Regulation Of PKR Research Articles

Grass carp (Ctenopharyngodon idella) Trans-Activation-Responsive RNA-binding protein 2 (TARBP2) inhibits apoptosis by decreasing PKR phosphorylation

PKR plays a significant role in IFN antiviral responses and in mediating apoptosis. Its activity is crucial for cellular antiviral and subsequent recovery. In mammalian cells, Protein Activator of the Interferon-induced Protein Kinase (PACT) and Trans-Activation-Responsive RNA-Binding Protein 2 (TARBP2) have the opposite effect on PKR activity in a dsRNA independent manner. There are some corresponding regulators of PKR in fish, too. In previous studies, we found that grass carp PACT can activate PKR in dsRNA independent manner. In this study, we tried to find out the effect of grass carp TARBP2 on PKR regulation. Grass carp TARBP2 expression is significantly increased at 6h post-poly I:C stimulation in CIK cells and grass carp tissues, indicating that it may play a role in poly I:C-mediated immune response. Then, we found that CiTARBP2 interacts with CiPKR and CiPACT, suggesting that it may regulate PKR activity by direct interaction with PKR or its regulators. Further, poly I:C promotes the phosphorylation of CiTARBP2 and enhances the interaction of CiTARBP2 and CiPKR. Finally, over-expression of CiTARBP2 decreases CiPKR phosphorylation and inhibits PKR-induced apoptosis. Therefore, our study reveals that CiTARBP2 can bind to CiPKR, CiPACT and CiTARBP2. The phosphorylated TARBP2 has stronger affinity to PKR, which results in the decrease of PKR phosphorylation and inhibition of cell apoptosis.

PKR activity modulation by phosphomimetic mutations of serine residues located three aminoacids upstream of double-stranded RNA binding motifs

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), better known as PKR, plays a key role in the response to viral infections and cellular homeostasis by regulating mRNA translation. Upon binding dsRNA, PKR is activated through homodimerization and subsequent autophosphorylation on residues Thr446 and Thr451. In this study, we identified a novel PKR phosphorylation site, Ser6, located 3 amino acids upstream of the first double-stranded RNA binding motif (DRBM1). Another Ser residue occurs in PKR at position 97, the very same position relative to the DRBM2. Ser or Thr residues also occur 3 amino acids upstream DRBMs of other proteins such as ADAR1 or DICER. Phosphoinhibiting mutations (Ser-to-Ala) introduced at Ser6 and Ser97 spontaneously activated PKR. In contrast, phosphomimetic mutations (Ser-to-Asp) inhibited PKR activation following either poly (I:C) transfection or virus infection. These mutations moderately affected dsRNA binding or dimerization, suggesting a model where negative charges occurring at position 6 and 97 tighten the interaction of DRBMs with the kinase domain, thus keeping PKR in an inactive closed conformation even in the presence of dsRNA. This study provides new insights on PKR regulation mechanisms and identifies Ser6 and Ser97 as potential targets to modulate PKR activity for therapeutic purposes.

Kisspeptin Inhibits Colorectal Cancer Cell Invasiveness by Activating PKR and PP2A.

The aim of the present study was to investigate the mechanism through which kisspeptin inhibits colorectal cancer metastasis. Colorectal cancer cells were treated with kisspeptin and then subjected to assays for cell viability, migration, invasion and anchorage-independent growth. Kisspeptin receptor (KISS1R) requirement was examined by siRNA-based gene silencing followed by western blot and invasion assays. Kisspeptin regulation of PKR and PP2A was examined by treating cells with inhibitors for PKR or PP2A. Kisspeptin inhibited colorectal cancer cell invasiveness without affecting cell proliferation. Kisspeptin required activation of KISS1R and resulted in activation of PKR and PP2A. PKR inhibitor blocked kisspeptin-induced PP2A phosphorylation, while PP2A inhibitor failed to block kisspeptin-induced PKR phosphorylation. Kisspeptin-mediated activation of PKR-PP2A inhibited colorectal cancer cell invasiveness.

Stress-induced TRBP phosphorylation enhances its interaction with PKR to regulate cellular survival

Transactivation response element RNA-binding protein (TRBP or TARBP2) initially identified to play an important role in human immunodeficiency virus (HIV) replication also has emerged as a regulator of microRNA biogenesis. In addition, TRBP functions in signaling pathways by negatively regulating the interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) during viral infections and cell stress. During cellular stress, PKR is activated and phosphorylates the α subunit of the eukaryotic translation factor eIF2, leading to the cessation of general protein synthesis. TRBP inhibits PKR activity by direct interaction as well as by binding to PKR’s two known activators, dsRNA and PACT, thus preventing their interaction with PKR. In this study, we demonstrate for the first time that TRBP is phosphorylated in response to oxidative stress and upon phosphorylation, inhibits PKR more efficiently promoting cell survival. These results establish that PKR regulation through stress-induced TRBP phosphorylation is an important mechanism ensuring cellular recovery and preventing apoptosis due to sustained PKR activation.

