Publications
132 results found
Gil J, Rullas J, Alcamí J, et al., 2001, MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-kappaB activation and apoptosis induced by PKR, J Gen Virol, Vol: 82, Pages: 3027-34
Molluscum contagiosum virus (MCV) is a human poxvirus that causes abnormal proliferation of epithelial cells. MCV encodes specific molecules to control host defences, such as MC159L, which as previously shown prevents apoptosis induced by death receptors. However, unlike most poxviruses, MCV lacks a homologue to the E3L and K3L proteins of vaccinia virus, which are involved in the control of the key antiviral and pro-apoptotic dsRNA-dependent protein kinase, PKR. In this study, we analysed the relationship of MC159L to PKR. We found that MC159L is not a direct inhibitor of PKR since it does not associate with PKR and cannot block PKR-induced phosphorylation of eIF-2alpha. However, expression of MC159L inhibits apoptosis triggered by PKR through death receptor-mediated pathways. In addition, MC159L inhibits NF-kappaB activation induced in response to PKR. Expression of MC159L cannot counteract the PKR-mediated antiviral action in the context of a poxvirus infection, despite its ability to affect these signalling events. These findings show that MC159L is able to interfere with downstream events triggered by PKR in the absence of a direct physical interaction, and assign a role to MC159L in the control of some PKR-mediated biological effects.
Gil J, Rullas J, Alcamí J, et al., 2001, MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-κB activation and apoptosis induced by PKR, Journal of General Virology, Vol: 82, Pages: 3027-3034, ISSN: 0022-1317
Molluscum contagiosum virus (MCV) is a human poxvirus that causes abnormal proliferation of epithelial cells. MCV encodes specific molecules to control host defences, such as MC 159L, which as previously shown prevents apoptosis induced by death receptors. However, unlike most poxviruses, MCV lacks a homologue to the E3L and K3L proteins of vaccinia virus, which are involved in the control of the key antiviral and pro-apoptotic dsRNA-dependent protein kinase, PKR. In this study, we analysed the relationship of MC159L to PKR. We found that MC159L is not a direct inhibitor of PKR since it does not associate with PKR and cannot block PKR-induced phosphorylation of elF-2α. However, expression of MC159L inhibits apoptosis triggered by PKR through death receptor-mediated pathways. In addition, MC159L inhibits NF-κB activation induced in response to PKR. Expression of MC159L cannot counteract the PKR-mediated antiviral action in the context of a poxvirus infection, despite its ability to affect these signalling events. These findings show that MC159L is able to interfere with downstream events triggered by PKR in the absence of a direct physical interaction, and assign a role to MC159L in the control of some PKR-mediated biological effects.
Gil J, Rullas J, García MA, et al., 2001, The catalytic activity of dsRNA-dependent protein kinase, PKR, is required for NF-kappaB activation, Oncogene, Vol: 20, Pages: 385-94
The double stranded RNA-dependent protein kinase (PKR), in addition to its role as a translational controlling factor, is a key transcriptional regulator exerting antiviral and antitumoral activities. We have previously shown that induction of NF-kappaB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. To gain insights into the mechanism of activation of NF-kappaB by PKR, we have analysed the domains of PKR involved in IKK activation and subsequent NF-kappaB induction. In PKR(0/0) cells infected with a collection of vaccinia virus (VV) recombinants expressing different mutant forms of PKR, we found that only PKR forms conserving the catalytic activity are able to activate NF-kappaB. An inactive PKR mutant (K296R), was unable to induce NF-kappaB activation despite full expression of the protein in a wide range of concentrations, as defined by Western blot, EMSA, IKK kinase activity and NF-kappaB transactivation assays. Moreover, the mutant PKR (K296R) acts as a dominant negative of PKR-induced eIF-2alpha phosphorylation and NF-kappaB activation. However, PKR mutants unable to activate NF-kappaB still retain their ability to associate with the IKK complex, as confirmed by immunoprecipitation analysis. We conclude that the catalytic activity of PKR and not only a protein-protein interaction with the IKK complex, is needed for activation of the transcription factor NF-kappaB.
Gil J, Esteban M, Roth D, 2000, In vivo regulation of the dsRNA-dependent protein kinase PKR by the cellular glycoprotein p67, Biochemistry, Vol: 39, Pages: 16016-25
Regulation of eIF2alpha phosphorylation is critical to the maintenance of cellular homeostasis, and eIF2alpha kinases are subject to complex and multidimensional controls. A cellular 67 kDa glycoprotein (p67) has been proposed to have an important role in regulating the activity of eIF2alpha kinases including the interferon-induced, dsRNA-stimulated protein kinase PKR. To dissect p67-PKR interactions and evaluate their significance in vivo, we have used a vaccinia virus (VV) expression system that successfully mimics PKR control pathways. Recombinant VV were constructed that constitutively express p67 and inducibly express PKR in BSC-40 cells. Stable expression of p67 reduced the PKR-mediated antiviral response and apoptosis. These effects correlated with decreased eIF2alpha phosphorylation, with rescue of PKR-mediated inhibition of protein synthesis, and with partial inhibition of PKR-triggered activation of NF-kappaB. The direct interaction between PKR and p67 was suggested by in vivo and in vitro analyses. These data demonstrate that in vivo p67 is an important modulator of PKR-mediated signal transduction pathways and may provide a useful tool to dissect the relative contributions of PKR to cell growth and stress response.
