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 α 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. The double-stranded (ds) RNA-dependent protein kinase (PKR) regulates protein synthesis by phosphorylating the α 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. Eukaryotic cells rapidly and reversibly halt protein synthesis in response to a variety of stresses including virus infection and cytotoxic chemical injury (1Duncan R.F. Hershey J.W. Arch. Biochem. Biophys. 1987; 256: 651-661Crossref PubMed Scopus (33) Google Scholar, 2Proud C.G. Curr. Top. Cell Regul. 1992; 32: 243-369Crossref PubMed Scopus (165) Google Scholar). This fundamental homeostatic mechanism is thought to involve the phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2α), 1The abbreviations used are: eIF2α, α subunit of eukaryotic initiation factor-2; As, sodium arsenite; IL-3, interleukin-3; PKR, double-stranded RNA-dependent protein kinase; TG, thapsigargin; ds, double-stranded; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin which regulates protein synthesis rate at translational initiation. Phosphorylation of eIF2α increases the stability of complexes formed between eIF2 and eIF2B, a guanine-nucleotide exchange factor. eIF2B converts eIF2-GDP to eIF2-GTP binary complex, which further forms a ternary complex with a Met-tRNA and becomes associated with the 40 S ribosomal subunit to initiate translation of mRNA. Because eIF2B exists in cells in relatively low molar quantities with respect to eIF2, phosphorylation of only a limited amount (i.e. 20–25%) of eIF2α is apparently sufficient to sequester virtually all of the eIF2B, resulting in inhibition of protein synthesis (3Proud C.G. Trends. Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar, 4Merrick W.C. Hershey J.W.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 31-69Google Scholar, 5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar). The interferon-inducible double-stranded (ds) RNA-dependent kinase (PKR) is an ubiquitously expressed eIF2α kinase, which was first identified as a component of the host defense mechanism induced by interferon (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar). In vitro, PKR can be activated by synthetic dsRNA, such as poly(I)·poly(C), and natural dsRNA forms, such as reovirus genomic RNA (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar). In interferon-treated cells, virus infection leads to activation of PKR by autophosphorylation, followed by eIF2α phosphorylation and inhibition of protein synthesis (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar, 6Mathews M.B. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 505-548Google Scholar). Thus, protein synthesis inhibition occurring after virus infection is thought to be due to the direct activation of PKR by dsRNA species. However, the mechanism of eIF2α phosphorylation in the absence of infection is not clear. We previously reported that in interleukin-3 (IL-3)-dependent cells, IL-3 deprivation induced activation of PKR in close association with a decreased rate of total protein synthesis (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). Recently, it has been reported that stresses such as Ca2+ depletion from the endoplasmic reticulum, sodium arsenite, or hydrogen peroxide treatments rapidly induce PKR activation (8Prostko C.R. Dholakia J.N. Brostrom M.A. Brostrom C.O. J. Biol. Chem. 1995; 270: 6211-6215Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 9Srivastava S.P. Davies M.V. Kaufman R.J. J. Biol. Chem. 1995; 270: 16619-16624Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 10Brostrom C.O. Prostko C.R. Kaufman R.J. Brostrom M.A. J. Biol. Chem. 1996; 271: 24995-25002Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). It was suggested that although PKR is activated by dsRNAin vitro, a dsRNA-independent activation mechanism may exist in cells because it is difficult to envision that such diverse stresses are likely to rapidly change the intracellular levels of dsRNA species. Thus a novel cellular regulator of PKR was sought. By screening a mouse cDNA library using the yeast two-hybrid interacting