Ctenopharyngodon idella p53 mediates between NF-κB and PKR at the transcriptional level

p53, NF-κB and PKR are well-known to be involved in antiviral response. Although p53 has been reported in fish, its role in the regulation of NF-κB and PKR is not well understood. Here, we cloned and characterized the full length of cDNA sequence of grass carp (Ctenopharyngodon idella) p53 (Cip53) and its promoter sequence. The full length cDNA of Cip53 was 1879 bp with an ORF of 1116 bp encoding a polypeptide of 371 amino acids. Phylogenetic tree analysis revealed that Cip53 shares high homology with Dario rerio p53 (Drp53). Similar to those of Cip65 and CiPKR, the expression of Cip53 in CIK cells was significantly up-regulated after stimulation with poly I:C. To further understand the roles of fish p53 in the transcriptional control of NF-κB and PKR, Cip53 and Cip65 were expressed in E. coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated that the high affinity interaction between Cip65 and Cip53 promoter. Similarly, Cip53 bound to CiPKR promoter with high affinity. Dual-luciferase reporter assays showed that Cip65 activated Cip53 promoter and Cip53 activated CiPKR promoter, respectively. In addition, the role of p53 in p65-p53-PKR transcription pathway was explored. When Cip53 was knockdown in CIK cells, the mRNA levels of Cip65 and CiPKR were decreased. Taken together, p53 may play pivotal roles in transcription pathway of NF-κB and PKR in fish.

Human noncoding RNA 886 (nc886) adopts two structurally distinct conformers that are functionally opposing regulators of PKR.

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) senses dsRNA produced during viral infection and halts cellular protein synthesis to block viral replication. How basal PKR activity is controlled in the absence of infection was unclear until the recent identification of a potential endogenous regulator, the cellular noncoding RNA 886 (nc886). However, nc886 adopts two distinct conformations for which the structural details and potential functional differences remain unclear. Here, we isolated and separately dissected the function of each form of nc886 to more clearly define the molecular mechanism of nc886-mediated PKR inhibition. We show that nc886 adopts two stable, noninterconverting RNA conformers that are functionally nonequivalent using complementary RNA structure probing and mutational analyses combined with PKR binding and activity assays. One conformer acts as a potent inhibitor, while the other is a pseudoinhibitor capable of weakly activating the kinase. We mapped the nc886 region necessary for high affinity binding and potent inhibition of PKR to an apical stem–loop structure present in only one conformer of the RNA. This structural feature is not only critical for inhibiting PKR autophosphorylation, but also the phosphorylation of its cellular substrate, the eukaryotic translation initiation factor 2α subunit. The identification of different activities of the nc886 conformers suggests a potential mechanism for producing a gradient of PKR regulation within the cell and reveals a way by which a cellular noncoding RNA can mask or present a structural feature to PKR for inhibition.

Regulation of PKR by RNA: Formation of Active and Inactive Dimers

PKR is a member of the eIF2α family of protein kinases that inhibit translational initiation in response to stress stimuli and functions as a key mediator of the interferon-induced antiviral response. PKR contains a dsRNA binding domain that bind to duplex regions present in viral RNAs, resulting in kinase activation and autophosphorylation. An emerging theme in the regulation of protein kinases is the allosteric linkage of dimerization and activation. The PKR kinase domain forms a back-to-back parallel dimer which is implicated in activation. We have developed a sensitive homo-FRET assay for kinase domain dimerization to directly probe the relationship between RNA binding, activation, and dimerization. In the case of perfect duplex RNAs, dimerization is correlated with activation and dsRNAs containing 30 bp or more efficiently induce kinase domain dimerization and activation. However, more complex duplex RNAs containing a 10-15 bp 2'-O-methyl RNA barrier produce kinase dimers but do not activate. Similarly, inactivating mutations within the PKR dimer interface that disrupt key electrostatic and hydrogen binding interactions fail to abolish dimerization. Our data support a model where activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration, providing an additional mechanism for distinguishing between host and pathogen RNA.

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR.

Regulation of PKR by RNA: formation of active and inactive dimers.

