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, theα-subunit of eukaryotic translation initiation factor-2 (eIF2α) 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 eIF2α in the developing cerebellum. The effect of ethanol on PKR/eIF2α 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 eIF2α phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2α phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2α 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. 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, theα-subunit of eukaryotic translation initiation factor-2 (eIF2α) 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 eIF2α in the developing cerebellum. The effect of ethanol on PKR/eIF2α 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 eIF2α phosphorylation was mediated by PKR. Overexpression of a wild-type RAX dramatically enhanced sensitivity to ethanol-induced PKR/eIF2α phosphorylation, as well as translational inhibition and cell death. In contrast, overexpression of a mutant (S18A) RAX inhibited ethanol-mediated PKR/eIF2α 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. Fetal alcohol syndrome is the most common non-hereditary cause of mental retardation (1May P.A. Gossage J.P. Alcohol Res. Health. 2001; 25: 159-167PubMed Google Scholar). Prenatal exposure to alcohol disrupts many events of neuronal development, including neurogenesis, migration, cell survival, protein synthesis, axonal growth, and synaptogenesis (2Diamond I. Gordon A.S. Physiol. Rev. 1997; 77: 1-20Crossref PubMed Scopus (323) Google Scholar, 3Goodlett C.R. Horn K.H. Alcohol Res. Health. 2001; 25: 175-184PubMed Google Scholar, 4Luo J. Miller M.W. Brain Res. Brain Res. Rev. 1998; 27: 157-167Crossref PubMed Scopus (151) Google Scholar, 5Olney J.W. Addict. Biol. 2004; 9: 137-149Crossref PubMed Scopus (87) Google Scholar). Neuronal death is a prominent pathologic effect of fetal alcohol exposure. This loss of neurons may underlie many of the behavioral deficits observed in fetal alcohol syndrome. The vulnerability of neurons to alcohol neurotoxicity differs among brain regions and changes with developmental stages (6Maier S.E. Miller J.A. Blackwell J.M. West J.R. Alcohol Clin. Exp. Res. 1999; 23: 726-734Crossref PubMed Google Scholar, 7Maier S.E. West J.R. Alcohol. 2001; 23: 49-57Crossref PubMed Scopus (140) Google Scholar). The causes for ethanol-induced neuronal loss remain incompletely elucidated, as are the cellular and molecular mechanisms underlying the spatiotemporal window of susceptibility. The double-stranded RNA (dsRNA) 2The abbreviations used are: dsRNA, double-stranded RNA; CNS, central nervous system; eIF2α, eukaryotic initiation factor-2α; HA, hemagglutinin; MEF, mouse embryonic fibroblasts; PACT, PKR-activating protein; PERK, PKR-like endoplasmic reticular kinase; PKR, double-stranded RNA-activated protein kinase; RAX, PKR activator X; W T, wild-type; 2-AP, 2-aminopurine; IL, interleukin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; p-, phosphorylated.-activated protein kinase (PKR) is a serine/threonine protein kinase ubiquitously expressed in mammalian cells (8Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11693-11695Crossref PubMed Scopus (133) Google Scholar, 9Levin D. London I.M. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 1121-1125Crossref PubMed Scopus (155) Google Scholar). PKR is initially identified as an interferon-induced protein that is activated in virus-infected cells by dsRNA produced during the virus life cycle (10Tan S.L. Katze M.G. J. Interferon Cytokine Res. 1999; 19: 543-554Crossref PubMed Scopus (99) Google Scholar, 11Ung T.L. Cao C. Lu J. Ozato K. Dever T.E. EMBO J. 2001; 20: 3728-3737Crossref PubMed Scopus (92) Google Scholar). PKR consists of two functionally distinct domains, an N-terminal dsRNA binding regulatory domain and a C-terminal catalytic domain. PKR is activated by dsRNA (11Ung T.L. Cao C. Lu J. Ozato K. Dever T.E. EMBO J. 2001; 20: 3728-3737Crossref PubMed Scopus (92) Google Scholar); interaction with dsRNA causes PKR to form homodimers and to autophosphorylate on multiple serine/threonine residues, including threonine 446 and 451 (12Williams B.R. Oncogene. 