cloning system, we discovered a PKR-associating protein RAX. RAX appears to be a mouse homologue of PACT, a recently isolated direct activator for PKR (11Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (379) Google Scholar). We now find that RAX can activate PKR in the absence of dsRNA in a cell-free system as reported for PACT. However, results here indicate that the RAX-PKR association and any resulting cellular activation of PKR may be regulated by an unique, stress-induced signaling mechanism featuring RAX phosphorylation. Using polymerase chain reaction-based mutagenesis, the mouse PKR cDNA (12Icely P.L. Gros P. Bergeron J.J. Devault A. Afar D.E. Bell J.C. J. Biol. Chem. 1991; 266: 16073-16077Abstract Full Text PDF PubMed Google Scholar) was first mutated at lysine 271 to arginine to generate the catalytically inactive PKR(K271R), the mouse equivalent to human PKR(K296R) mutant (13Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (417) Google Scholar), and then subcloned into pGBT9 (CLONTECH). A random primed NFS/N1.H7 cell cDNA library was generated in pGAD10 (CLONTECH). The yeast two-hybrid screening of the cDNA library was performed according to the Matchmaker Two-Hybrid System protocol (CLONTECH). A 405-base pair mouse RAX cDNA fragment (nucleic acid positions 943–1348) was amplified by polymerase chain reaction and radiolabeled using the Prime-A-Probe kit (Ambion, Inc., Austin, TX). A mouse multiple tissue blot was obtained fromCLONTECH, and hybridization was performed according to the manufacturer's instruction. The RAX cDNA was cloned into pRSET vector (Invitrogen) to generate a polyhistidine-tagged RAX and used to transform Escherichia coli BL21 (DE3) pLysS (Novagen, Madison, WI). The protein was induced and partially purified with TALON metal affinity resin according to the manufacturer's instruction (CLONTECH). The fraction containing RAX was incubated with poly(I·C)-agarose beads (Amersham Pharmacia Biotech), and the protein was dissociated by boiling in SDS-PAGE sample buffer and further purified by SDS-PAGE. The gel slice containing the recombinant RAX was used to immunize a rabbit to raise anti-RAX antiserum (COVANCE Research Products Inc., Denver, PA). The murine IL-3-dependent NFS/N1.H7 cells (14Boswell H.S. Mochizuki D.Y. Burgess G.S. Gillis S. Walker E.B. Anderson D. Williams D.E. Exp. Hematol. 1990; 18: 794-800PubMed Google Scholar) were maintained in RPMI 1640 medium supplemented with 20% WEHI-3B cell conditioned medium as described previously (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). The hemagglutinin (HA) epitope-tagged RAX cDNA was subcloned into the expression vector pcDEF3 (15Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar) and transfected into NFS/N1.H7 cells by electroporation. The clones stably expressing HA-RAX were selected as described previously (16Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Cells (1 × 107) were treated with 1 mm sodium arsenite (Sigma), 1 mm hydrogen peroxide (Sigma), or 1 μm thapsigargin (Calbiochem) and lysed in 1 ml of buffer A (10 mm HEPES, pH 7.2, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 20 mm sodium fluoride, 20 mm sodium pyrophosphate, 20 mmβ-glycerophosphate, 20 mm sodium molybdate, 20 μg/ml chymostatin, and 1 μm microcystin-LR). Immunoprecipitation and immunoblotting were performed using anti-PKR antibody (Ab-1) or anti-HA antibody (12CA5, Roche Molecular Biochemicals) using protein A-agarose beads, as described previously (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). For metabolic labeling, cells were incubated with 0.1 mCi/ml 32Porthophosphoric acid (ICN) at 1 × 107 cells/ml in phosphate-free RPMI 1640 medium for 2 h. Phosphoamino acid analysis was performed as described (17Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). 