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Binding of Double-stranded RNA to Protein Kinase PKR Is Required for Dimerization and Promotes Critical Autophosphorylation Events in the Activation Loop

2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês

10.1074/jbc.m102108200

ISSN

1083-351X

Autores

Fan Zhang, Patrick R. Romano, Tokiko Nagamura‐Inoue, Bin Tian, Thomas Dever, Michael B. Mathews, Keiko Ozato, Alan G. Hinnebusch,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2α to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA bindingin vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2α kinase function of PKR. Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2α to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA bindingin vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2α kinase function of PKR. double-stranded RNA dsRNA-binding motif X. laevis RNA-binding protein A second dsRNA binding domain whole cell extract complete protease inhibitor calf intestinal alkaline phosphatase polyacrylamide gel electrophoresis FLAG-tagged PKR triple hemagglutinin-tagged PKR interferon 3-aminotriazole The human double-stranded RNA (dsRNA)1-dependent protein kinase PKR is transcriptionally induced by interferon and activated in virus-infected cells by dsRNAs produced during the virus life cycle. PKR interferes with virus replication by phosphorylating the α subunit of translation initiation factor 2 (eIF2α), converting eIF2 from a substrate to an inhibitor of its guanine nucleotide exchange factor, eIF2B. This reduction in recycling of eIF2 by eIF2B leads to a general inhibition of translation that limits viral protein synthesis (1Mathews M.B. Semin. Virol. 1993; 4: 247-257Crossref Scopus (64) Google Scholar). The yeast Saccharomyces cerevisiaeharbors an eIF2α kinase known as GCN2 which is activated by uncharged tRNA when cells are starved for amino acids. Limited phosphorylation of eIF2α by GCN2 under starvation conditions leads to increased translation of GCN4 mRNA, encoding a transcriptional activator of amino acid biosynthetic genes (for review, see Ref. 2Hinnebusch A.G. Hershey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 199-244Google Scholar). Low level expression of PKR in yeast produces a moderate level of eIF2α phosphorylation that is sufficient to induce GCN4expression without inhibiting general protein synthesis (3Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4616-4620Crossref PubMed Scopus (187) Google Scholar). When PKR is expressed at higher levels, eIF2α phosphorylation increases to the point where general translation and yeast cell growth are strongly inhibited (3Dever T.E. Chen J.J. Barber G.N. Cigan A.M. Feng L. Donahue T.F. London I.M. Katze M.G. Hinnebusch A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4616-4620Crossref PubMed Scopus (187) Google Scholar, 4Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R.G. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (289) Google Scholar). PKR kinase activity is stimulated in vitro by dsRNA, and the N-terminal 168 amino acids of the protein contain two copies of a dsRNA-binding motif (DRBMs I and II; Fig.1 A) that is also present in other dsRNA-binding proteins (5Fierro-Monti I. Mathews M.B. Trends Biochem. Sci. 2000; 25: 241-246Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Point mutations in the DRBMs which impair dsRNA binding by PKRin vitro reduce the ability of PKR to phosphorylate eIF2α in yeast cells, supporting the idea that the DRBMs mediate the stimulatory effect of dsRNA on PKR kinase activity (4Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R.G. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (289) Google Scholar, 6Romano P.R. Green S.R. Barber G.N. Mathews M.B. