulatory factor 3 (IRF-3), and anti-inflamatory cytokine IL-10 through IRF-3 but not NF-kappaB signaling. EBERs also play critical roles in the growth transformation of B lymphocytes. Although EBER1 and EBER2 exhibit similarities in their primary (54%) and secondary structures, recent findings have shown that recombinant EBVs carrying only the EBER2 gene play a greater role in the growth transformation of B lymphocytes than EBVs carrying only the EBER1 gene. Thus, EBERs play multiple roles in various cell types, and we present a model that highlights the functions of EBERs in EBV-mediated oncogenesis in BL cells.

PKR and the ribosome compete for mRNA

The RNA-activated protein kinase PKR inhibits translation initiation by sensing long viral double-stranded RNA. A new report indicates that PKR is also activated by a cellular mRNA, but only when ribosomes are not initiating translation.

Mitogen-activated protein kinase-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.

Viral infections induce signaling pathways in mammalian cells that stimulate innate immune responses and affect cellular processes, such as apoptosis, mitosis, and differentiation. Here, we report that the ribosomal protein S6 kinase alpha 3 (RSK2), which is activated through the "classical" mitogen-activated protein kinase pathway, plays a role in innate immune responses to influenza virus infection. RSK2 functions in the regulation of cell growth and differentiation but was not known to play a role in the cellular antiviral response. We have found that knockdown of RSK2 enhanced viral polymerase activity and growth of influenza viruses. Influenza virus infection stimulates NK-kappaB- and beta interferon-dependent promoters. This stimulation was reduced in RSK2 knockdown cells, suggesting that RSK2 executes its effect through innate immune response pathways. Furthermore, RSK2 knockdown suppressed influenza virus-induced phosphorylation of the double-stranded RNA-activated protein kinase PKR, a known antiviral protein. These findings establish a role for RSK2 in the cellular antiviral response.

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein

As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.

Double-stranded RNA-activated protein kinase PKR of fishes and amphibians: Varying the number of double-stranded RNA binding domains and lineage-specific duplications

BackgroundDouble-stranded (ds) RNA, generated during viral infection, binds and activates the mammalian anti-viral protein kinase PKR, which phosphorylates the translation initiation factor eIF2α leading to the general inhibition of protein synthesis. Although PKR-like activity has been described in fish cells, the responsible enzymes eluded molecular characterization until the recent discovery of goldfish and zebrafish PKZ, which contain Z-DNA-binding domains instead of dsRNA-binding domains (dsRBDs). Fish and amphibian PKR genes have not been described so far.ResultsHere we report the cloning and identification of 13 PKR genes from 8 teleost fish and amphibian species, including zebrafish, demonstrating the coexistence of PKR and PKZ in this latter species. Analyses of their genomic organization revealed up to three tandemly arrayed PKR genes, which are arranged in head-to-tail orientation. At least five duplications occurred independently in fish and amphibian lineages. Phylogenetic analyses reveal that the kinase domains of fish PKR genes are more closely related to those of fish PKZ than to the PKR kinase domains of other vertebrate species. The duplication leading to fish PKR and PKZ genes occurred early during teleost fish evolution after the divergence of the tetrapod lineage. While two dsRBDs are found in mammalian and amphibian PKR, one, two or three dsRBDs are present in fish PKR. In zebrafish, both PKR and PKZ were strongly upregulated after immunostimulation with some tissue-specific expression differences. Using genetic and biochemical assays we demonstrate that both zebrafish PKR and PKZ can phosphorylate eIF2α in yeast.ConclusionConsidering the important role for PKR in host defense against viruses, the independent duplication and fixation of PKR genes in different lineages probably provided selective advantages by leading to the recognition of an extended spectrum of viral nucleic acid structures, including both dsRNA and Z-DNA/RNA, and perhaps by altering sensitivity to viral PKR inhibitors. Further implications of our findings for the evolution of the PKR family and for studying PKR/PKZ interactions with viral gene products and their roles in viral infections are discussed.