5 μg of polyhistidine-tagged RAX was incubated with 30 μl of poly(I·C)-agarose, native DNA-cellulose (Amersham Pharmacia Biotech), or poly(C)-agarose (Sigma) beads in Tris-buffered saline (50 mm Tris-HCl, pH 7.4, 150 mm NaCl) containing 1% Triton X-100 for 1 h at 4 °C. The beads were washed three times before boiling in Laemmli buffer. The eluted protein was loaded onto a 10% SDS-PAGE gel followed by Coomassie Blue R-250 staining. The phosphorylation state of eIF2α in NFS/N1.H7 cells was analyzed by vertical slab isoelectric focusing and immunoblotting essentially as described previously (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar, 18Maurides P.A. Akkaraju G.R. Jagus R. Anal. Biochem. 1989; 183: 144-151Crossref PubMed Scopus (42) Google Scholar). After treatment, cells (3 × 106) were lysed in 100 μl of buffer A. The acetone-precipitated extract (100 μg) was subjected to the isoelectric focusing and immunoblotting using ECL kit (Amersham Pharmacia Biotech) and a rabbit eIF2α polyclonal antibody raised against a KLH-coupled synthetic peptide corresponding to amino acid residues 298–315 of human eIF2α. For analysis of RAX phosphorylation, Bio-lyte 6/8 (Bio-Rad) and 10 mm ethylenediamine (Sigma) were used for the ampholytes and the cathode buffer, respectively. Before transfer, the gel was treated with 0.1 m Tris-HCl, pH 6.8, 2% SDS, and 10 mmdithiothreitol for 30 min at room temperature to enhance the elution efficiency. PKR was isolated from the NFS/N1.H7 cells by using anti-PKR antibody (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar, D. A Cold Spring Harbor Laboratory, Cold Spring Harbor, Scholar). PKR was by elution with the synthetic peptide (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). The HA-RAX was with anti-HA antibody as described and was from the by elution with 1 peptide Molecular After against Tris-buffered the purified HA-RAX was by immunoblotting using expressed RAX as a were incubated for 10 min at 30 in μl of reaction buffer mm Tris-HCl, pH 2 mm 2 mm 1 mm 20 μm 5 of 0.1 μg of rabbit were by SDS-PAGE and was by or directly by Cells × 106) were washed with Tris-buffered saline and lysed in 100 μl of buffer (10 mm Tris-HCl, pH 10 and Triton The was at for 10 and the was treated with 1 μg of A and 40 μg of for 2 h at respectively. The was with m and at and analyzed by in a 2% gel containing 10 μg/ml novel cellular that with and PKR, we a mouse IL-3-dependent NFS/N1.H7 cell cDNA library using the yeast two-hybrid cloning system S. 1989; PubMed Scopus Google Scholar). the mouse PKR(K271R), which is in kinase (13Meurs E.F. Galabru J. Barber G.N. Katze M.G. Hovanessian A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 232-236Crossref PubMed Scopus (417) Google Scholar), was used because PKR yeast growth Hovanessian A.G. Williams EMBO J. 1992; PubMed Scopus Google Scholar). 4 × yeast clones were The sequence analysis that clones are from the novel the cDNA and a acid by This is to as RAX protein and rapid of cDNA using RNA from NFS/N1.H7 cells that RAX cDNA is analysis that cDNA and amino acid of RAX are to of human and a recently identified PKR-associating protein (11Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (379) Google Scholar) 1 The that RAX is a murine of PACT. RAX also has homology to including a protein C.R. J. Cell Biol. 1997; PubMed Scopus Google Scholar) and the protein A. A. B. 1991; PubMed Scopus Google Scholar) and murine M.A. Biol. 1996; PubMed Scopus Google Scholar) in RAX also three in is not D. Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google Scholar) 1 recombinant RAX was to dsRNA because virtually to or RNA was blot analysis of RNA from mouse that RAX is expressed in all an polyclonal antibody raised against recombinant RAX a protein in the from cell from including mouse IL-3-dependent and cells 1 The at the SDS-PAGE gel well with a of RAX an was in cells after of RAX that the protein is RAX. The was in human and cell that the antibody can with human 1 results indicate that is an ubiquitously expressed and well between the species. the of we first transfected epitope-tagged RAX into IL-3-dependent NFS/N1.H7 cells (14Boswell H.S. Mochizuki D.Y. Burgess G.S. Gillis S. Walker E.B. Anderson D. Williams D.E. Exp. Hematol. 