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 365-378Crossref PubMed Google Scholar). dsRNA stimulates the autokinase activity of PKR (7Galabru J. Hovanessian A. J. Biol. Chem. 1987; 262: 15538-15544Abstract Full Text PDF PubMed Google Scholar, 8Galabru J. Katze M.G. Robert N. Hovanessian A.G. Eur. J. Biochem. 1989; 178: 581-589Crossref PubMed Scopus (118) Google Scholar, 9Kostura M. Mathews M.B. Mol. Cell. Biol. 1989; 9: 1576-1586Crossref PubMed Scopus (162) Google Scholar), and multiple autophosphorylation sites have been identified in the dsRNA binding and kinase domains of PKR (10Taylor D.R. Lee S.B. Romano P.R. Marshak D.R. Hinnebusch A.G. Esteban M. Mathews M.B. Mol. Cell. Biol. 1996; 16: 6295-6302Crossref PubMed Scopus (107) Google Scholar, 11Romano S.-S.P.R. Garcia-Barrio M.T. Zhang X. Wang Q. Taylor D.R. Zhang F. Herring C. Mathews M.B. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2282-2297Crossref PubMed Google Scholar, 12Zhang X. Herring C.J. Romano P.R. Szczepanowska J. Brzeska H. Hinnebusch A.G. Qin J. Anal. Chem. 1998; 70: 2050-2059Crossref PubMed Scopus (182) Google Scholar, 13Taylor D.R. Tian B. Romano P.R. Hinnebusch A.G. Lai M.M.C. Mathews M.B J. Virol. 2001; : 1265-1273Crossref PubMed Scopus (56) Google Scholar). Alanine substitution of the autophosphorylation site at Thr-258 (T258A), located between the DRBMs and kinase domain, produced a modest reduction in PKR function in yeast and mammalian cells (10Taylor D.R. Lee S.B. Romano P.R. Marshak D.R. Hinnebusch A.G. Esteban M. Mathews M.B. Mol. Cell. Biol. 1996; 16: 6295-6302Crossref PubMed Scopus (107) Google Scholar). In contrast, substitution of the autophosphorylation site at Thr-446 (T446A) in the activation loop of the kinase domain substantially reduced kinase activity. The T451A mutation in the activation loop completely destroyed kinase function, but the evidence that this site is autophosphorylated was inconclusive (11Romano S.-S.P.R. Garcia-Barrio M.T. Zhang X. Wang Q. Taylor D.R. Zhang F. Herring C. Mathews M.B. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2282-2297Crossref PubMed Google Scholar). It has been proposed that dsRNA binding overcomes a negative effect of the DRBMs on PKR catalytic function, based on the finding that extensive deletions in the N-terminal region of the protein that removed both DRBMs did not abolish (14Wu S. Kaufman R.J. J. Biol. Chem. 1996; 271: 1756-1763Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and even enhanced (15Zhu S. Romano P.R. Wek R.C. J. Biol. Chem. 1997; 272: 14434-14441Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) kinase activity. The N-terminal and C-terminal halves of PKR physically interacted in the two-hybrid assay (16Sharp T.V. Romashko A. Moonan F. Joshi B. Barber G.N. Jagus R. Virology. 1998; 250: 302-315Crossref PubMed Scopus (96) Google Scholar), and NMR data indicate that DRBM-II, but not DRBM-I, binds to the kinase domain (17Nanduri S. Rahman F. Williams B.R. Qin J. EMBO J. 2000; 19: 5567-5574Crossref PubMed Scopus (135) Google Scholar). Additionally, dsRNA binding induces a conformational change in PKR (18Manche L. Green S.R. Schmedt C. Mathews M.B. Mol. Cell. Biol. 1992; 12: 5238-5248Crossref PubMed Scopus (420) Google Scholar, 19Carpick B.W. Graziano V. Schneider D. Maitra R.K. Lee X. Williams B.R.G. J. Biol. Chem. 1997; 272: 9510-9516Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). These results have led to models in which the DRBMs (or just DRBM-II) bind to the kinase domain and interfere with its enzymatic function, and this inhibitory interaction is eliminated by a conformational change in PKR elicited by dsRNA binding to the DRBMs (17Nanduri S. Rahman F. Williams B.R. Qin J. EMBO J. 2000; 19: 5567-5574Crossref PubMed Scopus (135) Google Scholar, 20Wu S. Kaufman R.J. J. Biol. Chem. 1997; 272: 1291-1296Crossref PubMed Scopus (144) Google Scholar). There is also evidence that dsRNA activates PKR by promoting dimerization of the enzyme and intermolecular autophosphorylation. The fact that PKR activation exhibits