Increased interaction between PACT molecules in response to stress signals is required for PKR activation

PKR (protein kinase, RNA activated) is an interferon (IFN)-induced serine-threonine protein kinase and is one of the key mediators in IFN's cellular actions. Although double-stranded (ds) RNA is the most relevant PKR activator during viral infections, PACT acts as a stress-modulated activator of PKR and is an important regulator of PKR dependent signaling pathways in the absence of viral infections. Stress-induced phosphorylation of PACT is essential for PACT's association with PKR leading to PKR activation. PKR activation by PACT leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. In the present study, we have investigated the functional significance of PACT-PACT interaction in mediating PKR activation in response to cellular stress. Our results suggest that enhanced interaction between PACT molecules when PACT is phosphorylated in response to stress signals on serines 246 and 287 is essential for efficient PKR activation. Using a point mutant of PACT that is deficient in PACT-PACT interaction, we demonstrate that PACT-PACT interaction is essential for efficient PKR activation.     

Proapoptotic protein PACT is expressed at high levels in colonic epithelial cells in mice

The protein activator of RNA-activated protein kinase (PKR) is a proapoptotic protein called PACT. PKR is an interferon (IFN)-induced serine-threonine protein kinase that plays a central role in IFN's antiviral and antiproliferative activities. PKR activation in cells leads to phosphorylation of the alpha-subunit of the eukaryotic protein synthesis initiation factor (eIF)2alpha, inhibition of protein synthesis, and apoptosis. In the absence of viral infections, PKR is activated by its activator PACT, especially in response to diverse stress signals. Overexpression of PACT in cells causes enhanced sensitivity to stress-induced apoptosis. We examined PACT expression in different mouse tissues and evaluated its possible role in regulating apoptosis. PACT is expressed at high levels in colonic epithelial cells, especially as they exit the cell cycle and enter an apoptotic program. PACT expression also coincides with the presence of active PKR and phosphorylated eIF2alpha. These results suggest a possible role of PACT-mediated PKR activation in the regulation of epithelial cell apoptosis in mouse colon. In addition, transient overexpression of PACT in a nontransformed intestinal epithelial cell line leads to induction of apoptosis, further supporting PACT's role in inducing apoptosis.     

A truncated PACT protein resulting from a frameshift mutation reported in movement disorder DYT16 triggers caspase activation and apoptosis

P rotein Act ivator (PACT) activates the interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) in response to stress signals. Oxidative stress and endoplasmic reticulum (ER) stress causes PACT-mediated PKR activation, which leads to phosphorylation of translation initiation factor eIF2α, inhibition of protein synthesis, and apoptosis. A dominantly inherited form of early-onset dystonia 16 (DYT16) has been identified to arise due to a frameshift (FS) mutation in PACT. To examine the effect of the resulting truncated mutant PACT protein on the PKR pathway, we examined the biochemical properties of the mutant protein and its effect on mammalian cells. Our results indicate that the FS mutant protein loses its ability to bind dsRNA as well as its ability to interact with PKR while surprisingly retaining the ability to interact with PACT and PKR-inhibitory protein TRBP. The truncated FS mutant protein, when expressed as a fusion protein with a N-terminal fluorescent mCherry tag aggregates in mammalian cells to induce apoptosis via activation of caspases both in a PKR- and PACT-dependent as well as independent manner. Our results indicate that interaction of FS mutant protein with PKR inhibitor TRBP can dissociate PACT from the TRBP-PACT complex resulting in PKR activation and consequent apoptosis. These findings are relevant to diseases resulting from protein aggregation especially since the PKR activation is a characteristic of several neurodegenerative conditions.     

Altered Activation of Protein Kinase PKR and Enhanced Apoptosis in Dystonia Cells Carrying a Mutation in PKR Activator Protein PACT

PACT is a stress-modulated activator of the interferon-induced double-stranded RNA-activated protein kinase (PKR). Stress-induced phosphorylation of PACT is essential for PACT''s association with PKR leading to PKR activation. PKR activation leads to phosphorylation of translation initiation factor eIF2α inhibition of protein synthesis and apoptosis. A recessively inherited form of early-onset dystonia DYT16 has been recently identified to arise due to a homozygous missense mutation P222L in PACT. To examine if the mutant P222L protein alters the stress-response pathway, we examined the ability of mutant P222L to interact with and activate PKR. Our results indicate that the substitution mutant P222L activates PKR more robustly and for longer duration albeit with slower kinetics in response to the endoplasmic reticulum stress. In addition, the affinity of PACT-PACT and PACT-PKR interactions is enhanced in dystonia patient lymphoblasts, thereby leading to intensified PKR activation and enhanced cellular death. P222L mutation also changes the affinity of PACT-TRBP interaction after cellular stress, thereby offering a mechanism for the delayed PKR activation in response to stress. Our results demonstrate the impact of a dystonia-causing substitution mutation on stress-induced cellular apoptosis.     

TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress

The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTΔ13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTΔ13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTΔ13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2^−/− murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.     

Essential Role of PACT-Mediated PKR Activation in Tunicamycin-Induced Apoptosis

Cellular stresses such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds result in accumulation of misfolded proteins in the endoplasmic reticulum (ER) and lead 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 RNA (dsRNA)-activated protein kinase (PKR) and its protein activator PACT in tunicamycin-induced apoptosis. We demonstrate for the first time that PACT is phosphorylated in response to tunicamycin and is responsible for PKR activation by direct interaction. Furthermore, PACT-induced PKR activation is essential for tunicamycin-induced apoptosis, since PACT as well as PKR null cells are markedly resistant to tunicamycin and show defective eIF2α phosphorylation and C/EBP homologous protein (CHOP, also known as GADD153) induction especially at low concentrations of tunicamycin. Reconstitution of PKR and PACT expression in the null cells renders them sensitive to tunicamycin, thus demonstrating that PACT-induced PKR activation plays an essential function in induction of apoptosis.     

Phosphorylation of specific serine residues in the PKR activation domain of PACT is essential for its ability to mediate apoptosis

Activation of the latent protein kinase, PKR, by extracellular stresses and triggering of resultant cellular apoptosis are mediated by the protein, PACT, which itself gets phosphorylated in stressed cells. We have analyzed the underlying biochemical mechanism by carrying out alanine-scanning mutagenesis of the PKR activation domain of PACT. Among the indispensable residues identified were two serine residues, whose phosphorylation was essential for the cellular actions of PACT. Two-dimensional gel analysis, Western analysis using phosphoamino acid-specific antiserum, and in vivo 32P labeling of PACT demonstrated that constitutive phosphorylation of one of the two residues, Ser246, was required for stress-induced phosphorylation of the other, Ser287. Substitution of either of them by threonine or aspartic acid, but not alanine, was tolerated. Substitution of both residues with the phosphoserine mimetic, aspartic acid, produced a mutant PACT that, unlike the wild-type protein, caused PKR activation and apoptosis, even in unstressed cells. These results indicate that phosphorylation of specific serine residues in the activation domain of PACT is the major mode of transmission of cellular stress response to PKR.     

PACT, a stress-modulated cellular activator of interferon-induced double-stranded RNA-activated protein kinase, PKR

The interferon (IFN)-induced, double-stranded (ds)RNA-activated serine-threonine protein kinase, PKR, is a key mediator of the antiviral activities of IFNs. In addition, PKR activity is also involved in regulation of cell proliferation, apoptosis, and signal transduction. In virally infected cells, dsRNA has been shown to bind and activate PKR kinase function. Implication of PKR activity in normal cellular processes has invoked activators other than dsRNA because RNAs with perfectly duplexed regions of sufficient length that are able to activate PKR are absent in cellular RNAs. We have recently reported cloning of PACT, a novel protein activator of PKR. PACT heterodimerizes with PKR and activates it by direct protein-protein interaction. Overexpression of PACT in mammalian cells leads to phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha), the cellular substrate for PKR, and leads to inhibition of protein synthesis. Here, we present evidence that endogenous PACT acts as a protein activator of PKR in response to diverse stress signals such as serum starvation, and peroxide or arsenite treatment. Following exposure of cells to these stress agents, PACT is phosphorylated and associates with PKR with increased affinity. PACT-mediated activation of PKR leads to enhanced eIF2alpha phosphorylation followed by apoptosis. Based on the results presented here, we propose that PACT is a novel stress-modulated physiological activator of PKR.     

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.     