1990; 18: 794-800PubMed Google Scholar) and obtained 10 clones that stably levels of HA-RAX 2 was between the expression levels of HA-RAX and the cell growth that overexpression of RAX is not growth in cells in the presence of IL-3 we that HA-RAX overexpression cell of IL-3 and This appears to be the expression levels of the HA-RAX protein because high expressing clones the low expressing clones or vector results that RAX has a that may be in the presence of growth factor. from the yeast two-hybrid that RAX can to PKR. the of RAX PKR an in kinase was using isolated from HA-RAX was from cells The purified HA-RAX associated kinase that any associated PKR was first PKR was purified from NFS/N1.H7 We that HA-RAX activate PKR as by eIF2α phosphorylation and PKR in the absence of dsRNA of dsRNA are to the RAX activates PKR Thus, we find HA-RAX purified from cells can activate PKR in However, overexpression of HA-RAX apparently does not the growth of NFS/N1.H7 cells in the presence of IL-3, that the RAX in may not be cellular PKR the In of using anti-PKR and anti-HA that the RAX-PKR association is in cells expressed as in the with (i.e. the 4 However, an in the association the cells are of IL-3 for a by which protein synthesis is by PKR activation (7Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). and rapid RAX-PKR association is the cells are to various chemical stresses including sodium arsenite H2O2, or thapsigargin an of endoplasmic 4 known to inhibit protein synthesis (1Duncan R.F. Hershey J.W. Arch. Biochem. Biophys. 1987; 256: 651-661Crossref PubMed Scopus (33) Google Scholar, 10Brostrom C.O. Prostko C.R. Kaufman R.J. Brostrom M.A. J. Biol. Chem. 1996; 271: 24995-25002Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). the total amount of HA-RAX in the cell is not by any of stress that any association is not due to RAX expression as previously reported C.G. Curr. Top. Cell Regul. 1992; 32: 243-369Crossref PubMed Scopus (165) Google Scholar, T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar, C.O. Brostrom M.A. Biol. 1998; PubMed Scopus Google Scholar), the stress treatments were also to induce phosphorylation of eIF2α, that PKR, physiologic kinase, has been activated 4 any eIF2α phosphorylation be in transfected cells, that the overexpression of RAX is not sufficient to activate PKR and inhibit RAX-PKR association and activation of PKR cells 1 × 107) were incubated with 1 mm or 1 μm or 1 mm min at in the presence of IL-3. cells were from IL-3 for h. PKR was from the cell using anti-PKR antibody or and the associated RAX was analyzed by SDS-PAGE followed by immunoblotting using anti-HA antibody of cell were also the phosphorylation of eIF2α at A was analyzed by vertical slab isoelectric focusing and immunoblotting as described and results that RAX can directly to and activate PKR of stress The rapid RAX-PKR association after the stress treatments raised the that RAX may be as a of any stress by the analysis that NFS/N1.H7 cells only a of RAX with an of IL-3 deprivation or of cells with or induced the of RAX to 5 A RAX was also HA-RAX expressing cells were treated with As, TG, or or of IL-3 5 HA-RAX was RAX due to the of the peptide sequence and was to stress HA-RAX was from the cell and treated with before onto the the was of RAX purified from cells not change after indicate that cells only the the stress treatments induced phosphorylation of RAX. Phosphoamino acid analysis that RAX is or IL-3 deprivation 5 RAX and eIF2α phosphorylation were min after with and phosphorylation of RAX and is before that of eIF2α, that RAX phosphorylation PKR activation Because PKR was to directly HA-RAX in RAX does not to be a for PKR. cellular RAX be by PKR after we a for PKR T. R.J. Full Text PDF PubMed Scopus Google Scholar, A. L.A. T. PubMed Scopus Google Scholar), RAX phosphorylation. that the eIF2α phosphorylation by IL-3 deprivation was by or RAX phosphorylation results that RAX is by stress-activated, protein before it activates PKR. The for PKR activation viral infection that dsRNA activates regulator of protein synthesis, the mechanism of activation in the absence of viral infection is not clear. have that various stress can induce eIF2α phosphorylation C.G. Curr. Top. Cell Regul. 