second-order kinetics with respect to protein concentration suggests that the active form of PKR is a dimer (9Kostura M. Mathews M.B. Mol. Cell. Biol. 1989; 9: 1576-1586Crossref PubMed Scopus (162) Google Scholar), and the enzyme was purified as a phosphorylated dimer (21Langland J.O. Jacobs B.L. J. Biol. Chem. 1992; 267: 10729-10736Abstract Full Text PDF PubMed Google Scholar). The observation that two inactive PKR alleles containing deletions of DRBM-I or DRBM-II functionally complemented was most readily explained by formation of active heterodimers (6Romano P.R. Green S.R. Barber G.N. Mathews M.B. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 365-378Crossref PubMed Google Scholar). It was also shown that an N-terminal segment of PKR containing the DRBMs could be coimmunoprecipitated with full-length PKR from transfected COS cells (14Wu S. Kaufman R.J. J. Biol. Chem. 1996; 271: 1756-1763Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and N-terminal segments were found to interact with themselves and with full-length PKR in various interaction assays (22Cosentino G.P. Venkatesan S. Serluca F.C. Green S.R. Mathews M.B. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9445-9449Crossref PubMed Scopus (158) Google Scholar, 23Patel R.C. Stanton P. McMillan N.M.J. Williams B.R.G. Sen G.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8283-8287Crossref PubMed Scopus (144) Google Scholar, 24Patel R.C. Stanton P. Sen G.C. J. Biol. Chem. 1996; 271: 25657-25663Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 25Tan S.L. Gale M.J. Katze M.G. Mol. Cell. Biol. 1998; 18: 2431-2443Crossref PubMed Scopus (105) Google Scholar). PKR activation is inhibited by high concentrations of dsRNA, leading to a bell-shaped dsRNA activation curve (26Hunter T. Hunt T. Jackson R.J. Robertson H.D. J. Biol. Chem. 1975; 250: 409-417Abstract Full Text PDF PubMed Google Scholar). This behavior has been explained by proposing that dimerization requires binding of two PKR molecules to the same dsRNA, as high dsRNA concentrations would favor dissociation of dimers into inactive monomers bound to different dsRNA molecules (9Kostura M. Mathews M.B. Mol. Cell. Biol. 1989; 9: 1576-1586Crossref PubMed Scopus (162) Google Scholar). Consistently, high level activation of PKR requires a minimum length of dsRNA (∼40 base pairs) which is considerably larger than the binding site for two DRBMs in a single PKR molecule (11–16 base pairs) (18Manche L. Green S.R. Schmedt C. Mathews M.B. Mol. Cell. Biol. 1992; 12: 5238-5248Crossref PubMed Scopus (420) Google Scholar, 27Schmedt C. Green S.R. Manche L. Taylor D.R. Ma Y. Mathews M.B. J. Mol. Biol. 1995; 249: 29-44Crossref PubMed Scopus (101) Google Scholar, 28Bevilacqua P.C. Cech T.R. Biochemistry. 1996; 35: 9983-9994Crossref PubMed Scopus (202) Google Scholar). These data, combined with the observation that PKR can autophosphorylate in trans (29Thomis D.C. Samuel C.E. J. Virol. 1995; 69: 5195-5198Crossref PubMed Google Scholar,30Ortega L.G. McCotter M.D. Henry G.L. McCormack S.J. Thomis D.C. Samuel C.E. Virology. 1996; 215: 31-39Crossref PubMed Scopus (58) Google Scholar), have led to the idea that binding of two PKR molecules to the same dsRNA promotes dimerization and intermolecular autophosphorylation events required for substrate phosphorylation. Although there is some evidence that PKR dimerization is strongly dependent on dsRNA binding (19Carpick B.W. Graziano V. Schneider D. Maitra R.K. Lee X. Williams B.R.G. J. Biol. Chem. 1997; 272: 9510-9516Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 22Cosentino G.P. Venkatesan S. Serluca F.C. Green S.R. Mathews M.B. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9445-9449Crossref PubMed Scopus (158) Google Scholar), many studies suggest that dimerization is relatively unaffected by point mutations in the DRBMs that impair dsRNA binding in vitro (14Wu S. Kaufman R.J. J. Biol. Chem. 1996; 271: 1756-1763Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 22Cosentino G.P. Venkatesan S. Serluca F.C. Green S.R. Mathews M.B. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9445-9449Crossref PubMed Scopus (158) Google Scholar, 23Patel R.C. Stanton P. McMillan N.M.J. Williams B.R.G. Sen G.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8283-8287Crossref PubMed Scopus (144) Google Scholar, 24Patel R.C. Stanton P. Sen G.C. J. Biol. Chem. 1996; 271: 25657-25663Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 30Ortega L.G. McCotter M.D. Henry G.L. McCormack S.J. Thomis D.C. Samuel C.E. Virology. 1996; 215: 31-39Crossref PubMed Scopus (58) Google Scholar). The results of the latter studies suggest that protein-protein interactions can mediate PKR dimerization in the absence of dsRNA binding. Wu and Kaufman (20Wu S. Kaufman R.J. J. Biol. Chem. 1997; 272: 1291-1296Crossref PubMed Scopus (144) Google Scholar) found that dimerization by isolated DRBMs could occur independently of dsRNA binding but that dimerization of full-length PKR with the isolated DRBM was dsRNA-dependent. To account for these findings, they proposed that dsRNA binding to full-length PKR was required primarily to dissociate the DRBMs from the kinase domain and unmask a dimerization surface in the DRBMs. However, recent evidence that only DRBM-II interacts with the kinase domain implies that DRBM-I is exposed and available for dimerization constitutively (17Nanduri S. Rahman F. Williams B.R. Qin J. EMBO J. 2000; 19: 5567-5574Crossref PubMed Scopus (135) Google Scholar). A second dimerization domain in PKR was localized to the 244–296 interval, overlapping the N-terminal portion of the kinase domain (25Tan S.L. Gale M.J. Katze M.G. Mol. Cell. Biol. 1998; 18: 2431-2443Crossref PubMed Scopus (105) Google Scholar). Nanduriet al. (17Nanduri S. Rahman F. Williams B.R. Qin J. EMBO J. 2000; 19: 5567-5574Crossref PubMed Scopus (135) Google Scholar) proposed that this region becomes available for dimerization after dissociation of DRBM-II from the kinase domain upon dsRNA binding and provides the critical dimerization surface in the enzyme. However, the importance of the 244–296 interval for dimerization by full-length PKR has not been addressed experimentally. An alternative model for PKR activation was proposed by Patel and Sen (31Patel R.C. Sen G.C. Mol. Cell. Biol. 1998; 18: 7009-7019Crossref PubMed Scopus (48) Google Scholar) in which dimerization occurs independently of dsRNA binding, and dsRNA (or alternative stimulatory ligands) activate a preformed PKR dimer by producing a conformational change in the protein. It was found that mutating residue Ala-67 to Glu in DRBM-I abolished dimerization of the N-terminal domain of PKR without affecting dsRNA binding. This suggested that Ala-67 is required for a critical protein-protein contact between the dimerized DRBMs. The fact that A67E had a much greater effect on kinase function than did a mutation (K60A) that abolished dsRNA binding without impairing dimerization led these workers to propose that dimerization precedes dsRNA binding and that PKR dimers can be activated in yeast independently of dsRNA. The solution structure of the N-terminal domain of PKR showed that DRBMs I and II have topologies highly similar to that of other DRBMs (32Bycroft M. Grunert S. Murzin A.G. Proctor M. St. Johnston D. EMBO J. 1995; 14: 3563-3571Crossref PubMed Scopus (229) Google Scholar, 33Kharrat A. Macias M.J. Gibson T.J. Nilges M. Pastore A. EMBO J. 1995; 14: 3572-3584Crossref PubMed Scopus (246) Google Scholar), separated by a flexible linker of about 20 residues (34Nanduri S. Carpick B.W. Yang Y. Williams B.R. Qin J. EMBO J. 1998; 17: 5458-5465Crossref PubMed Scopus (249) Google Scholar). The crystal structure of a complex between dsRNA and a single DRBM from theXenopus laevis RNA-binding protein A (Xlrbpa-2) revealed