Proteins that interact with PKR

Biochimie (1994) 76, 779-791

© Soci6t6 fran~:aise de biochimie et biologie mol6culaire / Elsevier, Paris

779

Proteins that interact with PKR R Jagus, MM Gray Center of Marine Biotechnology, University of Maryland Biotechnology Centel; 600 E Lombard Street, Baltimore. MD 21202, USA

Summary D The in vitro activities of recombinant gene products of the vaccinia virus E3L and K3L genes have been compared. These proteins are both potent inhibitors of the dsRNA activated protein kinase (PKR) as assayed in cell-free translation systems or with purified PKR. The two gene products function at similar molar concentrations. Both proteins are expressed early in vaccinia virus infection suggesting that vaccinia virus maintains redundant mechanisms for the down regulation of PKR. The K3L gene product can be shown to be associated with PKR in vaccinia virus infected cells. The activities of the vaccinia virus PKR inhibitors are compared with other viral protein inhibitors of PKR. A variety of cellular proteins have also been identified by their ability to inhibit PKR activity or to prevent PKR activation. These cellular PKR interacting proteins have been uncovered from the studies of viral strategies to prevent PKR activation, as well as from studies looking at the effects of growth control, growth factors or oncogene expression on PKR activity. A picture emerges of PKR fulfilling a complex regulatory role in cell function with the regulation of its activity as part of a complex cascade interfacing with the signal transduction/cell cycle control machinery. PKR inhibitors / viral strategies / regulation cell proliferation

Introduction The d s R N A activated protein kinase, OKR [ 1], is currently the most studied m e m b e r o f the elF-2a-specific protein kinase subfamily [2--4]. This enzyme, a serine/ threonine protein kinase, has been variously referred to in the literature as elF-2~PKd~ [5], p68 kinase [6, 7], DAI [8] and dsRNA-PK [9]. Activation is accompanied by autophosphorylation on serine/threonine residues which may represent an inter- or intramolecular ! 10] reaction [11 ], with the kinase functioning either as a m o n o m e r [10, 1 I] or a dimer [8]. Although the majority o f work on the h u m a n kinase has focused on its participation in the inhibition of virus growth by interferon, P K R h a s recently been implicated in playing an important role in regulating cell ~ o w t h and gene expression, and seems to function as a tumor suppressor gene. PKR is expressed constitutively at

Abbreviations: HIV, human immunodeficiency virus; I-p58,

cellular inhibitor of p58; p58, cellular PKR inhibitor, activated by influenza virus; pE3, gene product of vaccinia virus E3L gene; pK3, gene product of vaccinia virus K3L gene; PDGF, platelet-derived growth factor; PKR, dsRNA activated, eIF-2cxspecific protein kinase; SKIE specific kinase inibitory factor from vaccinia virus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel e!ectrophoresis,

low levels in most cell types studied, but it is also inducible by interferon, the levels of PKR increasing 3-10-fold after exposure of cells to this cytokine 19]. PKR exists in an inactive form in rapidly growing and uninfected cells. Activation of PKR has been shown to follow the starvation of mouse N F S / N I . H 7 cells for IL-3 [12], upon terminal differentiation of 3T3-F422A fibroblasts to adipocytes [13, 14], and upon infection of a variety of cell types by vesicular stomatitis virus [15, 16], encephalo-myocarditis virus [17], reovirus [ 18, 191, mengovirus [20, 21 ] and poliovirus [22, 231. Proteins o f both viral and cellular origins have been described that interact with PKR to modulate its activity [24]. From the study of viruses that are able to grow in interferon treated cells, a variety of viral proteins have been identified that prevent PKR activity or activation (reviewed in [4, 10, 24-27]). In the first half of this article, the actions of two viral protein itflaibitors of PKR, the K3L and E3L gene products of vaccinia virus, will be described. Completing the article will be a review of a variety of cellular proteins identified through their interaction with PKR. The existence of these cellular P K R interacting proteins has been uncovered from a variety of studies. These include the elucidation of viral strategies to prevent PKR activation, as well as from studies looking at the relationship between PKR activity and proliferation rate, and

780 the effects of growth factors and oncogene expression on PKR activity.

Viral proteins that interact with PKR The susceptibility of a virus to the antiviral effects of interferon appears to correlate with its ability to down-

regulate PKR [28]. Several viruses have evolved strategies to down-regulate PKR, including adenovirus (reviewed in [25, 29]), influenza virus [30-33], human immunodeficiency virus (HIV) [34], poliovirus [22, 35] and vaccinia virus [5, 16]. The strategies used by viruses vary; some viruses down-regulate PKR by the generation of inhibitors of kinase function, others stimulate degradation of PKR. For in:-tance, in adeno-

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Fig 1. Levels and activation state of PKR during infection by vaccinia virus A. Activation state of PKR during vaccinia virus infection. HeLa cells were incubated with or without IS-interferon (50 units/ml) for 18 h prior to vaccinia virus or mock infection. At 2, 4, 6, or 8 h post infection, using a multiplicity of infection of 30, the cells were incubated in phosphate-free medium with 0.1% calf serum, containing 200 pCi/ml [32p]orthophosphate for 1 h, and subsequently harvested, lysed and PKR recovered by incubation with monoclonal antibody [96] cross-linked to CL-4B Sepharose beads [22]. As a positive control, the sample in the second lane is from interferon-treated cells incubated with 10 pg/ml poly(1):(C) for 4 h prior to harvest, and processed as above. The recovered proteins were analyzed by 10% SDS-PAGE. An autoradiograph is depicted here. B. Effect of vaccinia virus infection on activation of PKR by dsRNA. HeLa cells were incubated with [3-interferon (50 units/ml) for 18 h prior to treatment with or without 10 or 20 pg/ml poly(1):(C) for 4 h, with or without concurrent vaccinia virus infection. The cells were incubated in phosphate-free medium with 0.1% calf serum, contained with 200 pCi/ml [32p]orthophosphate for 1 h, and processed as above. C. Levels of PKR during vaccinia virus infection. Cells from interferon treated cells, uninfected, vaccinia virus infected, or mock infected, were lysed in SDS-PAGE sample buffer and the proteins fractionated by 10% SDSPAGE, transferred to lmmobilon-P, and subjected to immunoautography using monoclonal antibodies to PKR and Amersham's ECL reagents.