PKR is a member of the eIF2α family of protein kinases that inhibit translational initiation in response to stress stimuli and functions as a key mediator of the interferon-induced antiviral response. PKR contains a dsRNA binding domain that binds to duplex regions present in viral RNAs, resulting in kinase activation and autophosphorylation. An emerging theme in the regulation of protein kinases is the allosteric linkage of dimerization and activation. The PKR kinase domain forms a back-to-back parallel dimer that is implicated in activation. We have developed a sensitive homo-Förster resonance energy transfer assay for kinase domain dimerization to directly probe the relationship among RNA binding, activation, and dimerization. In the case of perfect duplex RNAs, dimerization is correlated with activation and dsRNAs containing 30 bp or more efficiently induce kinase domain dimerization and activation. However, more complex duplex RNAs containing a 10-15 bp 2'-O-methyl RNA barrier produce kinase dimers but do not activate. Similarly, inactivating mutations within the PKR dimer interface that disrupt key electrostatic and hydrogen binding interactions fail to abolish dimerization. Our data support a model in which activating RNAs induce formation of a back-to-back parallel PKR kinase dimer whereas nonactivating RNAs either fail to induce dimerization or produce an alternative, inactive dimer configuration, providing an additional mechanism for distinguishing between host and pathogen RNA.

Deletion of Human tarbp2 Reveals Cellular MicroRNA Targets and Cell-Cycle Function of TRBP

TRBP functions as both a Dicer cofactor and a PKR inhibitor. However, the role of TRBP in microRNA (miRNA) biogenesis is controversial and its regulation of PKR in mitosis remains unexplored. Here, we generate TRBP knockout cells and find altered Dicer-processing sites in a subset of miRNAs but no effect on Dicer stability, miRNA abundance, or Argonaute loading. By generating PACT, another Dicer interactor, and TRBP/PACT double knockout (KO) cells, we further show that TRBP and PACT do not functionally compensate for one another and that only TRBP contributes to Dicer processing. We also report that TRBP is hyperphosphorylated by JNK in M phase when PKR is activated by cellular double-stranded RNAs (dsRNAs). Hyperphosphorylation potentiates the inhibitory activity of TRBP on PKR, suppressing PKR in M-G1 transition. By generating human TRBP KO cells, our study clarifies the role of TRBP and unveils negative feedback regulation of PKR through TRBP phosphorylation.

Cyclophilin Inhibitors Reduce Phosphorylation of RNA-Dependent Protein Kinase to Restore Expression of IFN-Stimulated Genes in HCV-Infected Cells

Cyclophilin inhibitors are being developed for treatment of hepatitis C virus (HCV) infection. They are believed to inhibit the HCV replication complex. Weinvestigated whether cyclophilin inhibitors interact with interferon (IFN) signaling in cultured cells infected with HCV. We used immunoblot assays to compare expression of IFN-stimulated genes (ISGs) and of components of IFN signaling in HCV-infected and uninfected cells. Incubation with IFN alfa induced expression of ISGs in noninfected cells and, to a lesser extent, in HCV-infected cells; addition of the cyclophilin inhibitor SCY-635 restored expression ofISG products in HCV-infected cells. SCY-635 reduced phosphorylation of double-strand RNA-dependent protein kinase (PKR) and its downstream factor eIF2α; the phosphorylated forms of these proteins are negative regulators of ISG translation. Cyclophilin A interacted physically with PKR; this interaction was disrupted by SCY-635. SCY-635 also suppressed PKR-mediated formation of stress granules. Cyclophilin inhibitors were found to inhibit PKR phosphorylation and stress granule formation inHCV-infected and uninfected cells. In cultured cells, cyclophilin inhibitors reverse the attenuation of the IFN response by HCV, in addition to their effects on HCV replication complex. Cyclophilin A regulation of PKR has been proposed as a mechanism for observed effects of cyclophilin inhibitors on IFN signaling. We found that cyclophilin inhibitors reduce phosphorylation of PKR and eIF2α during HCV infection to allow for translation of ISG products. Proteins in this pathway might be developed as targets for treatment of HCV infection.

268 : Effect of dystonia-causing point mutation in PACT on activation of interferon-induced protein kinase PKR

PKR or Protein Kinase R is an interferon-induced, double stranded RNA (dsRNA)-activated, eIF2 α kinase that is a key regulator of cellular stress response pathways. PACT (also known as PRKRA, and DYT16) activates PKR in response to oxidative stress, endoplasmic reticulum (ER) stress, and serum starvation. Prolonged PKR activation in response to cellular stress leads to cell death by apoptosis. A recessively inherited form of early-onset dystonia has been recently identified to be associated with a homozygous missense mutation P222L in PACT. Dystonia is a neurological disorder defined by an occurrence of sustained involuntary muscle contractions, often leading to disabling and abnormal postures. Affected members from three unrelated Brazilian families have this P222L mutation in PACT’s coding region. The main hypothesis of this research is that in DYT16 patients the mutant PACT protein alters the normal functioning of the stress-response pathway thus leading to the disease phenotypes. We compared the ability of wild type PACT and mutant P222L proteins to interact with and activate PKR using in vitro biochemical assays. In addition, we compared activities of P222L and wt PACT during ER stress conditions in lymphoblast lines isolated from homozygous dystonia patients and controls. Our results indicate that as compared to the wild type PACT, substitution mutant P222L activates PKR with slower kinetics albeit more robustly and for longer duration. In addition, the affinity of PACT–PACT and consequently PACT–PKR interaction is enhanced in vivo in dystonia lymphoblast lines, thereby leading to intensified PKR activation and consequently enhanced cellular death. The implication of these results on development of dystonia due to enhanced apoptosis is unclear at present, since occurrence of neuronal apoptosis during dystonia progression remains unexplored. Our results elucidate new mechanistic details of PKR regulation by PACT and shed new light on its impact on stress induced cellular apoptosis.

