Brain ischemia and reperfusion activates the eukaryotic initiation factor 2alpha kinase, PERK

Reperfusion after global brain ischemia results initially in a widespread suppression of protein synthesis in neurons, which persists in vulnerable neurons, that is caused by the inhibition of translation initiation as a result of the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha). To identify kinases responsible for eIF2alpha phosphorylation [eIF2alpha(P)] during brain reperfusion, we induced ischemia by bilateral carotid artery occlusion followed by post-ischemic assessment of brain eIF2alpha(P) in mice with homozygous functional knockouts in the genes encoding the heme-regulated eIF2alpha kinase (HRI), or the amino acid-regulated eIF2alpha kinase (GCN2). A 10-fold increase in eIF2alpha(P) was observed in reperfused wild-type mice and in the HRI-/- or GCN2-/- mice. However, in all reperfused groups, the RNA-dependent protein kinase (PKR)-like endoplasmic reticulum eIF2alpha kinase (PERK) exhibited an isoform mobility shift on SDS-PAGE, consistent with the activation of the kinase. These data indicate that neither HRI nor GCN2 are required for the large increase in post-ischemic brain eIF2alpha(P), and in conjunction with our previous report that eIF2alpha(P) is produced in the brain of reperfused PKR-/- mice, provides evidence that PERK is the kinase responsible for eIF2alpha phosphorylation in the early post-ischemic brain.     

Renal ischemia and reperfusion activates the eIF 2 alpha kinase PERK

Inhibition of protein synthesis occurs in the post-ischemic reperfused kidney but the molecular mechanism of renal translation arrest is unknown. Several pathways have been identified whereby cell stress inhibits translation initiation via phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF 2 alpha, phospho-form eIF 2 alpha(P)]. Here, we report a 20-fold increase in eIF 2 alpha(P) in kidney homogenates following 10 min of cardiac arrest-induced ischemia and 10 min reperfusion. Using immunohistochemistry, we observed eIF 2 alpha(P) in tubular epithelial cells in both cortex and medulla, where the greatest eIF 2 alpha(P) staining was found in epithelial cells of the so-called watershed area at the corticomedullary junction. We further show that increased eIF 2 alpha(P) is accompanied by activation of the PKR-like endoplasmic reticulum eIF 2 alpha kinase (PERK). These observations indicate that renal ischemia and reperfusion induce stress to the endoplasmic reticulum and activate the unfolded protein response in renal epithelial cells. As the unfolded protein response can result alternatively in a pro-survival or pro-apoptotic outcome, the present study demonstrates an new additional mechanism involved in cell damage and/or repair in ischemic and reperfused kidney.     

Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival.

The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.     

Regulatory proteins of eukaryotic initiation factor 2-alpha subunit (eIF2 alpha) phosphatase, under ischemic reperfusion and tolerance

Phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), which is one of the substrates of protein phosphatase 1 (PP1), occurs rapidly during the first minutes of post-ischemic reperfusion after an episode of cerebral ischemia. In the present work, two experimental models of transient global ischemia and ischemic tolerance (IT) were used to study PP1 interacting/regulatory proteins following ischemic reperfusion. For that purpose we utilized PP1 purified by microcystin chromatography, as well as 2D DIGE of PP1α and PP1γ immunoprecipitates. The highest levels of phosphorylated eIF2α found after 30 min reperfusion in rats without IT, correlated with increased levels in PP1 immunoprecipitates of the inhibitor DARPP32 as well as GRP78 and HSC70 proteins. After 4 h reperfusion, the levels of these proteins in PP1c complexes had returned to control values, in parallel to a significant decrease in eIF2α phosphorylated levels. IT that promoted a decrease in eIF2α phosphorylated levels after 30 min reperfusion induced the association of GADD34 with PP1c, while prevented that of DARPP32, GRP78, and HSC70. Different levels of HSC70 and DARPP32 associated with PP1α and PP1γ isoforms, whereas GRP78 was only detected in PP1γ immunoprecipitates. Here we suggest that PP1, through different signaling complexes with their interacting proteins, may modulate the eIF2α phosphorylation/dephosphorylation during reperfusion after a transient global ischemia in the rat brain. Of particular interest is the potential role of GADD34/PP1c complexes after tolerance acquisition.     

Activation of signaling pathways and regulatory mechanisms of mRNA translation following myocardial ischemia-reperfusion.

Protein expression in the heart is altered following periods of myocardial ischemia. The changes in protein expression are associated with increased cell size that can be maladaptive. There is little information regarding the regulation of protein expression through the process of mRNA translation during ischemia and reperfusion in the heart. Therefore, the purpose of this study was to identify changes in signaling pathways and downstream regulatory mechanisms of mRNA translation in an in vivo model of myocardial ischemia and reperfusion. Hearts were collected from rats whose left main coronary arteries had either been occluded for 25 min or reversibly occluded for 25 min and subsequently reperfused for 15 min. Following reperfusion, both the phosphoinositide 3-kinase and mitogen-activated protein kinase pathways were activated, as evidenced by increased phosphorylation of Akt (PKB), extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase. Activation of Akt stimulated signaling through the protein kinase mammalian target of rapamycin, as evidenced by increased phosphorylation of two of its effectors, the ribosomal protein S6 kinase and the eukaryotic initiation factor eIF4E binding protein 1. Ischemia and reperfusion also resulted in increased phosphorylation of eIF2 and eIF2B. These changes in protein phosphorylation suggest that control of mRNA translation following ischemia and reperfusion is modulated through a number of signaling pathways and regulatory mechanisms.     

Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression

Translational repression induced during reperfusion of the ischaemic brain is significantly attenuated by ischaemic preconditioning. The present work was undertaken to identify the components of the translational machinery involved and to determine whether translational attenuation selectively modifies protein expression patterns during reperfusion. Wistar rats were preconditioned by 5-min sublethal ischaemia and 2 days later, 30-min lethal ischaemia was induced. Several parameters were studied after lethal ischaemia and reperfusion in rats with and without acquired ischaemic tolerance (IT). The phosphorylation pattern of the alpha subunit of eukaryotic initiation factor 2 (eIF2) in rats with IT was exactly the same as in rats without IT, reaching a peak after 30 min reperfusion and returning to control values within 4 h in both the cortex and hippocampus. The levels of phosphorylated eIF4E-binding protein after lethal ischaemia and eIF4E at 30 min reperfusion were higher in rats with IT, notably in the hippocampus. eIF4G levels diminished slightly after ischaemia and reperfusion, paralleling calpain-mediated alpha-spectrin proteolysis in rats with and without IT, but they did not show any further decrease after 30 min reperfusion in rats with IT. The phosphorylated levels of eIF4G, phosphatidylinositol 3-kinase-protein B (Akt) and extracellular signal-regulated kinases (ERKs) were very low after lethal ischaemia and increased following reperfusion. Ischaemic preconditioning did not modify the observed changes in eIF4G phosphorylation. All these results support that translation attenuation may occur through multiple targets. The levels of the glucose-regulated protein (78 kDa) remained unchanged in rats with and without IT. Conversely, our data establish a novel finding that ischaemia induces strong translation of growth arrest and DNA damage protein 34 (GADD34) after 4 h of reperfusion. GADD34 protein was slightly up-regulated after preconditioning, besides, as in rats without IT, GADD34 levels underwent a further clear-cut increase during reperfusion, this time as earlier as 30 min and coincident with translation attenuation.     

Phosphorylation of the alpha subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2alpha (alpha subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2alpha(P) (phosphorylated eIF2alpha) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191-194]. We show in the present study that one eIF2alpha kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2alpha(P) in these areas. In PKR-deficient animals subjected to SE, eIF2alpha phosphorylation was clearly evident coincident with activation of a secondary eIF2alpha kinase, PEK/PERK (pancreatic eIF2alpha kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2alpha phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2alpha(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2alpha contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2alpha phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.     

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.     

Caerulein-induced acute pancreatitis inhibits protein synthesis through effects on eIF2B and eIF4F

Acute pancreatitis (AP) has been shown in some studies to inhibit total protein synthesis in the pancreas, whereas in other studies, protein synthesis was not affected. Previous in vitro work has shown that high concentrations of cholecystokinin both inhibit protein synthesis and inhibit the activity of the guanine nucleotide exchange factor eukaryotic initiation factor (eIF)2B by increasing the phosphorylation of eIF2alpha. We therefore evaluated in C57BL/6 mice the effects of caerulein-induced AP on pancreatic protein synthesis, eIF2B activity and other protein translation regulatory mechanisms. Repetitive hourly injections of caerulein were administered at 50 microg/kg ip. Pancreatic protein synthesis was reduced 10 min after the initial caerulein administration and was further inhibited after three and five hourly injections. Caerulein inhibited the two major regulatory points of translation initiation: the activity of the guanine nucleotide exchange factor eIF2B (with an increase of eIF2alpha phosphorylation) and the formation of the eIF4F complex due, in part, to degradation of eIF4G. This inhibition was not accounted for by changes in the upstream stimulatory pathway, because caerulein activated Akt as well as phosphorylating the downstream effectors of mTOR, 4E-BP1, and ribosomal protein S6. Caerulein also decreased the phosphorylation of the eukaryotic elongation factor 2, implying that this translation factor was not inhibited in AP. Thus the inhibition of pancreatic protein synthesis in this model of AP most likely results from the inhibition of translation initiation as a result of increased eIF2alpha phosphorylation, reduction of eIF2B activity, and the inhibition of eIF4F complex formation.     

Global Brain Ischemia and Reperfusion: Modifications in Eukaryotic Initiation Factors Associated with Inhibition of Translation Initiation

Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.     

Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum

Exposure to ultraviolet light can cause inflammation, premature skin aging, and cancer. UV irradiation alters the expression of multiple genes that encode functions to repair DNA damage, arrest cell growth, and induce apoptosis. In addition, UV irradiation inhibits protein synthesis, although the mechanism is not known. In this report, we show that UV irradiation induces phosphorylation of eukaryotic translation initiation factor 2 on the alpha-subunit (eIF2alpha) and inhibits protein synthesis in a dosage- and time-dependent manner. The UV-induced phosphorylation of eIF2alpha was prevented by the overexpression of a non-phosphorylatable mutant of eIF2alpha (S51A). PERK is an eIF2alpha protein kinase localized to the endoplasmic reticulum that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. Expression of trans-dominant-negative mutants of PERK also prevented eIF2alpha phosphorylation upon UV treatment and protected from the associated translation attenuation. The luminal domain of dominant-negative mutant PERK formed heterodimers with endogenous PERK to inhibit the PERK signaling pathway. In contrast, eIF2alpha phosphorylation was not inhibited by overexpression of a trans-dominant-negative mutant kinase, PKR, supporting the theory that UV-induced eIF2alpha phosphorylation is specifically mediated by PERK. These results support a novel mechanism by which UV irradiation regulates translation via an endoplasmic reticulum-stress signaling pathway.     

Interaction of recombinant human eIF2 subunits with eIF2B and eIF2alpha kinases

The heterotrimeric eukaryotic initiation factor 2 (eIF2) plays a critical role in the mechanics and regulation of protein synthesis. Unlike yeast and archaeal eIF2, the purified baculovirus-expressed recombinant human eIF2 subunits used in these studies reveal that the alpha- and beta-subunits interact with each other. Consistent with this observation, the beta-subunit specifically interacts with the purified eIF2B in ELISA studies and this interaction is enhanced when wt eIF2alpha in the recombinant trimeric complex is phosphorylated or replaced by a mutant phosphomimetic eIF2alpha (S51D). These findings together with other observations raise the possibility that the beta-subunit plays a key role in the regulation and function of mammalian eIF2 complex. PERK, an eIF2alpha kinase, is found to interact with wt and mutants of eIF2alpha in which the serine 51 or 48 residue is replaced by alanine or aspartic acid thereby suggesting that the phosphorylation site in the substrate is not important for interaction. Fluorescence spectroscopic and fluorescence resonance energy transfer analyses reveal that the energy transfer occurs from PERK to eIF2alpha. The dissociation constant of alpha-subunit-PERK complex (Kd alpha-subunit) is 0.74 microM and the interaction is stoichiometric.     

Perk is essential for translational regulation and cell survival during the unfolded protein response

Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress-activated eIF2alpha kinase PERK abolishes the phosphorylation of eIF2alpha in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.     

Cellular energy status modulates translational control mechanisms in ischemic-reperfused rat hearts

Mechanisms regulating ischemia and reperfusion (I/R)-induced changes in mRNA translation in the heart are poorly defined, as are the factors that initiate these changes. Because cellular energy status affects mRNA translation under physiological conditions, it is plausible that I/R-induced changes in translation may in part be a result of altered cellular energy status. Therefore, the purpose of the studies described herein was to compare the effects of I/R with those of altered energy substrate availability on biomarkers of mRNA translation in the heart. Isolated adult rat hearts were perfused with glucose or a combination of glucose plus palmitate, and effects of I/R on various biomarkers of translation were subsequently analyzed. When compared with hearts perfused with glucose plus palmitate, hearts perfused with glucose alone exhibited increased phosphorylation of eukaryotic elongation factor (eEF)2, the alpha-subunit of eukaryotic initiation factor (eIF)2, and AMP-activated protein kinase (AMPK), and these hearts also exhibited enhanced association of eIF4E with eIF4E binding protein (4E-BP)1. Regardless of the energy substrate composition of the buffer, phosphorylation of eEF2 and AMPK was greater than control values after ischemia. Phosphorylation of eIF2alpha and eIF4E and the association of eIF4E with 4E-BP1 were also greater than control values after ischemia but only in hearts perfused with glucose plus palmitate. Reperfusion reversed the ischemia-induced increase in eEF2 phosphorylation in hearts perfused with glucose and reversed ischemia-induced changes in eIF4E, eEF2, and AMPK phosphorylation in hearts perfused with glucose plus palmitate. Because many ischemia-induced changes in mRNA translation are mimicked by the removal of a metabolic substrate under normal perfusion conditions, the results suggest that cellular energy status represents an important modulator of I/R-induced changes in mRNA translation.     

Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK

Regulated phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) by the endoplasmic reticulum (ER) stress-activated protein kinase PERK modulates protein synthesis and couples the production of ER client proteins with the organelle's capacity to fold and process them. PERK activation by ER stress is known to involve transautophosphorylation, which decorates its unusually long kinase insert loop with multiple phosphoserine and phosphothreonine residues. We report that PERK activation and phosphorylation selectively enhance its affinity for the nonphosphorylated eIF2 complex. This switch correlates with a marked change to the protease sensitivity pattern, which is indicative of a major conformational change in the PERK kinase domain upon activation. Although it is dispensable for catalytic activity, PERK's kinase insert loop is required for substrate binding and for eIF2alpha phosphorylation in vivo. Our findings suggest a novel mechanism for eIF2 recruitment by activated PERK and for unidirectional substrate flow in the phosphorylation reaction.