781 virus infected cells there is the production of a small RNA, VAI RNA, that binds to PKR and prevents its activation (reviewed in [29]). Influenza virus infection activates a cellular protein that inhibits PKR activation by preventing kinase autophosphorylation [31-33]. Conversely, in poliovirus infection, PKR is activated but rapidly degraded [22, 35], and in HIV infected cells PKR levels are reduced by an undescribed mechanism [34]. Viruses that encode PKR inhibitory proteins include reovirus and vaccinia virus. PKR is inhibited during reovirus serotype 1 infection due to the action of the o3 protein, the product of the reovirus $4 gene [36]. The ability of the o3 protein to down-regulate PKR activity is thought to reflect its ability to function as a dsRNA binding protein [36-381. Related to its ability to down-regulate PKR activity, co-expression of o3 enhances the translation of reporter genes in COS-I cells [39, 40]. Vaccinia virus is also able to grow in interferon treated cells by down-regulating PKR [5, 16] and encodes two gene products that are able to downregulate PKR activity (reviewed in [41]).

PKR inhibitory proteins from vaccinia virus Our own studies have shown that vaccinia virus infection prevents stimulation of PKR activity as measured by its in situ phosphorylation state. Since PKR levels remain constant after infection, it appears that vaccinia virus prevents activation of PKR rather than stimulating its breakdown. Figure 1A shows PKR immunoprecipitated from mock- or vaccinia virus-infected HeLa cells, pretreated or not with interferon for 18 h prior to infection, and incubated with [32p] orthophosphate. Although PKR autophosphorylation is increased in interferon treated cells, this reflects the higher PKR content of the cells. No increase in PKR autophosphorylation was observed after vaccinia virus infection over the first 8 h of infection, although incubation of interferon treated cells with 10 pg/ml poly(l).poly(C) for 4 h resulted in a dramatic increase in PKR autophosphorylation. Furthermore, figure 1B illustrates that vaccinia virus infection of HeLa cells is able to prevent the poly(I).polyC) induced increase in PKR autophosphorylation. Figure C is an immunoblot demonstrating that PKR levels are not changed by vaccinia virus infection. The two vaccinia virus early genes that have been implicated in the ability of vaccinia virus to prevent PKR activation are the K3L and E3L genes. The K3L gene product, pK3, is a homologue of the tx-subunit of elF-2 [42, 43]. The K3L gene encodes a polypeptide of 88 amino acid (10 kDa) that has 33% sequence identity and 68% similarity to the amino terminus of elF-2tx [42, 43]. The E3L gene encodes a polypepfide of 200 amino acids (28 kDa) that contains regions of

homology with one of the dsRNA binding domains in PKR [44, 45, 98]. Both PKR and pE3 belong to a recently described family of dsRNA binding proteins that include the Drosophila gene staufen, the Xenopus RNA binding protein, RNAse III, and other virus encoded proteins [46]. Vaccinia virus deletion mutants for either the K3L or E3L gene have been reported to have increased sensitivity to interferon [43, 47]. The K3L gene product, pK3, functions as a tightly binding pseudosubstrate, down-regulating PKR activity in in vitro reactions [48]. Transient expression of the K3L gene in COS-I cells has been shown to reduce PKR activity and eIF-2o~ phosphorylation state [41, 49]. This mechanism enables transient expression of the K3L gene to rescue the translational expression of a reporter gene mRNA [49]. The transfection studies have shown that pK3 does not exert its effects as a functional analogue of eIF-2o~ in translational initiation, since the presence of the K3L gene is not sufficient to by-pass a block in translation caused by a Ser51 to Asp51 mutation of eIF-2o~ [49]. The E3L gene product, pE3, corresponds to the activity found in vaccinia virus infected cells described as SKIF (specific kinase inhibitory factor) [5, 16]. pE3 functions as a dsRNA binding protein to sequester the dsRNA activator of PKR [5, 4 l, 44, 45, 98]. Transient expression of the E3L gene also stimulates translational expression of a reporter gene and reduces eIF-2o~ phosphorylation with an apparent potency higher than that of the K3L gene product [41 ]. In vitro studies using recombinant K3L and E3L

gene products pK3 has been shown to have potent PKR inhibitory effects in in vitro assays. Recombinant pK3, referred to as pK3r, is able to prevent activation of both PKR and the heme sensitive eIF-2o~-specific protein kinase in a reticulocyte translation system [48]. pK3r also prevents immunopurified PKR and partially purified eIF-2o~-specific protein kinase from phosphorylating the tx-subunit of purified rabbit eIF-2 [48]. Furthermore, pK3r is able to inhibit PKR activity whether added before or after activation of PKR by dsRNA [48]. Similarly, recombinant pE3, pE3,, has potent effects hl vitro. Figure 2 shows a comparison of the effects of pK3r and pE3r on translational inhibition by dsRNA in a rabbit reticulocyte translation system. Both are very potent in preventing translational inhibition by dsRNA caused by the activation of PKR. However, pK3, is functioning at molar concentrations 2-3-fold lower than pE3r. Although pK3, is able to prevent the inhibition of the heme sensitive eIF-2o~specific protein kinase [48], pE3r is not (data not shown). Figure 3 shows the different effects of increasing concentrations of dsRNA on the abilities of pK3r