Requirement for protein kinase R in interleukin-1α-stimulated effects in cartilage

Interleukin-1 (IL-1) has pleiotropic effects in cartilage. The interferon-induced, double stranded RNA-activated protein kinase PKR that phosphorylates eukaryotic initiation factor 2 (eIF2) α has been implicated in cytokine effects in chondrocytes. A compound was recently identified that potently suppresses PKR autophosphorylation (IC50 approximately 200 ηM) and partially restores PKR-inhibited translation in a cell-free system with significant effect in the nanomolar range. The objectives of this study were to exploit this potent PKR inhibitor to assess whether PKR kinase activity is required for catabolic and proinflammatory effects of IL-1α in cartilage and to determine whether IL-1α causes an increase in eIF2α phosphorylation that is antagonized by the PKR inhibitor. Cartilage explants were incubated with the PKR inhibitor and IL-1α. Culture media were assessed for sulfated glycosaminoglycan as an indicator of proteoglycan degradation and for prostaglandin E 2. Cartilage extracts were analyzed by Western blot for cyclooxygenase-2 and phosphorylated signaling molecules. Nanomolar concentrations of the PKR inhibitor suppressed proteoglycan degradation and cyclooxygenase-2 accumulation in IL-1α-activated cartilage. The PKR inhibitor stimulated or inhibited PGE 2 production with a biphasic dose response relationship. IL-1α increased the phosphorylation of both PKR and eIF2α, and nanomolar concentrations of PKR inhibitor suppressed the IL-1α-induced changes in phosphorylation. The results strongly support PKR involvement in pathways activated by IL-1α in chondrocytes.

Mapping of the interacting domains of hepatitis C virus core protein and the double-stranded RNA-activated protein kinase PKR

Hepatitis C virus (HCV) core protein has been shown to exhibit several biological properties which suggest an important role in liver pathogenesis and carcinogenesis. During a previous study, we showed that core mutants, isolated from tumour, could directly interact with PKR and maintain it in an activated form. In the present report, we have further investigated this interaction and mapped the core and PKR domains involved. Using glutathion S-transferase fusion protein harbouring the different domains of core or PKR, we determined that the N-terminal 1–58 amino acid (aa) of core protein and the N-terminal 1–180 aa of PKR are responsible for this direct interaction. Using this system we also confirmed that the core–PKR interaction induced PKR autophosphorylation. Furthermore, we found that core protein co-localized and co-immunoprecipitated with PKR in cells expressing a full-length HCV replicon, thus confirming that this interaction occurs when all HCV proteins are expressed. Considering that the activation of PKR has been observed in some cancer cell lines and tissues, it suggests that, depending on the cellular context, PKR may stimulate or inhibit cell proliferation. The precise mapping of core–PKR interaction provides new data to study the molecular mechanism underlying HCV pathogenesis.

Nck in a Complex Containing the Catalytic Subunit of Protein Phosphatase 1 Regulates Eukaryotic Initiation Factor 2α Signaling and Cell Survival to Endoplasmic Reticulum Stress

Stress imposed on the endoplasmic reticulum (ER) induces the phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 (eIF2) on Ser51. This results in transient inhibition of general translation initiation while concomitantly activating a signaling pathway that promotes the expression of genes whose products improve ER function. Conversely, dephosphorylation of eIF2alphaSer51 is accomplished by protein phosphatase 1 (PP1c) complexes containing either the protein CReP or GADD34, which target PP1c to eIF2. Here, we demonstrate that the Src homology (SH) domain-containing adaptor Nck is a key component of a molecular complex that controls eIF2alpha phosphorylation and signaling in response to ER stress. We show that overexpression of Nck decreases basal and ER stress-induced eIF2alpha phosphorylation and the attendant induction of ATF4 and CHOP. In contrast, we demonstrate that the mouse embryonic fibroblasts lacking both isoforms of Nck (Nck1-/-Nck2-/-) show higher levels of eIF2alpha phosphorylation and premature induction of ATF4, CHOP, and GADD34 in response to ER stress and finally, are more resistant to cell death induced by prolonged ER stress conditions. We establish that a significant amount of Nck protein localizes at the ER and is in a complex with eIF2 subunits. Further analysis of this complex revealed that it also contains the Ser/Thr phosphatase PP1c, its regulatory subunit CReP, and dephosphorylates eIF2alpha on Ser51 in vitro. Overall, we demonstrate that Nck as a component of the CReP/PP1c holophosphatase complex contributes to maintain eIF2alpha in a hypophosphorylated state. In this manner, Nck modulates translation and eIF2alpha signaling in response to ER stress.