Gil J, Esteban M, 2000, The interferon-induced protein kinase (PKR), triggers apoptosis through FADD-mediated activation of caspase 8 in a manner independent of Fas and TNF-alpha receptors, Oncogene, Vol: 19, Pages: 3665-74
The interferon-induced dsRNA-dependent protein kinase (PKR) induces apoptosis of mammalian cells. Apoptosis induction by PKR involves phosphorylation of the translational factor eIF-2alpha and activation of the transcriptional factor NF-kappaB, but caspase pathways activated by PKR are not known. Upregulation of Fas mRNA by PKR has been suggested to play a role in PKR-induced apoptosis. To learn how PKR induces apoptosis, we have analysed the role of molecules in death receptor pathways. We showed the involvement of the FADD-caspase 8 pathway on PKR-induced apoptosis based on four experimental findings: upregulation of caspase 8 activity during PKR-induced apoptosis, blocking of PKR-induced apoptosis by the use of a chemical inhibitor of caspase 8, and inhibition of PKR-induced apoptosis by expression of both a FADD dominant negative or a viral FLIP molecule. Significantly, despite the PKR-mediated upregulation of Fas mRNA expression, the Fas receptor-ligand pathway is not needed for PKR-induced apoptosis. Antibodies that inhibit TNFalpha-TNFR1 or Fas-FasL interactions were not able to block PKR-induced apoptosis. Taken together, our observations establish the involvement of caspase 8 in PKR-induced apoptosis and suggest that death receptors other than Fas or TNFR1 or, alternatively, a novel mechanism involving FADD independently of death receptors, are responsible for PKR-induced apoptosis.
Gil J, Esteban M, Roth D, 2000, In vivo regulation of protein synthesis by phosphorylation of the alpha subunit of wheat eukaryotic initiation factor 2, Biochemistry, Vol: 39, Pages: 7521-30
The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2alpha to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2alpha and inducibly express the eIF2alpha dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2alpha resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2alpha on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2alpha reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2alpha and the 51A mutant with mammalian eIF2gamma and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2alpha is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.
Gil J, Esteban M, 2000, Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action, Apoptosis, Vol: 5, Pages: 107-14
Interferons are a family of cytokines that exerts antiviral, antitumor and immunomodulatory actions by inducing a complex set of proteins. One of the best known IFN-induced protein is the dsRNA-dependent protein kinase (PKR), that mediates both antiviral and anticellular activities. PKR inhibits translation initiation through the phosphorylation of the alpha subunit of the initiation factor eIF-2 (eIF-2 alpha) and also controls the activation of several transcription factors such as NF-kappa B, p53, or STATs. In addition, PKR mediates apoptosis induced by many different stimuli, such as treatment with LPS, TNF-alpha, viral infection, or serum starvation. The mechanism of apoptosis induction by PKR involves phosphorylation of eIF-2 alpha and activation of NF-kappa B. In this way, expression of different genes is regulated by PKR. Among the genes upregulated in response to PKR are Fas, Bax and p53. The pathway of PKR-induced apoptosis involves FADD activation of caspase 8 by a mechanism independent of Fas and TNFR. Since IFNs are used as drugs for different disorders such as viral infection and cancer, understanding the pathway of apoptosis induction triggered by PKR should be useful in the rational design of IFN therapies.
Gil J, Alcamí J, Esteban M, 2000, Activation of NF-kappa B by the dsRNA-dependent protein kinase, PKR involves the I kappa B kinase complex, Oncogene, Vol: 19, Pages: 1369-78
Besides its known role as a translational controlling factor, the double stranded RNA-dependent protein kinase (PKR) is a key transcriptional regulator exerting antiviral and antitumoural activities. We have recently described that induction of NF-kappa B by PKR is involved in apoptosis commitment. To define how PKR mediates NF-kappa B activation by dsRNA, we have used two different approaches, one based on expression of PKR by a vaccinia virus (VV) recombinant and the other based on induction of endogenous PKR by poly I:C (pIC) treatment. We found that NF-kappa B complexes induced by PKR are composed primarily of p50-p65 heterodimers and also of c-rel-p50 heterodimers. As described for other stimuli, following pIC treatment, PKR phosphorylates the NF-kappa B inhibitor I kappa B alpha at serine 32 before degradation. Expression by VV recombinants of IKK1 or IKK2 dominant negative mutants together with PKR showed inhibition of PKR-induced NF-kappa B activation, as measured both by gel shift and luciferase reporter assays. Immunoprecipitation analysis revealed that PKR interacts with the IKK complex. Our findings demonstrate that physiological function(s) of PKR involve activation of the I kappa B kinase complex. Oncogene (2000) 19,1369 - 1378.
