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.     

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.     

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.     

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.     

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.     

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.     

Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).

During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in 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.     

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.     

RAX, a cellular activator for double-stranded RNA-dependent protein kinase during stress signaling.

The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the alpha subunit of eukaryotic initiation factor-2. PKR is activated by viral induced dsRNA and thought to be involved in the host antiviral defense mechanism. PKR is also activated by various nonviral stresses such as growth factor deprivation, although the mechanism is unknown. By screening a mouse cDNA expression library, we have identified an ubiquitously expressed PKR-associated protein, RAX. RAX has a high sequence homology to human PACT, which activates PKR in the absence of dsRNA. Although RAX also can directly activate PKR in vitro, overexpression of RAX does not induce PKR activation or inhibit growth of interleukin-3 (IL-3)-dependent cells in the presence of IL-3. However, IL-3 deprivation as well as diverse cell stress treatments including arsenite, thapsigargin, and H2O2, which are known to inhibit protein synthesis, induce the rapid phosphorylation of RAX followed by RAX-PKR association and activation of PKR. Therefore, cellular RAX may be a stress-activated, physiologic activator of PKR that couples transmembrane stress signals and protein synthesis.     

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.     

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.     

Resistance to vesicular stomatitis virus infection requires a functional cross talk between the eukaryotic translation initiation factor 2alpha kinases PERK and PKR

Phosphorylation of the alpha (alpha) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2alpha kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2alpha kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK(-/-) mice are more susceptible to VSV-mediated apoptosis than PERK(+/+) MEFs. The higher replication capacity of VSV in PERK(-/-) MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2alpha phosphorylation. We also show that VSV-infected PERK(-/-) MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2alpha kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.     

Involvement of double-stranded RNA-dependent protein kinase and phosphorylation of eukaryotic initiation factor-2alpha in neuronal degeneration

Inhibition of protein translation plays an important role in apoptosis. While double-stranded RNA-dependent protein kinase (PKR) is named as it is activated by double-stranded RNA produced by virus, its activation induces an inhibition of protein translation and apoptosis via the phosphorylation of the eukaryotic initiation factor 2alpha (eIF2alpha). PKR is also a stress kinase and its levels increase during ageing. Here we show that PKR activation and eIF2alpha phosphorylation play a significant role in apoptosis of neuroblastoma cells and primary neuronal cultures induced by the beta-amyloid (Abeta) peptides, the calcium ionophore A23187 and flavonoids. The phosphorylation of eIF2alpha and the number of apoptotic cells were enhanced in over-expressed wild-type PKR neuroblastoma cells exposed to Abeta peptide, while dominant-negative PKR reduced eIF2alpha phosphorylation and apoptosis induced by Abeta peptide. Primary cultured neurons from PKR knockout mice were also less sensitive to Abeta peptide toxicity. Activation of PKR and eIF2alpha pathway by Abeta peptide are triggered by an increase in intracellular calcium because the intracellular calcium chelator BAPTA-AM significantly reduced PKR phosphorylation. Taken together, these results reveal that PKR and eIF2alpha phosphorylation could be involved in the molecular signalling events leading to neuronal apoptosis and death and could be a new target in neuroprotection.     

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.     

trans-Autophosphorylation by the isolated kinase domain is not sufficient for dimerization or activation of the dsRNA-activated protein kinase PKR

The double-stranded (ds) RNA-activated protein kinase PKR phosphorylates the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits translation initiation. PKR contains two dsRNA binding domains in its amino terminus and a kinase domain in its carboxy terminus. dsRNA binding activates PKR from a latent state by inducing dimerization and trans-autophosphorylation. Recent studies show that PKR is also activated by caspase cleavage to remove the inhibitory dsRNA binding domains. In this report, we show that the isolated kinase domain of PKR is a constitutively active monomeric kinase that has an activity similar to that of wild-type PKR. We used a solid-phase kinase assay system to show that PKR does not transfer its own phosphate to either PKR or eIF2alpha but rather uses the gamma-phosphate from ATP. In addition, the isolated autophosphorylated kinase domain of PKR phosphorylated intact monomeric PKR in trans in a reaction that did not require dsRNA binding. However, this trans-phosphorylation did not occur at the critical Thr446/451 sites and was not sufficient to induce dimerization and/or activation of PKR. The results show that dsRNA binding domains of PKR are not only required for dimerization of PKR but also required for phosphorylation of Thr446/451 sites of PKR. The results imply that even though the isolated kinase domain of PKR phosphorylates intact PKR and eIF2alpha, it is unable to activate PKR.     

A novel function of eIF2alpha kinases as inducers of the phosphoinositide-3 kinase signaling pathway

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.