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rabbit reticulocytes were incubated under standard conditions, as described [48] in the absence ( i ) or presence ( e ) of poly(l):(C), 125 nghul, and in the absence or presence of recombinant pK3 (A) or pE3 (B), as indicated. Protein synthetic activity was measured by [~4C]valine incorporation into trichloracetic acid-precipitable material after a 30 min incubation at 30°C. pK3, was purified after expression of the K3L gene in bacteria, using the vector, pETI ld, as described [48]. pE3r was purified after expression in bacteria, also using the vector, pET I i d, followed by Cibacron-Blue chromatography. and pE3r to prevent translational inhibition. The effects of 5 pmol pK3, per 100 pl reaction volume is independent of the concentration of dsRNA, consistent with its functioning as a pseudosubstrate. In contrast, pE3, (15 pmol/100 pl) shifts the dsRNA concentration dependency to the right, consistent with its dsRNA binding function, The relative potencies of purified recombinant pK3 and pE3 in in vitro assays are not consistent with their apparent activity in transient expression systems, in which pE3 appears to be more potent than pK3 in supporting both translation of a reporter gene and in reducing the phosph0rylation state of eIF-2o~ [41 ]. This could reflect two possibilities. Firstly, the levels of the two PKR inhibitors in transiently transfected cells have not been determined, although the synthesis of pE3 was faster than that for pK3 and the levels of pE3 probably correspondingly higher [41]. Secondly, it is difficult to equate a

poly(1).poly(C) concentration in a reticulocyte translation system with concentrations of dsRNA in transfected cells and the pE3 requirement is reflective of dsRNA concentration. Figure 4 shows a comparison of the effects of pK3r and pE3r on the activity of immunopurified PKR from interferon-treated 293 cells. Both pK3, and pE3, reduce eIF-2o~ phosphorylation, although at the poly(l).poly(C) concentration used (200 ng/ml), pK3, was more potent than pE3r. In addition, the ability of pE3r to prevent eIF-2o~ phosphorylation is overcome by increasing the poly(I).poly(C) concentration to 1 pg/ ml. Although the effects of pK3~ and pE3~ on the ability of PKR to phosphorylate eIF-2o~ are clear, the effect of both on PKR autophosphorylation is slight. This is consistent with previous observations from several laboratories that in vitro autophosphorylation, as measured by the incorporation of [~32p] ATP into

783 immunopurifed PKR in vitro does not always reflect the activation state of PKR [48, 50]. This anomaly is thought to reflect differences in the autophosphorylation state of PKR as isolated from interferon-treated cells• The ability of pK3 to bind tightly to PKR is illustrated in figure 5, in which psS]methionine-radiolabelled pK3r produced in the reticulocyte translation system, can be co-immunoprecipitated with PKR and antibodies to the kinase. The binding of [35S]methionine-radiolabelled pK3, to PKR can be decreased by increasing levels of non-radiolabelled pK3,, giving an estimated Kd of 10- ~2 M. In contrast to this, stable binding of [35S]methionine-radiolabelled elF-2ot to PKR is hard to demonstrate (data not shown), reflecting the Kd of PKR for elF-2tx, measured as 6 x 10-7 M [51 ]. The natural substrate should not be expected to bind with great stability to PKR since this would decrease the enzyme's turnover number, and thereby

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its efficiency. The addition of purified elF-2o~ also decreases the binding of [3sS]methionine-radiolabelled pK3, to PKR somewhat, but only at very high molar excess• These data support the idea that pK3 and elF-2ot compete for the same site(s) on PKR. Not only does pK3 bind to PKR, but it is also found associated with ribosomes. Using [35S]methionine radiolabeled pK3r added to a reticulocyte translation system, and subsequently subjected to conditions that block translational initiation, pK3r is found associated with the small ribosomal subunit (Jagus and Witzel, unpublished observations). Expression of K3L and E3L genes during vaccinia virus infection The summary from the in vitro studies is that the products of the K3L and E3L genes are both potent inhi-

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poly (I):poly (C) (ng/ml) Fig 3. Effect of increasing dsRNA concentrations on the abilities of pK3r and pE3r to prevent translational inhibition by dsRNA. Cell-free extracts of rabbit reticulocytes were incubated under standard conditions, as described [48] in the absence (B) or presence ( • ) of pK3, (5 pmol/100 pl) (A) or pE3r (15 pmol/100 lal) (B), over a range of poly(I):(C) concentrations, as indicated. Protein synthetic activity was measured by [14C]valine incorporation into trichloracetic acid-precipitable material after a 30 min incubation at 30°C.

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bitors of PKR that act through different mechanisms. In addition, pK3 can be found associated with PKR and ribosomes. It is important to ask if these results correspond with events occurring in vivo during vaccinia virus infection. Figure 6 shows the synthesis of pK3 and pE3 during vaccinia virus infection in the presence and absence of cytosine arabinoside. Both gene products are produced at early times, the synthesis of both can be maintained by inhibiting viral DNA synthesis which prevents transcription of the late genes. Although [35Slmethionine incorporation into pE3 is higher by approximately two-fold, pE3 contains more methionine residues (six in pE3 compared with four in pK3), suggesting little difference in their synthetic rates. The involvement of pK3 in the down-regulation of PKR during vaccinia virus infection is supported by the finding that endogenous pK3 can be co-immunoprecipitated with endogenous PKR from vaccinia virus infected cells, using monocional antibody to PKR. Figure 7 shows that [35S]methionine radiolabeled pK3 accumulated in HeLa cells during vaccinia virus infection can also be co-immunoprecipitated with PKR and monoclonal antibodies to PKR bound to CL-4B Sepharose beads. The co-precipitation of the endogenous [35SImethionine radiolabeled pK3 can be reduced by inclusion of an excess of non-radioactive pK3r. Note that the gel system used for this experiment did not allow entry of PKR, These studies demonstrate that pK3 is able to bind to PKR in vivo as well as in vitro. The E3L gene product has been reported to have both nuclear and cytoplasmic locations hinting at multiple functions of this PKR inhibitor in the vaccinia virus life cycle I981. None of the above studies tells us whether one gene product is more important than the other, and whether