1992; 32: 243-369Crossref PubMed Scopus (165) Google Scholar, C.O. Brostrom M.A. Biol. 1998; PubMed Scopus Google Scholar), that activation of PKR may as a of stress dsRNA is involved is not results indicate that RAX may be a activator of PKR. The high sequence between mouse RAX and human that RAX is a mouse homologue of PACT, which was recently identified using a cloning (11Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (379) Google Scholar). the in with purified RAX indicate that PACT, can directly activate PKR, that PKR can be activated by cellular in the absence of dsRNA. The of PKR activation that of PKR to a dsRNA to which results in (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar). and activation of PKR that RAX may also PKR to of PKR. We also that RAX can activate PKR in the presence of dsRNA an mechanism. Although further are the for a between RAX and dsRNA with respect to PKR is high because PKR is associated with dsRNA may be (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar, S. R.C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). PKR activation leads to protein synthesis inhibition and decreased cell growth (5Clemens M.J. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 139-172Google Scholar, Hovanessian A.G. Williams EMBO J. 1992; PubMed Scopus Google Scholar, 1994; PubMed Scopus Google Scholar). Because RAX can activate PKR, RAX-PKR association cell growth be regulated to activation of PKR. that the RAX-PKR can be by stress The unknown. We a between PKR and the phosphorylation state of RAX after various stress Because RAX phosphorylation PKR the that the of RAX may be involved in the RAX-PKR association and activation of PKR in Phosphoamino acid analysis that only is that RAX is the for a thapsigargin, hydrogen and IL-3 all have been reported to activate the protein kinase that protein kinase and kinase J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. Curr. Cell Biol. 1997; PubMed Scopus Google Scholar). RAX is a for further RAX in cells is not RAX can apparently activate PKR in Therefore, results that RAX phosphorylation is not for PKR activation may the RAX-PKR association and PKR by the affinity or of RAX to PKR. However, the for RAX phosphorylation and the mechanism by which RAX can activate PKR to be Because PKR is a ribosomal protein S. R.C. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, S. Kaufman R.J. 1998; PubMed Scopus Google Scholar), is that phosphorylation may ribosomal of RAX and association with PKR. is that RAX may have a affinity for PKR PKR such as N. S. J. Katze M.G. Biol. 1994; PubMed Google Scholar) and protein Davies M.V. J. Kaufman R.J. S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: PubMed Scopus Google Scholar). of the phosphorylation and analysis of RAX are to and Sen (11Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (379) Google Scholar) have reported that of the cDNA the of eIF2α phosphorylation in cells, and the of cell stably was it was that overexpression of activates PKR and cell By RAX can be in IL-3-dependent NFS/N1.H7 cells with PKR or cell at in the presence of IL-3. Although the for the between and RAX with respect to cell growth is not it be due to the cell in the absence of IL-3, in direct to the expression levels Therefore, it likely that growth such as IL-3 at in stress signals stress kinase by the association between RAX and PKR. It be to can be in NFS/N1.H7 cells and not in cell in the presence of IL-3. In to PKR, eIF2α kinase, D. PubMed Scopus Google Scholar) or R. J. J. R.C. Biol. 1998; 18: PubMed Google Scholar), has been recently is associated with endoplasmic and can be activated by endoplasmic stresses such as However, is not activated by stresses including arsenite, and D. PubMed Scopus Google Scholar). In indicate that arsenite IL-3 deprivation activates in NFS/N1.H7 D. and S. Thus, stresses may activate only PKR, may activate eIF2α In we that RAX is of a novel signaling that inhibition of protein synthesis that as a of the direct between RAX and PKR. Because PKR activation has been in and the stresses here can all induce cell activated RAX appears to be a regulator of cell growth and We R. Jagus for purified eIF2α, J. A. Langer for and D. A. and S. P. for