Serine 18 phosphorylation of RAX, the PKR activator, is required for PKR activation and consequent translation inhibition

It is now apparent that the double-stranded (ds)RNA-dependent protein kinase, PKR, is a regulator of diverse cellular responses to stress. Recently, the murine dsRNA-binding protein RAX and its human ortholog PACT were identified as cellular activators of PKR. Previous reports demonstrate that following stress, RAX/PACT associates with and activates PKR resulting in eIF2alpha phosphorylation, consequent translation inhibition, and cell death via apoptosis. Although RAX/PACT is phosphorylated during stress, any regulatory role for this post-translational modification has been uncertain. Now we have discovered that RAX is phosphorylated on serine 18 in both human and mouse cells. The non-phosphorylatable form of RAX, RAX(S18A), although still able to bind dsRNA and associate with PKR, fails to activate PKR following stress. Furthermore, stable expression of RAX(S18A) results in a dominant-negative effect characterized by deficiency of eukaryotic initiation factor 2 alpha subunit phosphorylation, delay of translation inhibition, and failure to undergo rapid apoptosis following removal of interleukin-3. We propose that the ability of RAX to activate PKR is regulated by a sequential mechanism featuring RAX association with PKR, RAX phosphorylation at serine 18, and activation of PKR.     

Inhibition of PACT-mediated activation of PKR by the herpes simplex virus type 1 Us11 protein

PACT, a protein activator of PKR, can cause inhibition of cellular protein synthesis and apoptosis. Here, we report that the Us11 protein of herpes simplex virus type 1 can block PKR activation by PACT both in vitro and in vivo. Although Us11 can bind to both PKR and PACT, mutational analyses revealed that the binding of Us11 to PKR, and not to PACT, was essential for its inhibitory action. Similar analyses also revealed that the inhibitory effect was mediated by an interaction between the C-terminal half of Us11 and the N-terminal domain of PKR. The binding of Us11 to PKR did not block the binding of PKR to PACT but prevented its activation. Us11 is the first example of a viral protein that can inhibit the action of PACT on PKR.     

The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation,leading to G1 arrest

Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACTdependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the 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 the desumoylase SENP1 fails to induce such a G1 arrest. Furthermore, co-expression of p53, RAX/PACT and the dominantnegative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G₁ growth arrest and expression of RAX/PACT in pkr+/+ but not pkr-/- MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/ PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G₁ cell cycle arrest.     

Translation inhibition in apoptosis: caspase-dependent PKR activation and eIF2-alpha phosphorylation

The protein kinase PKR is a major player in the cellular antiviral response, acting mainly by phosphorylation of the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2-alpha) to block de novo protein synthesis. PKR activation requires binding of double-stranded RNA or PACT/RAX proteins to its regulatory domain. Since several reports have demonstrated that translation is inhibited in apoptosis, we investigated whether PKR and eIF2-alpha phosphorylation contribute to this process. We show that PKR is proteolysed and that eIF2-alpha is phosphorylated at the early stages of apoptosis induced by various stimuli. Both events coincide with the onset of caspase activity and are prevented by caspase inhibitors. Using site-directed mutagenesis we show that PKR is specifically proteolysed at Asp(251) during cellular apoptosis. This site is cleaved in vitro by recombinant caspase-3, caspase-7, and caspase-8 and not by the proinflammatory caspase-1 and caspase-11. The released kinase domain efficiently phosphorylates eIF2-alpha at the cognate Ser(51) residue, and its overexpression in mammalian cells impairs the translation of its own mRNA and of reporter mRNAs. Our results demonstrate a new and caspase-dependent activation mode for PKR, leading to eIF2-alpha phosphorylation and translation inhibition in apoptosis.     

Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells

The double‐stranded RNA‐dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double‐stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF‐κB to the nucleus after 30 min, and inhibition of NF‐κB decreased the PACT–PKR interaction induced by LPS. The level of pro‐inflammatory cytokine mRNA, including interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro‐inflammatory cytokines was not affected by RNAi‐mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF‐κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT–PKR association in Sa3 cells. Our results also suggest that NF‐κB is involved in the PACT–PKR interaction and the production of pro‐inflammatory cytokines in periodontitis. J. Cell. Biochem. 113: 165–173, 2012. © 2011 Wiley Periodicals, Inc.     