Fig 4. Effects of pK3r and pE3r on the activity of immunopurified PKR. PKR was immunopurified from 293 cell extracts and incubated with [¥32PlATP (final ATP concentration, 20 IaM), essentially as described 130], in the presence of 4 pmoi of elF-2, and the presence or absence of poly(l):(C) (200 ng/ml or I pg.dml),pK3, (5 pmol), pE3, (15 pmoi), as indicated. Samples were subjected to 12.5% SDS-PAGE and autoradiography.

both are necessary to prevent PKR activity during infection, or are illustrative of redundancy in the antidefense strategy of vaccinia virus. Vaccinia virus deletion mutants for both K3L and E3L have been developed and both decrease the ability of the virus to grow in interferon treated cells [43, 47[. However, investigations from other laboratories have indicated that the results from the virus deletion mutants are not consistent, Furthermore, the results are complicated by the fact that not only is there an apparent reduncancy in PKR inactivation mechanisms, but also a redundancy in the effects of interferon on the shut down of the host translational machine~. An alternate strategy to the use of vaccinia virus deletion mutants is to develop cells that stably express either the K3L or the E3L genes and to determine their ability to support the growth of interferon-sensitive viruses. We currently have both 3T3 and HeLa cell lines stably expressing these two genes and are characterizing their growth characteristics and their response to virus infection and incubation with poly(1).poly(C). Our conclusion so far is that inactivation of PKR seems of such importance to vaccinia virus that redundant strategies are maintained. There are precedents for viruses using multiple mechanisms to dominate the cellular machinery and overcome defense mechanisms, with pox viruses in particular exhibiting a wide range of antidefense strategies [521. For instance, cow pox virus has two mechanisms for decommissioning IL-1; a gene for a soluble IL-1 receptor, that binds up IL-i in the circulation [53], as well as a gene for a protease inhibitor that prevents the maturation of IL-1 to the mature, processed form [54]. It would seem that the activation of PKR poses a sufficient hazard to vaccinia virus growth that more than one mechanism is maintained to prevent its activation.

785

The relationship between PKR activity, elF-2ct phosphorylation state and cell proliferation state The inverse relationship between elF-2ot phosphorylation and cell proliferation rate has long been apparent (reviewed in [55, 561). Starvation of cultured cells for serum and/or growth factors leads to the phosphorylation of elF-2o~ along with a reduction in protein synthetic rates !12, 57, 58]. Conversely, the re-addition of serum or growth factors to cultured cells results in decreases in elF-2o~ phosphorylation, as well as the stimulation of protein synthesis [ 12, 59]. The identity of PKR as the elF-2tx-specific protein kinase involved in the action of serum and growth factors has only recently become apparent. An accumulation of evidence points to a role of PKR in the control of growth and transformation, and it is in this area that most unanswered questions on PKR function and regulation remain. All members of the elF-2o~-specific protein kinase subfamily show close homology to protein kinases involved in cell cycle regulation [601. Several laboratories have shown that expression of recombinant wild-type PKR suppresses growth in yeast [61, 62], although expression of catalytically inactive PKR mutants do not [62]. The decreased growth rate in yeast reflects increased phosphorylation of elF-2o~ [62]. Wild-type PKR is also toxic in mouse and insect cells. There is additional evidence for a role for PKR in regulating cell growth, by regulating the induction of certain proto-oncogenes and growth factors [12, 63--66]. PKR has been implicated as part of the IL-3, PDGF and I$-interferon signal transduction pathways [12, 65--68]. Similarly, transient PKR activation accompanies the terminal differentiation of 3T3F422A fibroblasts to adipocytes [ 13, 14]. The most definitive evidence for a role for PKR in the control of cell growth and proliferation has arisen from the stable high level expression of mutant, nonfunctional forms of PKR in murine NIH/3T3 cells (reviewed in [69-711). Using the retrovirus expression system vector, pMV7, expression of a PKR mutant, the A6 mutant, that encodes a polypeptide lacking the six conserved amino acids L F I Q M E , between catalytic domains V and Vl, yielded NIH/3T3 cells displaying decreased generation times, changed morphology, an ability to grow in soft agar, and an ability to cause tumors when injected into nude mice [72]. Similar results were obtained using a mutant PKR, the domain II mutant (DII), that encodes a polypeptide in which the invariant lysine at position 296 (mutant K296R) is converted to arginine using transfection with a pCDNA1/neo construct instead of retroviral mediated gene transfer [73]. An example of a spontaneous PKR mutation has been reported in a mouse lymphoblastic cell line. An in-frame deletion in the murine PKR results in the

expression of an inactive kinase and is believed to underly the transformed nature of these cells (Abraham, Jaramillo, Duocan, Methot, Icely, Barber, Bell, submitted). Recently, the gene for PKR has been mapped to human chromosome 22P21-22, known to be a site of rearrangements in myeloproliferative disorders [74, 75]. Furthermore, an inverse correlation has been noted in head and neck squamous cell carcinomas between PKR levels and patient survival rates [76, 77]. These data not only implicate PKR in cell growth, bat suggest that this kinase has potent tumor suppressor activity, consistent with the role of PKR as an intracellular transducer of extracel!ular signals. The inverse correlations between eIF-2tx phosphorylation and cell proliferation rate, along with a similar correlation between PKR activity and proliferation rate, have been combined into a model suggesting that the non-functional PKR mutants exert their effects on proliferation rate through inactivation of endogenous PKR leading to increased amounts of