1999; 18: 6112-6120Crossref PubMed Scopus (706) Google Scholar, 13Wu S. Kaufman R.J. J. Biol. Chem. 1997; 272: 1291-1296Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 14Zhang F. Romano P.R. Nagamura-Inoue T. Tian B. Dever T.E. Mathews M.B. Ozato K. Hinnebusch A.G. J. Biol. Chem. 2001; 276: 24946-24958Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). PKR is a component of signal transduction pathways mediating many important cellular functions, such as survival, proliferation, differentiation, and stress responses (10Tan S.L. Katze M.G. J. Interferon Cytokine Res. 1999; 19: 543-554Crossref PubMed Scopus (99) Google Scholar, 12Williams B.R. Oncogene. 1999; 18: 6112-6120Crossref PubMed Scopus (706) Google Scholar, 15Barber G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar, 16Williams B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). In addition to dsRNA, PKR can be activated by cytokines, growth factors, serum deprivation, bacterial products, or physiochemical stress (12Williams B.R. Oncogene. 1999; 18: 6112-6120Crossref PubMed Scopus (706) Google Scholar, 16Williams B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar, 17Gil J. Esteban M. Apoptosis. 2000; 5: 107-114Crossref PubMed Scopus (331) Google Scholar). Recent studies indicate that dsRNA-independent activation may be mediated by protein activators of PKR, PACT, and its mouse homologue, RAX (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (384) Google Scholar, 20Patel C.V. Handy I. Goldsmith T. Patel R.C. J. Biol. Chem. 2000; 275: 37993-37998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Following autophosphorylation, PKR catalyzes the phosphorylation of target substrates, the most well characterized being the α-subunit of eukaryotic initiation factor-2 (eIF2α) (12Williams B.R. Oncogene. 1999; 18: 6112-6120Crossref PubMed Scopus (706) Google Scholar). Phosphorylated eIF2α sequesters eIF2B, a rate-limiting component of translation, leading to an inhibition of protein synthesis in the cells. In some cases, eIF2α phosphorylation leads to cell death (21Saelens X. Kalai M. Vandenabeele P. J. Biol. Chem. 2001; 276: 41620-41628Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In this study, we have demonstrated that ethanol promotes PKR/RAX interaction; this interaction in turn activates PKR and induces eIF2α phosphorylation. The levels of RAX expression determine the cellular sensitivity to ethanol. These findings provide important insight into the mechanisms of ethanol-induced damage to the CNS. Materials—Sprague-Dawley rats were obtained from Hilltop Laboratory Inc. (Scottdale, PA). 2-Aminopurine (2-AP) was purchased from Sigma, and a selective PKR inhibitor was purchased from Calbiochem (La Jolla, CA; catalogue number 527450). All antibodies, except anti-PACT and anti-ATF4, were obtained from Cell Signaling Technology (Beverly, MA). The anti-PACT and anti-ATF4 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture and Treatment—Human neuroblastoma cells (SH-SY5Y and SK-N-MC cells) and a human embryonic kidney cell line (HEK293) were obtained from the American Type Culture Collection. These cells were grown in minimum Eagle's medium containing 10% fetal bovine serum, 2 mm l-glutamine, and 25 μg/ml gentamycin at 37 with and mouse embryonic were obtained from for and in Eagle's medium with 10% fetal bovine serum and at 37 with neurons were from the of were cultured for to ethanol in The for and of has been C. S. J. J. 2004; 18: PubMed Scopus Google Scholar, M. J. Brain Res. Brain Res. 2001; PubMed Scopus Google Scholar). Ethanol to the of a was used to ethanol levels in the medium J. Miller M.W. Alcohol Clin. Exp. Res. 1997; Google Scholar). this ethanol in the medium be In ethanol exposure was as by T. C.R. Brain Res. PubMed Scopus Google Scholar). two and two of a were ethanol by two by 2 The alcohol produced by the is two and two of the the ethanol exposure of ethanol exposure were a ethanol was on a of ethanol was during the the were and were were and at All were by the and of West and with the of for and number of cells in was by the as C. S. J. J. 2004; 18: PubMed Scopus Google Scholar). Cell and of RAX and S18A mutant of to by were from May of May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Cell was with to the cell expressing RAX were by the of for were verified by the expression of as well as the overexpression of RAX. The expressing the of RAX were synthesis was by the of catalogue number cells were in cell at the of ethanol for the of was to the and at 37 for and the medium was the cells were with of mm mm mm and were by the addition