Gene therapy withE2F-1up-regulates the protein kinase PKR and inhibits growth of leiomyosarcomain vivo

Overexpression of the transcription factor E2F-1 induces apoptosis in a variety of carcinoma cells and inactivates murine double minute protein 2, a factor associated with poor prognosis in soft tissue sarcomas. We have shown previously that the double-stranded RNA-activated protein kinase PKR plays an important role in mediating this apoptotic response in carcinoma cells to E2F-1. We sought to evaluate the potential of E2F-1 gene therapy in soft tissue sarcomas and to study the involvement of PKR in the response to E2F-1 overexpression in mesenchymal cells. A replication-deficient adenovirus carrying the E2F-1 gene (Ad5E2F) was used to induce E2F-1 overexpression in the p53 mutated leiomyosarcoma cell line, SKLMS-1. Western blot analysis confirmed E2F-1 overexpression and up-regulation of the antiapoptotic factor Bcl-2 48 hours following infection with Ad5E2F. Apoptosis in Ad5E2F-treated cells was confirmed by fluorescence-activated cell sorting analysis and by poly(ADP-ribose) polymerase cleavage and DNA fragmentation assays. Vector-dependent up-regulation of PKR correlated with the amount of Ad5E2F-induced apoptosis. In vivo treatment of SKLMS-1 tumor-bearing BALB/c mice with intratumoral injections of Ad5E2F at a dose of 2 x 10(10) viral particles resulted in significant inhibition in tumor growth compared with control-treated animals (P < 0.016). Complete disappearance of all tumors was seen in two of seven mice in the Ad5E2F-treated animals. Immunohistochemical analysis of tumor specimens showed overexpression of E2F-1 and up-regulation of PKR in Ad5E2F-treated tumors. These findings show that adenovirus-mediated overexpression of E2F-1 results in up-regulation of PKR and significant growth suppression of leiomyosarcomas in vivo. Taken together, these data suggest that E2F-1 gene therapy and PKR modulation might be a promising treatment strategy for these tumors that are highly resistant to conventional therapies.

Melanoma differentiation-associated gene-7 protein physically associates with the double-stranded RNA-activated protein kinase PKR

We previously reported that adenoviral-mediated overexpression of the melanoma differentiation-associated gene-7 (Ad-mda7; approved gene symbol IL24) leads to the rapid induction of PKR and activation of its downstream targets, resulting in apoptosis induction in human lung cancer cells. To evaluate the mechanism by which Ad-mda7 activates PKR, we studied the interaction between MDA-7 and PKR proteins. Following Ad-mda7 transduction of lung cancer cells, intracellular and extracellular MDA-7 protein was generated, leading to dose- and time-dependent PKR induction. Purified MDA-7 protein administered extracellularly did not induce PKR or apoptosis, suggesting that Ad-mda7-mediated PKR activation and apoptosis were not dependent on extracellular MDA-7 protein. Following Ad-mda7 transduction, RT-PCR demonstrated no increase in PKR mRNA levels despite increased levels of PKR protein, suggesting posttranscriptional regulation of PKR by MDA-7. Immunofluorescence and coimmunoprecipitation studies demonstrated that MDA-7 protein physically interacts with PKR. Transduction of PKR+/+ and PKR-/- transformed MEFs with Ad-mda7 demonstrated phosphorylated MDA-7 and PKR proteins in the lysates of PKR+/+ but not PKR-/- cells. These findings identify the first binding partner for MDA-7 and suggest that direct interaction between PKR and MDA-7 may be important for PKR activation and apoptosis induction, possibly through MDA-7 phosphorylation or activation of other downstream targets.

Protein synthesis persists during necrotic cell death

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-α. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-α phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.