three different contact regions in the DRBM which interact with two successive minor grooves and the intervening major groove on one face of the dsRNA molecule (35Ryter J.M. Schultz S.C. EMBO J. 1998; 17: 7505-7513Crossref PubMed Scopus (393) Google Scholar). The same three RNA contact regions were identified in the NMR structure of the DRBM-3 of DrosophilaStaufen bound to an RNA stem-loop (36Ramos A. Grunert S. Adams J. Micklem D.R. Proctor M.R. Freund S. Bycroft M. St. Johnston D. Varani G. EMBO J. 2000; 19: 997-1009Crossref PubMed Scopus (295) Google Scholar). In view of these findings, we considered the possibility that certain PKRmutations studied previously which altered only one of three RNA contact regions in DRBM-I (e.g. K60A) might not completely impair dsRNA binding. In this event, conclusions about the dependence of dimerization and kinase activation on dsRNA binding based on such mutations would be questionable. In addition, the conclusion that dimerization by full-length PKR is dsRNA-dependent derived from analysis of a single mutation (K64E) in DRBM-I (20Wu S. Kaufman R.J. J. Biol. Chem. 1997; 272: 1291-1296Crossref PubMed Scopus (144) Google Scholar). With the finding that only DRBM-II interacts with the kinase domain (17Nanduri S. Rahman F. Williams B.R. Qin J. EMBO J. 2000; 19: 5567-5574Crossref PubMed Scopus (135) Google Scholar), we wished to determine whether dsRNA binding by DRBM-II is required for dimerization. Accordingly, we constructed point mutations designed to destroy side chain interactions in one, two, or all three predicted dsRNA contact regions in DRBMs I and II and examined their effects on dimerization by full-length PKR molecules expressed in yeast cells. Our results provide strong support for the idea that dimerization by full-length PKR is dependent on dsRNA binding, but they show that it occurs independently of the dimerization domain between residues 244 and 296. We also employed the DRBM point mutants to investigate whether dsRNA binding and dimerization stimulate autophosphorylation in the PKR activation loop. Using an antibody specific for phosphothreonine 451, we provide evidence that phosphorylation of this residue is stimulated by dsRNA binding. Finally, we show that Ser-33 and multiple autophosphorylation sites in the flexible linker between the DRBMs, and within kinase subdomain V, are largely dispensable for PKR function in yeast. Our data provide strong support for a model in which dsRNA binding enables dimer formation, enhancing autophosphorylation of Thr-451 in the activation loop with attendant activation of the eIF2α kinase function of PKR. Descriptions of the plasmids employed are given in Table I, and details of their construction will be provided upon request.Table IPlasmids used in this studyPlasmidsDescriptionSource (Ref.)pEMBLyex4High copyURA3 yeast expression vector with CYC-GALpromoter55Cesareni G. Murray J.A.H. Setlow J.K. Hollaender A. Genetic Engineering: Principles and Methods. 9. Plenum Press, New York1987: 135-154Google ScholarpRS314Low copy TRP1 yeast vector56Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholarp1469Wild type PKR in pEMBLyex46Romano P.R. Green S.R. Barber G.N. Mathews M.B. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 365-378Crossref PubMed Google Scholarp1470PKR-K296Rin pEMBLyex46Romano P.R. Green S.R. Barber G.N. Mathews M.B. Hinnebusch A.G. Mol. Cell. Biol. 1995; 15: 365-378Crossref PubMed Google Scholarp1689PKR-T446A in pEMBLyex457Romano S.-S.P.R. Garcia-Barrio M.T. Zhang X. Wang Q. Taylor D.R. Zhang F. Herring C. Mathews M.B. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2282-2297Crossref PubMed Google Scholarp1895PKR-Δ14–257 in pEMBLyex415Zhu S. Romano P.R. Wek R.C. J. Biol. Chem. 1997; 272: 14434-14441Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar and this studyp2111PKR-T451A in pEMBLyex457Romano S.