of of The were on mm and was by a were at a of a and at The expression of RAX in the was by a anti-PACT number Santa Cruz The for has been J. J. 2005; PubMed Scopus Google Scholar). The of this has been by were by the and for has been C. S. J. J. 2004; 18: PubMed Scopus Google Scholar). was as J. Miller M.W. J. 1999; 19: PubMed Google Scholar). an of cell containing of protein was with or anti-PACT at of protein to (Santa Cruz were to the and the was for at were by at for The was with of the were in and for the expression of by The of protein expression was with a the among were of in were In were between were with Ethanol of PKR and effect of ethanol on the phosphorylation of PKR and eIF2α was in cells. Ethanol and increased phosphorylation of PKR and at a phosphorylation of PKR and eIF2α a the expression of a of was by ethanol exposure. We the expression of PACT/RAX in neuronal cells (SH-SY5Y and SK-N-MC neuroblastoma cells and and a cell line kidney cell line with findings the expression of PACT/RAX as in mammalian cells (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (384) Google Scholar, J. Sen G.C. Biol. 2001; PubMed Scopus Google cells expressed with from rats of the expression of RAX cells with the PACT/RAX were most to PKR phosphorylation and The developing has been used for ethanol-induced of a window of vulnerability to ethanol exposure West J.R. Alcohol Clin. Exp. Res. PubMed Scopus Google Scholar, C.R. West J.R. Alcohol. PubMed Scopus Google Scholar, C.R. West J.R. PubMed Scopus Google Scholar). We the expression of RAX, PKR, and eIF2α in the developing cerebellum. in RAX expression was observed during RAX expression from and The form of PKR and eIF2α increased dramatically from and to remain at a We the expression of eIF2α kinase, PKR-like endoplasmic reticular kinase the of was during demonstrated a RAX in the and cell in the of cells were in the determine in exposure to ethanol PKR/eIF2α we ethanol to shown in ethanol exposure increased the phosphorylation of PKR and eIF2α in the of rats Ethanol not the expression of RAX, PKR, and results were observed exposure to ethanol not of ethanol on the developing cerebellum. of a were to ethanol as a the ethanol exposure the ethanol the were and were The expression of RAX and as well as PKR and eIF2α was by a from an The was In addition to PKR, eIF2α can PERK, inhibitor and B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). We to determine ethanol-induced eIF2α phosphorylation was mediated by PKR. shown in two selective inhibitors of PKR and blocked ethanol-induced eIF2α phosphorylation in that PKR was in ethanol-induced eIF2α phosphorylation. The was by an PKR shown in ethanol promoted eIF2α phosphorylation in but not in cells. ethanol increased expression in cells Overexpression of PACT/RAX to between PACT/RAX expression and ethanol-induced PKR/eIF2α phosphorylation to that PACT/RAX a role in the of ethanol. determine a expression of PACT/RAX ethanol-induced PKR/eIF2α phosphorylation, we increased the levels of PACT/RAX expression by SK-N-MC cells with a wild-type or a mutant (S18A) RAX It has been shown that phosphorylation of RAX at plays an role in PKR activation. Overexpression of RAX was verified by and anti-PACT antibodies shown in overexpression of RAX in SK-N-MC cells dramatically enhanced PKR and eIF2α phosphorylation, with cells. In contrast, overexpression of S18A RAX inhibited ethanol-induced PKR/eIF2α phosphorylation. We or S18A RAX and the effect of ethanol on PKR and eIF2α results were obtained The was not to neuronal overexpression of RAX cells to ethanol-induced PKR/eIF2α phosphorylation These findings that a expression of PACT/RAX ethanol-mediated PKR/eIF2α phosphorylation. Ethanol the between PKR and to determine ethanol promoted the interaction between PKR and We the effect of ethanol on the association between PKR and PACT/RAX in SK-N-MC cells with RAX, S18A RAX, or an shown in the association between RAX and PKR was demonstrated by In cells RAX, ethanol promoted the association between PKR and PKR and S18A RAX were ethanol to the association between PKR and PACT/RAX in cells S18A RAX of PKR and PACT/RAX was in the cells expressing This was of the of PACT/RAX in SK-N-MC cells. We observed that ethanol enhanced PKR and PACT/RAX association in cells RAX not In cases, ethanol not PKR binding to RAX in cells S18A RAX. Overexpression of PACT/RAX and Cell of