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Fig 5. Co-immunoprecipitation of pK3 with PKR. PKR wa~ immunopurified from 293 cell extracts using monoclonal antibody to PKR covalently bound to CL--4B Sepharose, and incubated at 30°C for 5 min with in vin'o translated [35S] methionine-radiolabelled pK3 at 400 mM NaCI, as described [48], in the absence or presence of non-radiolabelled pK3r or eIF-2, as indicated. The resin was rinsed ext,ensively with buffer containing 400 mM NaCI. Material remaining bound to the resin was eluted with SDS-PAGE sample buffer and subjected to 17.5% SDS-PAGE using a Tris/Tricine buffer system [97] followed by fluorography.

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Fig 6. Synthesis of pK3 and pE3 during vaccinia virus infection. HeLa cells were infected with vaccinia virus at a multiplicity of infection of 30, and incubated in the presence or absence of cytosine arabinoside (40 pg/ml) for 0-18 h. 1 h prior to harvest the cells weie transferred to methionine-free medium containing 0.1% calf serum and 250 pCi/ml psS]methionine. Cells were lysed and incubated with either antibody to pK3 pre-bound to protein-G Sepharose (A), or to dsRNA agarose [99] (B). The resin was rinsed extensively with buffer cortaining 400 mM NaCI. Material remaining bound to the resin was eluted with SDS-PAGE sample buffer and subjected to 17.5% SDS-PAGE using a Tris/Tricine buffer system [97] and autoradiography. The first two lanes are of unfractionated extracts from cells mock or vaccinia virus iafected in the presence of cytosine arabinoside

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787 functional eIFo2. This in turn could lead to higher protein synthetic rates and unregulated cell proliferation. Such a model takes into account the necessity for protein synthesis and cell proliferation rate to be coordinated because of the synthesis of structural and regulatory proteins involved in growth. This model is consistent with the characteristics of the growth suppressive effects of wild-type PKR in yeast [61, 62]. It is also supported by the finding that IL-3 stimulates protein synthesis (and increased proliferation) by regulating PKR activity and eIF-2cx phosphorylation state [ 12]. An additional supporting correlation is the increased proliferation rate, along with decreased PKR activity and eIF-2ct phosphorylation state, of cells stably expressing the cellular inhibitor of PKR, p58 (see below) [78]. Although this evidence is compelling, there has been no unequivocal demonstration that inactivation of PKR by non-functional mutants exerts its effects on proliferation rate through changing the phosphorylation state of eIF-2ot. It remains a formal possibility that the effects of PKR on proliferation rate are mediated through other substrates and that the effects on translation are coincidental. There is also a body of indirect evidence suggesting that the activation of PKR may be involved in the regulation of dsRNA-activated genes, dsRNA treatment of osteosarcoma cells induces the expression of c-fos, c-myc and IFN-[3 which is blocked at the transcriptional level by 2-aminopurine, a known inhibitor of PKR activity [63]. Similarly, 2-aminopurine blocks the induction of IFN in both mouse L-cells and chick embryo cells [79]. Furthermore, dsRNA activates PKR and induces the transcription of mRNAs for the cellular adhesion molecules, VCAM, ICAM and Eselectin in human umbilical vein endothelial cells (Ofl~rman, Shaw, Medford, Jagus, submitted). These data have led to the suggestion that PKR may have an alternate substrate(s) that functions in the regulation of transcription. The induction of genes by dsRNA requires the assembly of a complex set of transcription factors on responsive DNA elements and functions through activation of the transcription factor, NF-gB [80-82]. It has recently been shown that PKR can phosphorylate IKB, an inhibitor of NF-rLB, and in doing so activates the NF-:r,B DNA binding activity in vitro [83]. The ability of activated PKR induce transcription of specific mRNAs raises the possibility that the effects of non-functional PKR mutants on proliferation rate could function through t ~ s mechanism. The mechanism(s) by which catalytically inactive variants of PKR inhibit the activity of the wildtype enzyme is not clear° but possible mechanisms that have been considered include: a) the sequestration of activator dsRNA; b) the formation of a hybrid dimerized PKR which is functionally inactive; or c) the binding of non,functional variants to eIF-2o~, protec-

44 31 18 12.8 pK3 MI VV MI W MI VV co-IP

Fig 7. Co-immunoprecipitation of pK3 and PKR from vaccinia virus infected cells. HeLa cells were mock-infected or infected with vaccinia virus at a multiplicity of infection of 30, and incubated in the presence of cytosine arabinoside (40 pg/nll) for 18 h. 1 h prior to harvest the cells were transferred to methionine-free medium containing 0.1% calf serum and 250 pCi/ml [35S]methionine. Cells were lysed and incubated with monoclonal antibody to PKR covalently bound to CL--4B Sepharose, in buffer containing 400 mM NaCI [48]. The specificity of binding was determined by the addition of l IJg of non-radiolabelled pK3r during the immunoprecipitation step, to the extracts shown in the last two lanes. The fi~st two lanes are of unfractionated extracts from cells mock- or vaccinia virus-infected in the presence of cytosine arabinoside (40 lig/ml) for 18 h. Material remaining bound to the resin after extensive rinsing was eluted with SDS-PAGE sample buffer and subjected to 17.5% SDS-PAGE using a Tris/Tricine buffer system [97] and fluorography. ting it from phosphorylation by the wild-type enzyme. Recent studies using recombinant DII variant [84] and recombinant wild-type and DII variants (Barber, Katze, Jagus, submitted) in in vitro reactions have eliminated a role for non-functional variants in 'sparing' eIF-2a. However, non-functional PKR variants seem able to function in vitro both by binding dsRNA [84] (Barber, Katze, Jagus, submitted) and by direct interaction with the wild-type enzyme [85] (Barber, Katze, Jagus, submitted). Difficulties arise in the interpretation of these data due to the present uncertainty regarding the mechanism of PKR activation; whether