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.

Regulation of Double-Stranded RNA Dependent Protein Kinase Expression and Attenuation of Protein Synthesis Induced by Bacterial Toll-Like Receptors Agonists in the Absence of Interferon

Double-stranded RNA dependent protein kinase (PKR) is a host defense enzyme whose expression is up-regulated in response to interferons (IFNs) and during viral infections. Increased levels of PKR can result in its activation, which, in turn, inhibits global cellular protein synthesis. Despite growing evidence suggesting the involvement of PKR in bacterial infections, little is known about its expression, regulation and cellular role in nonviral infections. The aim of this work was to determine the expression and regulation of PKR in response to stimulation of human THP-1 monocytes with bacterial agonists of TLR2/4. Treatment of cells with Pam3CSK4 or lipopolyssacharide (LPS) resulted in an increase in PKR mRNA and protein levels. Robust PKR expression at later times correlated with a decrease in global protein synthesis. PKR was also required to regulate the inhibition of protein synthesis triggered by LPS in mouse splenocytes. Surprisingly, no increase of IFN-β or IFN-α mRNA levels was detected after treatment of THP-1 cells with toll-like receptor (TLR) agonists. In accordance with this, the supernatants from LPS or Pam3CSK4-treated cells lacked the ability to activate the PKR and ISG56 promoters in gene reporter assays carried out in HEK293T cells. The expression of PKR induced by TLRs agonists was dramatically impaired when cells were treated in the presence of tosyl-phenylalanyl chloromethylketone or Mithramycin, suggesting that NF-κB and Sp1 transcription factors, but not those activated by IFNs, regulate the expression of PKR in human monocytes.

Viral Double-stranded RNAs from Vaccinia Virus Early or Intermediate Gene Transcripts Possess PKR Activating Function, Resulting in NF-κB Activation, When the K1 Protein Is Absent or Mutated

PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58IPK mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.

Regulation of PKR By Viral RNAS

PKR is an interferon-induced kinase that plays a key role in the innate immunity response to viral infection. Upon binding dsRNA, PKR undergoes autophosphorylation reactions that activate the kinase. We have investigated the mechanism of PKR activation by two viral RNAs that regulate PKR. HIV-I TAR is a 23 bp RNA hairpin with three bulges that is known to dimerize. A single PKR binds with moderate affinity to TAR monomer whereas dimers bind two PKRs. TAR dimers activate PKR whereas monomers do not. The secondary structure defects in the TAR RNA stem function as antideterminants to PKR binding and activation. Our results support a model where dimerization of the TAR RNA hairpin facilitates sequential binding of two PKR monomers, leading to protein dimerization and subsequent activation. Adenovirus VAI is a 160 nt highly structured RNA that inhibits activation of PKR by dsRNA. The stoichiometry and affinity of PKR binding to VAI are regulated by Mg2+. In the presence of 5 mM Mg2+, PKR binds similarly to VAI and to a truncation mutant lacking the terminal stem, indicating that this region of VAI is dispensable for regulation of PKR activation.View Large Image | View Hi-Res Image | Download PowerPoint Slide

PRELIMINARY RESULTS OF A PHASE I STUDY WITH INTRAVESICAL ADMINISTRATION OF ADENOVIRAL-MEDIATED INTERFERON-α FOR PATIENTS WITH BCG REFRACTORY TRANSITIONAL CELL CARCINOMA OF THE BLADDER

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.

PKR Regulates B56α-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56alpha is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56alpha at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56alpha in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56alpha-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56alpha phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56alpha proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56alpha protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2alpha activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56alpha, because wild-type but not mutant S28A B56alpha promoted mitochondrial PP2A activity. Cells expressing wild-type B56alpha but not S28A B56alpha were sensitized to etoposide. These results suggest that PKR regulates B56alpha-mediated PP2A signaling in REH cells.

79 Viral Regulation of the PKR Innate Response by Specific Proteolytic Targeting

79 Viral Regulation of the PKR Innate Response by Specific Proteolytic Targeting