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Non-coding transcript in T cells (NTT): Antisense transcript activates PKR and NF-kappa B in human lymphocytes

ANDRE, Nayara Delgado; LUCCA, Femando L. De
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
ENG
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37.39%
T cell activation is a complex process involving many steps and the role played by the non-protein-coding RNAs (ncRNAs) in this phenomenon is still unclear. The non-coding T cells transcript (NTT) is differentially expressed during human T cells activation, but its function is unknown. Here, we detected a 426 m NTT transcript by RT-PCR using RNA of human lymphocytes activated with a synthetic peptide of HIV-1. After cloning, the sense and antisense 426 nt NTT transcripts were obtained by in vitro transcription and were sequenced. We found that both transcripts are highly structured and are able to activate PKR. A striking observation was that the antisense 426 nt NTT transcript is significantly more effective in activating PKR than the corresponding sense transcript. The transcription factor NF-kappa B is activated by PKR through phosphorylation and subsequent degradation of its inhibitor I-kappa B beta. We also found that the antisense 426 nt NTT transcript induces more efficiently the degradation Of I-kappa B beta than the sense transcript. Thus, this study suggests that the role played by NTT in the activation of lymphocytes can be mediated by PKR through NF-kappa B activation. However, the physiological significance of the activity of the antisense 426 nt NTT transcript remains unknown. (c) 2007 Elsevier Inc. All rights reserved.

Induction of Apoptosis by Double-Stranded-RNA-Dependent Protein Kinase (PKR) Involves the α Subunit of Eukaryotic Translation Initiation Factor 2 and NF-κB

Gil, Jesús; Alcamí, José; Esteban, Mariano
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /07/1999 EN
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27.64%
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-2α (α subunit of eukaryotic translation initiation factor 2) and IκBα, the inhibitor of the transcription factor NF-κB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-κB and eIF-2α 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-2α or a repressor form of IκBα. 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-2α (Ser-51→Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IκBα (Ser-32,36-Ala) also leads to the inhibition of apoptosis by abolishing NF-κB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IκBα 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-κB binding and transactivation. In addition...

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

Gale, Michael; Blakely, Collin M.; Kwieciszewski, Bart; Tan, Seng-Lai; Dossett, Michelle; Tang, Norina M.; Korth, Marcus J.; Polyak, Stephen J.; Gretch, David R.; Katze, Michael G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /09/1998 EN
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27.68%
The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A...

Inhibition of Double-Stranded RNA-Dependent Protein Kinase PKR by Vaccinia Virus E3: Role of Complex Formation and the E3 N-Terminal Domain

Romano, Patrick R.; Zhang, Fan; Tan, Seng-Lai; Garcia-Barrio, Minerva T.; Katze, Michael G.; Dever, Thomas E.; Hinnebusch, Alan G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/1998 EN
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27.66%
The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2α (eIF2α). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2α, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments...

Double-Stranded RNA-Independent Dimerization of Interferon-Induced Protein Kinase PKR and Inhibition of Dimerization by the Cellular P58IPK Inhibitor

Tan, Seng-Lai; Gale, Michael J.; Katze, Michael G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1998 EN
Relevância na Pesquisa
27.64%
The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the λ repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the λ repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors...

Hepatitis C Virus Envelope Protein E2 Does Not Inhibit PKR by Simple Competition with Autophosphorylation Sites in the RNA-Binding Domain

Taylor, Deborah R.; Tian, Bin; Romano, Patrick R.; Hinnebusch, Alan G.; Lai, Michael M. C.; Mathews, Michael B.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/2001 EN
Relevância na Pesquisa
27.66%
Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide...

Double-Stranded RNA-Activated Protein Kinase (PKR) Is Negatively Regulated by 60S Ribosomal Subunit Protein L18

Kumar, Kotlo U.; Srivastava, Sri P.; Kaufman, Randal J.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /02/1999 EN
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27.67%
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2α), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2α in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2α phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity...

Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

Romano, Patrick R.; Garcia-Barrio, Minerva T.; Zhang, Xiaolong; Wang, Qizhi; Taylor, Deborah R.; Zhang, Fan; Herring, Christopher; Mathews, Michael B.; Qin, Jun; Hinnebusch, Alan G.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /04/1998 EN
Relevância na Pesquisa
27.64%
The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop...

Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase.

Thomis, D C; Samuel, C E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1995 EN
Relevância na Pesquisa
27.66%
The interferon-inducible, RNA-dependent protein kinase (PKR) is activated by autophosphorylation, a process mediated by double-stranded RNA. A catalytically deficient, histidine-tagged mutant PKR protein [His-PKR(K296R)] was used as the substrate for characterization of the intermolecular phosphorylation catalyzed by purified wild-type PKR [PKR(Wt)]. The intermolecular autophosphorylation of His-PKR(K296R) by PKR(Wt) was RNA dependent. Excess His-PKR(K296R) substrate inhibited both the auto- and the trans-phosphorylation activities of PKR(Wt). Inhibition of PKR(Wt) by His-PKR(K296R) was relieved by higher concentrations of activator double-stranded RNA. Phosphopeptide analysis revealed that the sites of intermolecular autophosphorylation in His-PKR(K296R) were very similar, if not identical, to the sites that were autophosphorylated in PKR(Wt) and suggest a multiple of four major phosphorylation sites per PKR molecule.

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Gale, M; Tan, S L; Wambach, M; Katze, M G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1996 EN
Relevância na Pesquisa
27.67%
Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK ...

Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR.

Thomis, D C; Samuel, C E
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1993 EN
Relevância na Pesquisa
27.69%
The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R) was a substrate for the purified wild-type human PKR kinase. This intermolecular phosphorylation of mutant PKR(K296R) by wild-type PKR was dependent on double-stranded RNA and was inhibited by 2-aminopurine. Finally, PKR mRNA was capable of mediating an autoactivation of wild-type PKR kinase autophosphorylation in vitro.

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

García, M. A.; Gil, J.; Ventoso, I.; Guerra, S.; Domingo, E.; Rivas, C.; Esteban, M.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /12/2006 EN
Relevância na Pesquisa
27.66%
The double-stranded RNA-dependent protein kinase PKR is a critical mediator of the antiproliferative and antiviral effects exerted by interferons. Not only is PKR an effector molecule on the cellular response to double-stranded RNA, but it also integrates signals in response to Toll-like receptor activation, growth factors, and diverse cellular stresses. In this review, we provide a detailed picture on how signaling downstream of PKR unfolds and what are the ultimate consequences for the cell fate. PKR activation affects both transcription and translation. PKR phosphorylation of the alpha subunit of eukaryotic initiation factor 2 results in a blockade on translation initiation. However, PKR cannot avoid the translation of some cellular and viral mRNAs bearing special features in their 5′ untranslated regions. In addition, PKR affects diverse transcriptional factors such as interferon regulatory factor 1, STATs, p53, activating transcription factor 3, and NF-κB. In particular, how PKR triggers a cascade of events involving IKK phosphorylation of IκB and NF-κB nuclear translocation has been intensively studied. At the cellular and organism levels PKR exerts antiproliferative effects, and it is a key antiviral agent. A point of convergence in both effects is that PKR activation results in apoptosis induction. The extent and strength of the antiviral action of PKR are clearly understood by the findings that unrelated viral proteins of animal viruses have evolved to inhibit PKR action by using diverse strategies. The case for the pathological consequences of the antiproliferative action of PKR is less understood...