788 autophosphorylation is intramolecular or intermolecular, and whether the functional form of the kinase is a monomer or a dimer (see [10]).

Cellular proteins that interact with PKR In addition to the above data, the increasing number of cellular proteins that interact with and regulate PKR are also indicative of a complex regulatory role for PKR in the cell 1241. The best characerized of these is p58, a cellular PKR inhibitor of molecular mass 58 000, activated by influenza virus infection [24, 31-331. Recombinant p58 inhibits both PKR autophosphorylation and elF-2ct phosphorylation in vin'o by an undescribed mechanism that seems to involve a direct interaction with PKR [321. It is highly conserved across species [331. Furthermore, both p58 expression and activity appear to be highly regulated [331. The activity of p58 is regulated by a potent cellular inhibitor of p58 activity, termed the anti-inhibitor (I-p58), that can be separated from p58 by ammonium sulfate fractionation in extracts from uninfected cells [241. Except for influenza virus infection, the mechanisms that regulate p58 and I-p58 activities are not understood. The gene encoding p58 has been cloned and sequenced and found to be a member of the tetratricopeptide repeat (TPR) family of proteins [24, 33, 78]. TPR proteins, which contain several internal 34 amino acid repeats, are thought to play a role in protein-protein interactions [861. Other members of the family including CDC 16, CDC23, and Bim A, are implicated in the regulation of mitosis and the cell cycle 187-89], although only p58 has been ascribed a specific biochemical function [24, 33, 781. p58 atso contains regions of homology to the DnaJ family of proteins and to a lesser extent to the N-terminal of elF-2ct [331. Mutagenesis studies, coupled with in vim, assays of recombinant p58 variants have shown that the PKR inhibitory domain of the molecule contains some of the TPR motifs, the region of homology to eIF-2~, but not the DnaJ motif [33]. Like cells stably expressing non-functional PKR variants, cell liaes overexpressing o58 exhibit a transformed phe'aotype, proliferating at higher rates and to higher saturation densities, compared with vector only transformed cells, and producing tumors in nude mice [78]. Furthermore, PKR activity and eIF-2ct phosphoryiation are decreased in p58 expressing cell lines compared to control cells [781. In addition to p58, there are reports of several cellular proteiq~ that down-regulate PKR activity (reviewed in [241). In rapidly growing 3T3-F422 preadipocyte fibroblasts, another cellular inhibitor of PKR has been purified as a 15 kDa protein that prevents the interaction of dsRNA with PKR [901. This

inhibitor, termed dRE is not a dsRNA binding protein, but rather serves to prevent ATP binding to PKR, thus inhibiting both dsRNA binding and autophosphorylation [911. Another inhibitor of dsRNA responsiveness has been isolated from human FL amnion cells [92]. This inhibitor is not well characterized, but can be isolated as a 160 kDa complex, the activity of which is decreased by interferon treatment or Sindbis virus infection [93]. Another relatively uncharacterized PKR inhibitor is found in Balbc-3T3 cells after the introduction of an activated ras gene (Ki-v-ras) ([661 see this volume). The relationship of the above proteins to each other and p58/I-p58 is not known, but the picture emerges of PKR fulfilling a complex regulatory role in cell function with the regulation of its activity as part of a complex cascade interfacing with the signal transduction/cell cycle control machinery.

Coordination of signal transduction events and activation/inactivation of PKR: involvement of a phosphotyrosine-containing protein The first direct evidence of a growth factor mediating its effects on protein synthesis through PKR comes from a study of mouse IL-3-dependent NSF/NI.H7 cells [12]. In these cells, IL-3 deprivation induces the activation of PKR as measured by autophosphorylation and in vitro PKR activity, as well as increased phosphorylation of elF-2o~ [ 12]. This is accompanied by the rapid inhibition of protein synthesis. Re-addition of IL-3 elicits a rapid decrease in PKR and elF-2tx phosphorylation, followed by the recovery of protein synthesis [121. The molecular mechanisms by which growth factors such as IL-3 might couple to and down-regulate PKR activity are not known. An important question that arises about the effects of IL-3 is whether PKR responds to the accumulation of cellular dsRNA, perhaps present as secondary structure within cellular mRNAs, or whether PKR responds to regulatory molecules other than dsRNA. Prior to the effects on PKR and elF-2tx phosphorylation, the re-addition of IL-3 results in a rapid but transient association of PKR with a 97 kDa phosphotyrosine and phosphoserine containing protein [ 12]. If phosphorylation of the 97 KDa protein is blocked with the tyrosine kinase inhibitor, genistein, neither the IL-3 triggered reduction in PKR autophosphorylation, nor the increase in protein synthetic activity occurs [ 12]. A phosphoprotein of similar size to p97 can be seen to co-immunoprecipitate with PKR and antibodies to PKR in extracts from 3T3-preadipocytes [90], suggesting that this protein may be involved in regulation of PKR activity in many cell types. Furthermore, a 90 kDa phosphoprotein can be co-immunoprecipitated with PKR and antibodies to PKR from rabbit reticulo-

789 cyte lysate [94]. This is a ribosome associated protein that binds dsRNA and serves as a substrate for PKR. Although it is not yet known if this reticulocyte 90 kDa protein represents the same protein as the mouse p97, it raises the possibility of the existence of a PKR regulatory protein responsive to more than one kinase activity. Thus the studies with IL-3 are beginning to uncover pieces of the cascade interfacing PKR function with the signal transduction/cell cycle control machinery.

