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.     

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.     

Stress-induced phosphorylation of PACT reduces its interaction with TRBP and leads to PKR activation

PACT is a stress-modulated activator of interferon (IFN)-induced double-stranded (ds) RNA-activated protein kinase (PKR) and is an important regulator of PKR-dependent signaling pathways. 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 addition to positive regulation by PACT, PKR activity in cells is also negatively regulated by TRBP. In this study, we demonstrate for the first time that stress-induced phosphorylation at serine 287 significantly increases PACT's ability to activate PKR by weakening PACT's interaction with TRBP. A non-phosphorylatable alanine substitution mutant at this position causes enhanced interaction of PACT with TRBP and leads to a loss of PKR activation. Furthermore, TRBP overexpression in cells is unable to block apoptosis induced by a phospho-mimetic, constitutively active PACT mutant. These results demonstrate for the first time that stress-induced PACT phosphorylation functions to free PACT from the inhibitory interaction with TRBP and also to enhance its interaction with PKR.     

PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by Newcastle disease virus

Virus infection triggers innate responses to host cells including production of type I interferon (IFN). Since IFN production is also induced by treatment with poly(I:C), viral double-stranded (ds) RNA has been postulated to play a direct role in the process. In the present study, we investigated the effect of dsRNA binding proteins on virus-induced activation of the IFN-beta gene. We found that PACT, originally identified as protein activator for dsRNA-dependent protein kinase (PKR) and implicated in the regulation of translation, augmented IFN-beta gene activation induced by Newcastle disease virus. Concomitantly with the augmented activity of IFN-beta enhancer, increased activity of NF-kappaB and IRF-3 and IRF-7 was observed. For the observed effect, the dsRNA-binding activity of PACT was essential. We identified residues of PACT that interact with a presumptive target molecule to exert its function. Furthermore, PACT colocalized with viral replication complex in the infected cells. Thus the observed effect of PACT is novel and PACT is involved in the regulation of viral replication and results in a marked increase of cellular IFN-beta gene expression.     

PACT, a protein activator of the interferon-induced protein kinase, PKR.

PKR, a latent protein kinase, mediates the antiviral actions of interferon. It is also involved in cellular signal transduction, apoptosis, growth regulation and differentiation. Although in virus-infected cells, viral double-stranded (ds) RNA can serve as a PKR activator, cellular activators have remained obscure. Here, we report the cloning of PACT, a cellular protein activator of PKR. PACT heterodimerized with PKR and activated it in vitro in the absence of dsRNA. In mammalian cells, overexpression of PACT caused PKR activation and, in yeast, co-expression of PACT enhanced the anti-growth effect of PKR. Thus, PACT has the hallmarks of a direct activator of PKR.     

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.     

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.     

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.     

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.     

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.     

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.     

Modular structure of PACT: distinct domains for binding and activating PKR

PACT is a 35-kDa human protein that can directly bind and activate the latent protein kinase, PKR. Here we report that PKR activation by PACT causes cellular apoptosis in addition to PKR autophosphorylation and translation inhibition. We analyzed the structure-function relationship of PACT by measuring its ability to bind and activate PKR in vitro and in vivo. Our studies revealed that among three domains of PACT, the presence of either domain 1 or domain 2 was sufficient for high-affinity binding of PACT to PKR. On the other hand, domain 3, consisting of 66 residues, was absolutely required for PKR activation in vitro and in vivo. When fused to maltose-binding protein, domain 3 was also sufficient for efficiently activating PKR in vitro. However, it bound poorly to PKR at the physiological salt concentration and consequently could not activate it properly in vivo. As anticipated, activation of PKR by domain 3 in vivo could be restored by attaching it to a heterologous PKR-binding domain. These results demonstrated that the structure of PACT is modular: it is composed of a distinct PKR-activation domain and two mutually redundant PKR-interacting domains.     

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.     

Double-stranded RNA-dependent protein kinase (PKR) is downregulated by phorbol ester

The double-stranded RNA-dependent protein kinase (PKR) is one of the key mediators of interferon (IFN) action against certain viruses. PKR also plays an important role in signal transduction and immunomodulation. Understanding the regulation of PKR activity is important for the use of PKR as a tool to discover and develop novel therapeutics for viral infections, cancer and immune dysfunction. We found that phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), decreased the level of autophosphorylated PKR in a dose- and time-dependent manner in IFN-treated mouse fibroblast cells. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced the activity of PKR induced by IFN, but did not overcome the PMA-induced reduction of PKR autophosphorylation. Western blot analysis with a monoclonal antibody to mouse PKR revealed that the decrease of PKR autophosphorylation in cells by PMA was a result of PKR protein degradation. Selective PKC inhibitors blocked the degradation of PKR stimulated by PMA, indicating that PKC activity was required for the effect. Furthermore, we also found that proteasome inhibitors prevented PMA-induced down regulation of PKR, indicating that an active proteasome is required. Our results identify a novel mechanism for the post-translational regulation of PKR.     

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.     

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.     

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

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, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.