The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation,leading to G1 arrest

Cellular stresses, including growth factor deprivation, inflammatory cytokines or viral infection promote RAX/PACT-dependent activation of the double-stranded RNA-dependent protein kinase, PKR, to phosphorylate eIF2α, resulting in translation inhibition and apoptosis. In addition, PKR has been reported to regulate p53, STAT1 and NFκB. Here, we report that RAX/PACT interacts with the SUMO E2 ligase Ubc9 to stimulate p53-Ubc9 association and reversible p53 sumoylation on lysine 386. In addition, expression of RAX/PACT in a variety of cell lines promotes p53 stability and activity to increase p53 target gene expression. Significantly, while the 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 the desumoylase SENP1 fails to induce such a G₁ arrest. Furthermore, co-expression of p53, RAX/PACT and the dominant-negative PKR(K296R) mutant inhibits RAX/PACT-induced, p53-dependent G₁ growth arrest and expression of RAX/PACT in pkr^+/+ but not pkr^−/− MEF cells promotes p53 and p21 expression following gamma irradiation. Significantly, p53 stability is decreased in cells with reduced RAX/PACT or PKR following doxorubicin treatment, and expression of exogenous RAX/PACT promotes phosphorylation of wild-type but not p53(K386R) on serine 392. Collectively, results indicate that, in response to stress, the RAX/PACT-PKR signaling pathway may inhibit p53 protein turnover by a sumoylation-dependent mechanism with promotion of p53 phosphorylation and translational activation leading to G₁ cell cycle arrest.Key words: p53, PKR, RAX, PACT, Ubc9, sumoylation     

The double-strand RNA-dependent protein kinase PKR plays a significant role in a sustained ER stress-induced apoptosis

Sustained ER stress leads to apoptosis. However, the exact mechanism still remains to be elucidated. Here, we demonstrate that the double strand RNA-dependent protein kinase (PKR) is involved in the ER stress-mediated signaling pathway. ER stress rapidly activated PKR, inducing the phosphorylation of eIF2alpha, followed by the activation of the ATF4/CHOP pathway. ER-stress-mediated eIF2alpha/ATF4/CHOP signaling and associated cell death was markedly reduced by PKR knockdown. We also found that PKR activation was mediated by PACT, the expression of which was elevated by ER-stress. These results indicate that the ER-stress-mediated eIF2alpha/ATF4/CHOP/cell death pathway is, to some degree, dependent on PACT-mediated PKR activation apart from the PERK pathway.     

Interaction between RAX and PKR modulates the effect of ethanol on protein synthesis and survival of neurons

Ethanol exposure inhibits protein synthesis and causes cell death in the developing central nervous system. The double-stranded RNA (dsRNA)-activated protein kinase (PKR), a serine/threonine protein kinase, plays an important role in translational regulation and cell survival. PKR has been well known for its anti-viral response. Upon activation by viral infection or dsRNA, PKR phosphorylates its substrate, the alpha-subunit of eukaryotic translation initiation factor-2 (eIF2alpha) leading to inhibition of translation initiation. It has recently been shown that, in the absence of a virus or dsRNA, PKR can be activated by direct interactions with its protein activators, PACT, or its mouse homologue, RAX. We have demonstrated that exposure to ethanol increased the phosphorylation of PKR and eIF2alpha in the developing cerebellum. The effect of ethanol on PKR/eIF2alpha phosphorylation positively correlated to the expression of PACT/RAX in cultured neuronal cells. Using PKR inhibitors and PKR null mouse fibroblasts, we verified that ethanol-induced eIF2alpha phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2alpha phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2alpha activation. Ethanol promoted PKR and RAX association in cells expressing wild-type RAX but not in cells expressing S18A RAX. S18A RAX functioned as a dominant negative protein and blocked ethanol-induced inhibition of protein synthesis and cell death. Our results suggest that the interactions between PKR and PACT/RAX modulate the effect of ethanol on protein synthesis and cell survival in the central nervous system.     

Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing

During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer–TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer–dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.     

The Multiple Functions of TRBP,at the Hub of Cell Responses to Viruses,Stress, and Cancer

Summary: The TAR RNA binding protein (TRBP) has emerged as a key player in many cellular processes. First identified as a cellular protein that facilitates the replication of human immunodeficiency virus, TRBP has since been shown to inhibit the activation of protein kinase R (PKR), a protein involved in innate immune responses and the cellular response to stress. It also binds to the PKR activator PACT and regulates its function. TRBP also contributes to RNA interference as an integral part of the minimal RNA-induced silencing complex with Dicer and Argonaute proteins. Due to its multiple functions in the cell, TRBP is involved in oncogenesis when its sequence is mutated or its expression is deregulated. The depletion or overexpression of TRBP results in malignancy, suggesting that the balance of TRBP expression is key to normal cellular function. These studies show that TRBP is multifunctional and mediates cross talk between different pathways. Its activities at the molecular level impact the cellular function from normal development to cancer and the response to infections.