Problems in determining PKR activation state The emerging work PKR activity and growth control has focused attention on the current methodologies for assessing PKR activation state. One of the customary methods has been to look at the degree of in vivo phosphorylation of PKR by incubating cells with [32p] orthophosphate and looking at incorporation into immunopurified PKR [30]. This method is not amenable to a quantitative assessment of PKR activation/ phosphorylation state. Such studies are often combined with an assessment of PKR activity by its ability to phosphorylate purified eIF-2tz in in vitro reactions [78]. This is also impossible to quantitate and although adequate for the large changes in PKR activity observed after virus infection, it is not so useful for the smaller changes observed over a range of proliferation rates [78]. In addition, the conditions used for immunoprecipitation of PKR can give rise to differing conclusions since different conditions can lead to removal, or not, of PKR regulatory proteins. A few investigators have reported that phosphorylated form(s) of PKR can be separated from the non-phosphorylated form on low percentage polyacrylamide gels, using high bisacrylamide:acrylamide ratios [7, 12, 85, 95]. Coupled with immunoblotting, this can give a sensitive and potentially quantitative assessment of PKR phosphorylation/activation state. However, such a method is dependent on knowing the number of authentic phosphorylation sites involved in activation. Although the catalytic and regulatory domains of PKR have been defined, little information is currently available on the phosphorylation sites.

Concluding remarks The varied roles played by PKR in the regulation of cellular function are only just becoming apparent. Similarly, the methods by which PKR activity is controlled are still incompletely understood. From the study of growth factor action, the effects of non-functional PKR mutants and the varied strategies of viruses to down-regulate PKR, the identities of protein components of a PKR regulatory cascade are begin-

ning to emerge. It remains to elucidate the relationships of these proteins to each other, and to fit them into the regulatory cascades controlling cell proliferation and gene expression.

Acknowledgments The work presented from the authors' laboratory was supported by NSF grant MCB 91-05451 to RJ with an REU supplement for MMG. We are grateful to Drs Glen Barber, Taka Ito, Michael Katze, Stratford May, Harry Mellor, and Margaret K Offerman, for helpful discussions and/or for providing manuscripts prior to publication. We thank Dr Ara Hovanessian for PKR monoclonal antibody, and Dr Bernard Moss for the vaccinia virus E3L gene.

References 1 Clemens M, Hershey JWB, Hovanessian AG, Jacobs BL, Katze MG, Kanfman R, Lengyei P, Samuel CE, Sen G, Williams BRG (1993) PKR: proposed nomenclature for RNA-dependent protein kinase induced by interferon. J Interferon Res 13, 241 2 Meurs E, Chong K, Galabru J, Thoma NM, Kerr IM, Williams BRG, Hovanessian AG (1990) Molecular cloning and characterization of dsRNA-PK induced by interferon. Cell 62, 379-390 3 Hinnebusch A, Wek RC, Dever 1", Cigan M, Feng L, Donohue T (1993) Regulation of GCN4 expression in yeast. Translational regulation of gene expression (Ilan J, ed) Plenum Plublishing, New York, 87-115 4 Samuel CE (1993) The elF-2cx protein kinases, regulation of translation from yeasts to eukaryoles. J Biol Chem 268, 7603-7606 5 Akkaraju GR, Whitaker-Dowling P, Youngner JS. Jagus R (1989) Vaccinia virus SKIF prevents translational inhibition by dsRNA in reticulocyte lysate. J Bioi Chem 264, 10321-10325 6 Galabru J, Hovanessian AG (1985) Two interferon-induced proteins are involved in PK complex dependent on dsRNA. Cell 43,685-694 7 Galabru J, Hovanessian AG (1987) Autophosphorylation of dsRNA-dependent protein kinase. J Biol Chem 262, ! 5538-15544 8 Kostura M, Mathews MB (1989) Purification and activation of dsRNA-dependent eIF-2a-specific protein kinase. J Mol Biol 9, 1576-1586 9 Hovanessian AG (1989) dsRNA-PK induced by interferon. J Interferon Res 9, 641--647 10 Mathews MB (1993) Viral evasion of cellular defense mechanisms: regulation of PKR by RNA effectors. Sen: Virol 4, 247- 257 II Galabru J, Katze MG, Robert N, Hovanessian AG 0989) The binding of dsRNA and adenovirus VA I RNA to PKR. Eur J Biochem 178.581-589 12 lto T, Jagus R, May S (1994) IL-3 stimulates protein synthesis by regulating dsRNA-dependent protein kinase (PKR). Proe Natl Acad Sci USA 91, 7455-7459 13 Petryshyn R, Chen JJo London IM (1984) Growth-reared expression of a dsRNA-dependent protein kinase in 31"3 cells. J Bioi Chem 259, 14736--14742 14 Petryshyn R, Chen JJ, London IM (1988) Detection of activated dsRNAdependent protein kinase in 3T3-F4.42A cells. Proc Natl Acad Sci USA 85, 1427-1431 15 Whitaker-Dowling P, Youngner JS (1983) Vacinia virus rescue of VSV from interferon induced resistance. Virology 131,128-139 16 Whitaker-Dowling P, Youngner JS (i 984) Characteristics of a specific kinase inhibitory factor produced by vaccinia virus that inhibits interferon induced protein kinase. Virology 37, 171-181 ! 7 Rice A, Duncan R, Hershey JWB, Kerr IM (1985) dsRNA-dependent protein kinase activated in virus infected HeLa cells. J Viro! 54. 894-898 18 Gupta SL, Holmes 5L, Mehra L (1982) Interferon action against reovirus: activation of PKR ,_'nmouse L929 cells. Virology 120. 495-499 19 Nilsen TW, Moroney PG. Baglioni C (1982) Inhibition of protein synthesis in reovirus infected HeLa cells with elevated PKR levels. J Biol Chem 259, 14593- i 4596