Protein Kinase PKR Plays a Stimulus- and Virus-Dependent Role in Apoptotic Death and Virus Multiplication in Human Cells▿

Zhang, Ping; Samuel, Charles E.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.68%
The protein kinase regulated by double-stranded RNA (dsRNA), PKR, is implicated in a range of biologic processes, including apoptotic death and interferon antiviral responses, based in part on studies with mouse cells genetically deficient in Pkr. To test the role of the PKR protein in human cells, an RNA interference silencing strategy was used to generate stable HeLa cell lines with less than 2% of the PKR protein (PKR deficient) compared to either parental or control knockdown HeLa lines. Phosphorylation of the α subunit of eukaryotic initiation factor 2 on serine 51 was not detectably increased in response to dsRNA in PKR-deficient HeLa cells but was elevated severalfold in PKR-sufficient cells. PKR-deficient cells displayed reduced dsRNA-induced apoptosis compared to PKR-sufficient cell lines, whereas tumor necrosis factor alpha (TNF-α)-induced apoptosis was comparable between the HeLa lines. NF-κB was activated to a comparable extent in PKR-deficient and PKR-sufficient HeLa cells upon treatment with either dsRNA or TNF-α. The antiviral response against vesicular stomatitis virus was reduced in interferon-treated PKR-deficient compared to PKR-sufficient HeLa cells. However, the growth of two human viruses, adenovirus and reovirus...

Loss of Protein Kinase PKR Expression in Human HeLa Cells Complements the Vaccinia Virus E3L Deletion Mutant Phenotype by Restoration of Viral Protein Synthesis▿

Zhang, Ping; Jacobs, Bertram L.; Samuel, Charles E.
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.68%
The E3L proteins encoded by vaccinia virus bind double-stranded RNA and mediate interferon resistance, promote virus growth, and impair virus-mediated apoptosis. Among the cellular proteins implicated as targets of E3L is the protein kinase regulated by RNA (PKR). To test in human cells the role of PKR in conferring the E3L mutant phenotype, HeLa cells stably deficient in PKR generated by an RNA interference-silencing strategy were compared to parental and control knockdown cells following infection with either an E3L deletion mutant (ΔE3L) or wild-type (WT) virus. The growth yields of WT virus were comparable in PKR-sufficient and -deficient cells. By contrast, the single-cycle yield of ΔE3L virus was increased by nearly 2 log10 in PKR-deficient cells over the impaired growth in PKR-sufficient cells. Furthermore, virus-induced apoptosis characteristic of the ΔE3L mutant in PKR-sufficient cells was effectively abolished in PKR-deficient HeLa cells. The viral protein synthesis pattern was altered in ΔE3L-infected PKR-sufficient cells, characterized by an inhibition of late viral protein expression, whereas in PKR-deficient cells, late protein accumulation was restored. Phosphorylation of both PKR and the α subunit of protein synthesis initiation factor 2 (eIF-2α) was elevated severalfold in ΔE3L-infected PKR-sufficient...

Magnesium-Dependent Interaction of PKR with Adenovirus VAI RNA

Launer-Felty, Katherine; Wong, C. Jason; Wahid, Ahmed M.; Conn, Graeme L.; Cole, James L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.64%
PKR is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the alpha subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in viral infected cells. Viruses have evolved diverse PKR inhibitory strategies to evade the antiviral response. Adenovirus encodes VAI, a highly-structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg2+. Two PKR monomers bind in the absence of Mg2+ but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

Increased Expression of the dsRNA-Activated Protein Kinase PKR in Breast Cancer Promotes Sensitivity to Doxorubicin

Bennett, Richard L.; Carruthers, Aubrey L.; Hui, Teng; Kerney, Krystal R.; Liu, Xiangfei; May, W. Stratford
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 24/09/2012 EN
Relevância na Pesquisa
27.68%
It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H2O2-induced toxicity compared to control cells. In addition...

The Protein Kinase Double-Stranded RNA-Dependent (PKR) Enhances Protection against Disease Cause by a Non-Viral Pathogen

Ogolla, Pauline Sebby; Portillo, Jose-Andres C.; White, Christine L.; Patel, Krupen; Lamb, Bruce; Sen, Ganes C.; Subauste, Carlos S.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.66%
PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy...