eIF2α protein we that eIF2α phosphorylation was enhanced by ethanol in a we to determine protein synthesis on the of PACT/RAX shown in ethanol exposure the of protein synthesis by in SK-N-MC inhibited the of protein synthesis by in cells RAX. Ethanol protein synthesis in cells with S18A RAX. was observed exposure to ethanol for this of exposure ethanol not cell number not the in the of protein synthesis not from an in cell We PACT/RAX the effect of ethanol on cell survival. shown in ethanol the number of SK-N-MC cells with an by the in SK-N-MC cells with RAX, ethanol-induced cell loss was increased to Ethanol to the number of cells S18A RAX. cells were in medium and not the in cell number have from ethanol-induced cell death. of was to ethanol-induced cell loss The effect of PKR inhibition was observed selective PKR inhibitor not This that the effect of ethanol was mediated by PKR. PKR/RAX is a ubiquitously expressed serine/threonine PKR has been in the of the anti-viral G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar, 16Williams B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). PKR two dsRNA binding and is activated by viral dsRNA, in the phosphorylation of a of PKR, eIF2α, and translation In addition to its role in translation, PKR has been as a signal in pathways B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). the of factors, such as and is by PKR B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). PKR is an of by and growth B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). PKR in the of the protein kinase and kinase in to cell B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). PKR can be activated many that of This dsRNA-independent activation is to be mediated by the PKR-activating protein and its mouse RAX (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (384) Google Scholar, May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and RAX are the known cellular activators for PKR are in and dsRNA binding It has been demonstrated that PACT/RAX can PKR in a (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (384) Google Scholar). in activation is on stress to the cells (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 20Patel C.V. Handy I. Goldsmith T. Patel R.C. J. Biol. Chem. 2000; 275: 37993-37998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, F. X. May W.S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Our that PACT/RAX is an important of the of ethanol. ethanol induces a activation of PKR/eIF2α in cells expressing a expression of PACT/RAX dramatically ethanol-induced PKR/eIF2α phosphorylation. Ethanol not the expression of promotes the association between PACT/RAX and PKR. Ethanol of RAX and PKR in cells In we not this This is of the of PACT/RAX in cells. to studies that stress by serum and can PACT/RAX and PKR leads to PKR activation and eIF2α phosphorylation (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 20Patel C.V. Handy I. Goldsmith T. Patel R.C. J. Biol. Chem. 2000; 275: 37993-37998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of RAX at for its effect on PKR activation. May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google that association and RAX phosphorylation at in cells. Overexpression of the mutant of RAX, S18A RAX, inhibits PKR activation. results suggest that association RAX phosphorylation and PKR activation. on May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google a In this PACT/RAX with PKR and is at phosphorylation of PACT/RAX at may a in PACT/RAX that its C-terminal dsRNA binding domain to and PKR. The S18A RAX, to with PKR, to PKR Our neuronal cells the that phosphorylation of RAX in is for PKR activation in to overexpression of S18A RAX inhibits ethanol-induced PKR/eIF2α phosphorylation. S18A RAX as a negative and ethanol-mediated S18A RAX and PKR are in the absence of that RAX phosphorylation at is to PKR results that ethanol the association between S18A RAX and PKR. phosphorylation at is important for association in to ethanol exposure. Ethanol eIF2α phosphorylation in a In addition to PKR, eIF2α These PERK, and B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). Using selective PKR we have demonstrated that PKR ethanol-induced eIF2α phosphorylation. The is by the ethanol to eIF2α phosphorylation in and Cell has been shown that ethanol inhibits protein synthesis S. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cell Biol. 2001; PubMed Scopus Google Scholar). the underlying mechanisms remain incompletely D. J. Physiol. 1999; Google that ethanol and eIF2α phosphorylation in In the ethanol inhibits initiation increased phosphorylation of eIF2α S. PubMed Scopus Google Scholar, J.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Our results the that eIF2α is we have identified that PKR, an eIF2α kinase, is a target of ethanol. We have that the of PACT/RAX expression in a cell sensitivity to ethanol-induced translational The of PACT/RAX for translational regulation has been demonstrated in to stress overexpression of PACT/RAX is shown to translational inhibition by exposure or May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, F. X. May W.S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). Our an insight into the mechanisms of ethanol-induced translational Ethanol exposure induces the death of of cells some J.W. Addict. Biol. 2004; 9: 137-149Crossref PubMed Scopus (87) Google Scholar, M. J. Brain Res. Brain Res. 2001; PubMed Scopus Google Scholar, J. West J.R. Alcohol Clin. Exp. Res. 1997; PubMed Scopus Google Scholar, J. West J.R. Alcohol Clin. Exp. Res. 1999; 23: PubMed Google Scholar). Our results indicate that the PKR/eIF2α plays a role in ethanol-mediated cell death. PKR and eIF2α have been to have in mechanisms G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar, 17Gil J. Esteban M. Apoptosis. 2000; 5: 107-114Crossref PubMed Scopus (331) Google Scholar). Our that the of PACT/RAX expression is important in to ethanol. Overexpression of RAX ethanol-mediated PKR activation and cell death. In contrast, S18A RAX has a dominant negative effect and ethanol-induced cell death. phosphorylation of RAX at is for PKR activation and cell death by ethanol exposure. Overexpression of in and cells induces and cell death by stress by and C.V. Handy I. Goldsmith T. Patel R.C. J. Biol. Chem. 2000; 275: 37993-37998Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). neuronal cells as well as cells indicate that overexpression of RAX is not to cause cell death (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, May W.S. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of RAX cell death by and or not expression of PACT/RAX induces cell death is cell of PKR causes cell death or translational mechanisms G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar). number of cell mechanisms and have been in cell death G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar, 16Williams B.R. Science's STKE. 2001; 2001: RE2Crossref PubMed Scopus (367) Google Scholar). the inhibition of translation may or the cell death by (21Saelens X. Kalai M. Vandenabeele P. J. Biol. Chem. 2001; 276: 41620-41628Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Exp. Cell Res. PubMed Scopus Google Scholar). phosphorylation of eIF2α causes a in protein synthesis, translation of functions, such as has been G.N. Cell Death Differ. 2005; 12: 563-570Crossref PubMed Scopus (71) Google Scholar, S. K. G.N. EMBO J. 1998; 17: PubMed Scopus Google Scholar). It to be of PKR/eIF2α are for ethanol-induced cell death. in to the that the of ethanol-induced PKR/eIF2α phosphorylation to the expression of PACT/RAX in a cell Overexpression of PACT/RAX the effect of ethanol on PKR/eIF2α phosphorylation as well as protein synthesis and cell survival. and RAX are and ubiquitously PKR, and RAX are not by or dsRNA (18Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 19Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (384) Google Scholar, J. Sen G.C. Biol. 2001; PubMed Scopus Google Scholar). The is of the regions most to of an is to the in is used for ethanol-induced damage J.R. J. PubMed Scopus Google Scholar). ethanol exposure causes a in and protein of the C.R. West J.R. PubMed Scopus Google Scholar, C. J. Brain Res. PubMed Scopus Google Scholar, S. 9: PubMed Scopus Google Scholar, M.W. Brain Res. 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Exp. 2004; PubMed Scopus Google Scholar). PKR is to be in the in and of PKR is observed in the of neurons by T. T. T. K. M. 2005; PubMed Scopus Google Scholar). PKR to mutant RNA and activated PKR is in the brain of 2001; PubMed Google Scholar). in the may not underlie ethanol neurotoxicity but be in the by We at for in the on We May of for the RAX for and for in the of

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