Rivas C, Gil J, Esteban M, 1999, Identification of functional domains of the interferon-induced enzyme PKR in cells lacking endogenous PKR, J Interferon Cytokine Res, Vol: 19, Pages: 1229-36
The interferon (IFN)-induced, double-stranded RNA (dsRNA)-activated human protein kinase (PKR) has been shown to exert antiviral and antiproliferative effects. Activation of the enzyme in mammalian cells results in protein synthesis inhibition and cell death by apoptosis. Previous studies on the structure-function relationship of PKR have been based on vectors expressing the enzyme in mammalian cells containing endogenous PKR. As exogenously expressed PKR can form heterodimers with endogenous PKR, the results obtained on the functional characterization of mutant forms of PKR have been taken with caution. To address the natural consequences of heterodimer formation between endogenous and exogenous PKR, we have analyzed the structure-function relationship of PKR ectopically expressed from vaccinia virus (VV) recombinants in cells lacking the endogenous enzyme. We demonstrate that PKR-mediated inhibition of protein synthesis and induction of apoptosis is not dependent on the presence of endogenous PKR. Further, PKR activity is independent of the presence of dsRNA binding motifs (dsRBM). Moreover, single-point mutations of the third basic domain decreased PKR activation. Our findings demonstrate that PKR can be activated in the absence of its N-terminal domain (amino acids 1-232) and that the third basic domain is important for its biologic function.
Gil J, Alcamí J, Esteban M, 1999, Induction of apoptosis by double-stranded-RNA-dependent protein kinase (PKR) involves the alpha subunit of eukaryotic translation initiation factor 2 and NF-kappaB, Mol Cell Biol, Vol: 19, Pages: 4653-63
The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2alpha (alpha subunit of eukaryotic translation initiation factor 2) and IkappaBalpha, the inhibitor of the transcription factor NF-kappaB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-kappaB and eIF-2alpha in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2alpha or a repressor form of IkappaBalpha. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2alpha (Ser-51–>Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IkappaBalpha (Ser-32, 36-Ala) also leads to the inhibition of apoptosis by abolishing NF-kappaB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IkappaBalpha degradation, prevented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-kappaB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2alpha and NF-kappaB in the induction of apoptosis by PKR.
Gil J, Esteban M, 1998, Involvement of eIF2a and I-kBa in apoptosis mediated by the ds-RNA dependent protein kinase PKR, Publisher: JOHN LIBBEY EUROTEXT LTD, Pages: 307-307, ISSN: 1148-5493
Rivas C, Gil J, elková ZMU, et al., 1998, Vaccinia virus E3L protein is an inhibitor of the interferon (i.f.n.)-induced 2-5A synthetase enzyme, Virology, Vol: 243, Pages: 406-14
Induction of apoptosis in mammalian cells by double-stranded (ds) RNA-dependent enzymes, protein kinase (PKR), and 2-5A-synthetase/RNase L (referred to as the 2-5A system) might be a mechanism mediating anticellular and antiviral actions of interferon (i.f.n.). To counteract the effect of i.f.n., animal viruses have acquired genes that block specific i.f.n. pathways. Among poxviruses, vaccinia virus (VV) encodes E3L, a dsRNA-binding protein, which inhibits activation of i.f.n.-induced PKR. It has been proposed that E3L might also block activation of the 2-5A system, but direct proof is lacking. To establish if E3L inhibits the 2-5A system, we have developed a method to assay apoptosis induced by increased production of enzymes in the 2-5A pathway, as well as of their putative modulators. This assay is based on the use of cells derived from homozygous PKR knockout mice (Pkr-/-) infected with a VV mutant lacking E3L (delta E3L) and transiently transfected with a luciferase reporter gene together with plasmid vectors expressing 2-5A-synthetase, RNase L, or E3L, all controlled by the same inducible promoter. We found that expression of 2-5A-synthetase inhibited luciferase activity in a dose-response manner, reaching inhibition values of 80% relative to transfections with control plasmids. Similar results were obtained by transfection with an RNase L vector, although in this case the extent of inhibition was further enhanced upon coexpression of 2-5A-synthetase and RNase L. Inhibition of protein synthesis mediated by the 2-5A system correlated well with induction of apoptosis. Transfection of cells with a plasmid vector expressing E3L together with 2-5A-synthetase completely prevented apoptosis induced by this enzyme. We conclude that VV E3L acts as an inhibitor of the i.f.n.-induced 2-5A-synthetase enzyme.
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