Domain interactions in Adenovirus VAI RNA mediate high affinity PKR binding

Launer-Felty, Katherine; Cole, James L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
27.64%
Protein kinase R (PKR) is a component of the innate immunity antiviral pathway. PKR is activated upon binding to dsRNA to undergo dimerization and autophosphorylation. Adenovirus virus-associated RNA I (VAI) is a short, non-coding transcript whose major function is to inhibit the activity of PKR. VAI contains three domains: an apical stem-loop, a highly structured central domain, and a terminal stem. Previous studies have localized PKR binding to the apical stem and central domain. However, the molecular mechanism for inhibition of PKR is not known. We have characterized the stoichiometry and affinity of PKR binding to VAI and several domain constructs using analytical ultracentrifugation and correlated VAI binding and PKR inhibition. Although PKR binding to simple dsRNAs is not regulated by divalent ion, analysis of the interaction of the isolated dsRNA binding domain with VAI reveals that the binding affinity is enhanced by divalent ion. Dissection of VAI into its constituent domains indicates that none of the isolated domains retains the PKR binding affinity or inhibitory potency of the full length RNA. PKR is capable of binding the isolated terminal stem, but deletion of this domain from VAI does not affect PKR binding or inhibition. These results indicate that the apical stem and the central domain are both required to form a high affinity PKR binding site. Our data support a model whereby VAI functions as a PKR inhibitor because it binds a monomer tightly but does not facilitate dimerization.

A atuação da proteína quinase dependente de dsRNA (PKR) no desenvolvimento de tumor de cólon em camundongos obesos; The role of dsRNA dependent protein kinase (PKR) on colon tumor development in obese mice

Guilherme Zweig Rocha
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 09/05/2014 PT
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27.68%
Embora a obesidade seja reconhecida como importante causa de diabetes e doença cardiovascular, a associação entre obesidade e diferentes tipos de câncer tem recebido muito menos atenção. A associação entre obesidade e o desenvolvimento de câncer de cólon representa um dos principais avanços conceituais na patogênese do câncer de cólon da última década. Recentemente a atuação da inflamação subclínica da obesidade na carcinogênese ganhou destaque. Mecanisticamente acredita-se que a obesidade atue como promotor tumoral, e seus efeitos pró-tumorigênicos dependam principalmente da resposta inflamatória de baixo grau ocasionada pela obesidade que envolve a produção de citocinas inflamatórias e pró-tumorigênicas (TNF e IL-6). Uma das principais características da inflamação induzida por obesidade é a infiltração de macrófagos no tecido adiposo, produzindo citocinas inflamatórias e outros mediadores que interferem na sinalização insulínica. Inflamação e estresse de retículo que são conectadas em diversos níveis, são sistemas adaptativos de curto período de expressão necessárias para a função e sobrevivência do organismo, e ambas são prejudiciais quando ativadas cronicamente. Neste sentido...

The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR).

Huang, Xu; Hutchins, Brian; Patel, Rekha C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/08/2002 EN
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One of the key mediators of the antiviral and antiproliferative actions of interferon is double-stranded-RNA-dependent protein kinase (PKR). PKR activity is also involved in the regulation of cell proliferation, apoptosis and signal transduction. We have recently identified PACT, a novel protein activator of PKR, as an important modulator of PKR activity in cells in the absence of viral infection. PACT heterodimerizes with PKR and activates it by direct protein-protein interactions. Endogenous PACT acts as an activator of PKR in response to diverse stress signals, such as serum starvation and peroxide or arsenite treatment, and is therefore a novel, stress-modulated physiological activator of PKR. In this study, we have characterized the functional domains of PACT that are required for PKR activation. Our results have shown that, unlike the N-terminal conserved domains 1 and 2, the third conserved domain of PACT is dispensable for its binding of double-stranded RNA and inter action with PKR. However, a deletion of domain 3 results in a loss of PKR activation ability, in spite of a normal interaction with PKR, thereby indicating that domain 3 plays an essential role in PKR activation. Purified recombinant domain 3 could also activate PKR efficiently in vitro. Our results indicate that...