-S.P.R. Garcia-Barrio M.T. Zhang X. Wang Q. Taylor D.R. Zhang F. Herring C. Mathews M.B. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2282-2297Crossref PubMed Google Scholarp2130PKR-S242A,T255A,T258A in pEMBLyex410Taylor D.R. Lee S.B. Romano P.R. Marshak D.R. Hinnebusch A.G. Esteban M. Mathews M.B. Mol. Cell. Biol. 1996; 16: 6295-6302Crossref PubMed Scopus (107) Google Scholarp2587PKR-S242A,T255A,T258A,T446Ain pEMBLyex457Romano S.-S.P.R. Garcia-Barrio M.T. Zhang X. Wang Q. Taylor D.R. Zhang F. Herring C. Mathews M.B. Qin J. Hinnebusch A.G. Mol. Cell. Biol. 1998; 18: 2282-2297Crossref PubMed Google Scholarp2640PKR-S33A in pEMBLyex4This studyp2642PKR-S33A,T446A in pEMBLyex4This studyp2734FLAG, His6-tagged wild type PKRin pEMBLyex4This studyp2828FLAG, His6-taggedPKR-K296R in pEMBLyex4This studyp2831PKR-T336A,S337A,S340A,S343A,S344A,S351A,S354A,S355A,S357A,T359Ain pEMBLyex4This studyp2832PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97Ain pEMBLyex4This studyp2965PKR-T336A,S337A,S340A,S343A,S344A,S351A,S354A,S355A,S357A,T359A,T446Ain pEMBLyex4This studyp2966PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97A,T446Ain pEMBLyex4This studyp2967PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97A,T336A,S337A,S340A, S343A,S344A,S351A,S354A,S355A,S357A,T359Ain pEMBLyex4This studyp2968PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97A,T336A,S337A,S340A, S343A,S344A,S351A,S354A,S355A,S357A,T359A,T446A in pEMBLyex4This studyp2969PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97A,S242A,T255A,T258A, T336A,S337A,S340A,S343A,S344A,S351A,S354A,S355A,S357A, T359Ain pEMBLyex4This studyp2970PKR-S83A,T88A,T89A,T90A,S92A,S93A,S97A,S242A,T255A,T258A, T336A,S337A,S340A,S343A,S344A,S351A,S354A,S355A,S357A, T359A,T446Ain pEMBLyex4This studyp2971FLAG, His6-taggedPKR-A67E in pEMBLyex4This studyp2975FLAG, His6-tagged PKR-S59A,K60A in pEMBLyex4This studyp2976FLAG, His6-taggedPKR-H37A,S59A,K60A in pEMBLyex4This studyp2977FLAG, His6-taggedPKR-T149A,K150A in pEMBLyex4This studyp2978FLAG, His6-taggedPKR-H126A,T149A,K150A in pEMBLyex4This studyp2979FLAG, His6-taggedPKR-S59A,K60A,T149A,K150Ain pEMBLyex4This studyp2980FLAG, His6-taggedPKR-H37A,S59A,K60A,H126A,T149A,K150A in pEMBLyex4This studyp2981FLAG, His6-taggedPKR-N15A,T16A,H37A,S59A,K60A,K61A in pEMBLyex4This studyp2982FLAG, His6-taggedPKR-N106A,R107A,H126A,T149A,K150A,Q151A in pEMBLyex4This studyp2983FLAG, His6-taggedPKR-N15A,T16A,H37A,S59A,K60A,K61A,N106A, R107A,H126A,T149A,K150A,Q151Ain pEMBLyex4This studyp2984HA3-tagged wild typePKR in pRS314This studyp3105FLAG, His6-tagged PKR-Δ244–296 in pEMBLyex4This study Open table in a new tab Transformants of strains H1894 (a ura3–52 leu2–3, -112, gcn2Δ trp1-Δ63) (37Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (565) Google Scholar), J82 (a ura3–52 leu2–3, -112 gcn2Δ trp1-Δ63 sui2Δ p1098 [SUI2-S51A LEU2] (37Dever T.E. Feng L. Wek R.C. Cigan A.M. Donahue T.D. Hinnebusch A.G. Cell. 1992; 68: 585-596Abstract Full Text PDF PubMed Scopus (565) Google Scholar), and GP3299 (a ura3–52 leu2–3, -112 gcn2Δ trp1-Δ63 gcd2Δ::hisG pAV1033 [GCD2-K627T TRP1] (38Pavitt G.D. Yang W. Hinnebusch A.G. Mol. Cell. Biol. 1997; 17: 1298-1313Crossref PubMed Scopus (107) Google Scholar) containing different PKR alleles were grown in SC medium overnight, diluted 1:50 in SC medium (39Sherman F. Fink G.R. Lawrence C.W. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1974Google Scholar) and grown toA 600 of 0.6–1.0, and then shifted to synthetic medium containing 10% galactose and 2% raffinose (SGAL) for∼12 h. Whole cell extracts (WCEs) were prepared by breaking cells with glass beads in lysis buffer (20 mmTris, pH 8.0, 50 mm KCl, 400 mm NaCl, 20% glycerol, 0.5 mm EDTA, 0.1% Triton X-100) supplemented with 1 mm phenylmethylsulfonyl fluoride, 10 mm2-aminopurine, 10 mm NaF, 50 mmβ-glycerolphosphate, 125 μm sodium orthovanadate, and complete protease inhibitor (CPI) mixture (Roche). For analysis of phosphatase-treated proteins, samples of WCE containing 25 μg of protein, prepared as above except that 10 mm NaF, 50 mm β-glycerolphosphate, and 125 μm sodium orthovanadate were omitted from the lysis buffer, were treated with 2 units of calf intestinal alkaline phosphatase (CIP) (New England BioLabs) for 30 min at 37 °C. A phosphatase inhibitor mixture containing 10 mm sodium pyrophosphate, 5 mmEDTA, 5 mm EGTA, and 125 μm sodium orthovanadate was added to selected samples prior to CIP treatment. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes probed with pT451 antibodies were blocked in TBS-T (20 mm Tris, pH 8.0, 150 mm NaCl, 0.1% Tween 20) containing 3% bovine serum albumin, whereas all other membranes where blocked in a solution of TBS-T containing 5% non-fat dry milk. Immunodetection of FLAG-tagged PKR (FL-PKR), triple HA-tagged PKR (HA3-PKR), and untagged PKR was conducted as indicated in the figure legends using a monoclonal antibody against the FLAG epitope (Sigma), polyclonal antibodies against the HA-epitope (BabCo), and polyclonal antibodies specific for the N terminus (N-18) or C terminus (K-17) of PKR (Santa Cruz Biotechnology). Immunodetection of PKR phosphorylated at Thr-451 was conducted using phosphospecific polyclonal antibodies (BIOSOURCE International) in blocking solution containing 3% bovine serum albumin. Immunoblot analysis of eIF2α phosphorylation was conducted using polyclonal antibodies specific for pS51 (Research Genetics) and polyclonal antibodies against total eIF2α (CM-217) (40Cigan A.M. Pabich E.K. Feng L. Donahue T.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2784-2788Crossref PubMed Scopus (145) Google Scholar). Immune complexes were visualized with the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech) according to the vendor's instructions and quantified by video image densitometry of the resulting autoradiograms using NIH Image 1.61 software. Yeast WCEs were prepared by breaking cells with glass beads in the yeast lysis buffer described above supplemented with CPI and phosphatase inhibitor mixtures. Samples of WCEs containing 200 μg of protein were incubated with poly(I-C)-agarose beads (Amersham Pharmacia Biotech) in 200 μl of binding buffer (150 mm KCl, 20 mm Hepes, 10% glycerol, 5 mm magnesium acetate) plus CPI mixture and phosphatase inhibitors for 1 h at 4 °C. The beads were collected by centrifugation, washed three times with lysis buffer, resuspended in 30 μl of 2 × Laemmli sample buffer (41Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar), and boiled for 5 min. Proteins were resolved by SDS-PAGE and subjected to immunoblot analysis as described above. Yeast WCEs were prepared by breaking yeast cells with glass beads in immunoprecipitation lysis buffer (IP buffer) (20 mm sodium phosphate, pH 7.0, 500 mmNaCl, 0.1% Triton X-100) supplemented with CPI and phosphatase inhibitor mixtures. Aliquots containing 500 μg of protein were diluted to 100 μl in IP buffer and incubated with anti-FLAG M2-agarose (Sigma) at 4 °C overnight with rocking. The beads were collected by centrifugation, washed three times with IP buffer, resuspended in 30 μl of 2 × Laemmli sample buffer, and boiled for 5 min. The proteins were resolved by SDS-PAGE and subjected to immunoblot analysis as described above. HeLa cells grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum were treated with interferons (IFN) α/β and poly(I-C) as described in Fig. 6 A. Cells were washed twice with cold phosphate-buffered saline containing CPI and phosphatase inhibitor mixtures described above and lysed in extraction buffer (20 mm Tris, pH 7.5, 150 mmNaCl, 1% Triton X-100) containing CPI mixture and phosphatase inhibitors. For immunopurification of PKR, an aliquot of WCE containing 500 μg of protein was incubated with PKR monoclonal antibody 71/10 (Ribogene/Questcor) for 2

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