790 20 Pani A. Julian M. Lucas-Lennard J (1986) A kinase able to phosphorylate elF-2ot is present in exracts of Mengovirus infected L-cells. J Viro160. 10121017 21 DeStefano J. Olmsted E, Panniers R, Lucas-Lenard J (1990) The ot subunit of elF-2 is phosphorylated in Mengovirus infected L cells. J Virol 641 44454453 22 Black TL. Safer B, Hovanessian AG. Katze MG (1989) Cellular PKR is highly phosphorylated and activated yet significantly degraded during poliovirus infection. J Viro163. 2244-225 i 23 O'Niell RE. Racaniello VR (1989) Inhibition of translation in cells infected with polio virus 2Apro mutant correlates with phosphorylation of elF-2ct. J Viro163. 5069-5075 24 Lee TG. Katze MG (1993) Cellular inhibitors of PKR. Progr Mol Subcel Biol 14, 48-65 25 Sonenberg N (1990) Measures and countermeasures in the modulation of initiation factor activities by viruses. New Biol 2. 4022-4029 26 Katze MG (1992) The war against PKR: can viruses win? J Interferon ICes 12.241-248 27 Katze MG (1993) Games viruses play: a strategic initiative against PKR. Semin Viroi 4. 259-268 28 Meurs E. Wahanabe Y. Kadereit S. Barber GN. Katze MG, Chong K, Williams BRG (1992) Constitutive expression of human PKR in murine cells mediates phospborylation of elF-2ct and partial resistance to EMC virus growth. J Viro166. 5805-5814 29 Mathews MB. Shenk T (1991) Adenovirus associated RNA and translation. J Viro165.5657-5662 30 Katze MG. Tomita J, Black T. Safer B. Hovanessian AG (1988) Influenza virus regulates protein synthesis by repressing the autophosphorylation of PKR. J Viro162. 3710-3717 31 Lee TG. Tomita J. Hovanessian AG, Katze MG (1990) Purification and partial characterization of an inhibitor of PKR during influenza virus infection. Proc Nail Acad Sci USA 8~. 6208-6212 32 Lee TG. Tomita J, Hovanessian AG. Katze MG (1992) Characterization and regulation of the 58 kDa cellular inhibitor of PKR. J Biol Chem 2~7. 14238-14243 33 Lee TG. Tang N. Thompson S. Miller J. Katze MG (1994) The 58-kDa cellular inhibitor of PKR is a member of the TPR family of proteins. Mol Cel Bioi 14. 2331-2342 34 Roy S, Katze MG. Parkin NT. Edery I. Hovanessian AG. Sonenberg N (1990) Control of the interferon induced p68 protein kinas¢ by HIV-I tat gene. Science 247. 1216.- 1219 35 Black TL. Barber GN, Katze MG (1993) Degradation of PKR by poliovirus requires RNA and protein. J Viro167. 791-800 36 lmani F. Jacobs BL (1988) Inhibitory activity for the interferon-induced protein kinase is associated with reovirus serotype I 03 protein. Proc Natl Acad Sci USA 85.7887-7891 37 Huismans H, Joklik WK (1976) Reovirus-coded polypcptides in infected cells: isolation of two monomerie polypeptides with affinity for singlestranded and double-stranded RNA. Virology 70. 411-424 38 Schiff LA, Nibert ML, Co MS, Brown EG. Fields BN (1988) Distinct binding sites for zinc and dsRNA in the reovirus outer capsid protein 03. Mol Cei Biol 8, 273-283 39 Giantini M, Shatkin AJ (1989) Stimulation of CAT mRNA translation by reovirus capsid polypeptide t73 in cotransfected COS cells. J Virol 63, 2415242 ! 40 Lloyd RM. Shatkin AJ (1992) Translational stimulation by reovirus polypeptide t~3: substitution for VA RNA and inhibition of phosphorTlation of elF-2~. J Viro166, 6878--6884 41 Davies MV, Chang H-W. Jacobs BL, Kaufman RJ (1993) The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of PKR by different mechanisms. J Viro167, 1688-1692 42 Goebel SJ, Johnson GP. Parkus ME, Davis SW, Winslow JP, Paoletti E (1990) The complete sequence of vaccinia virus. Virology 179, 247-266 43 Beattie E, Tattaglia J, Paoletti E (1991) Vaccinia virus eIF-2ct homologue abrogates the antiviral effects of interferon. Virology 183,419-422 44 Chang HW, Watson JL, Jacobs BL (1992) The E3L gene of vaccinia virus encodes an inhibitor of PKR. Proc Nail Acad Sci USA 89, 4825-4829 ,;5 Chang H-W, Jacobs BL (1993) Identification of a conserved motif that is necessary for binding of the vaccinia virus E3L gene product to dsRNA. Virology 194, 537-547

46 St Jol'mson D. Brown N. Gall JG. Jantsch M (1992) A conserved dsRNA binding domain. Proc Nail Acad Sci USA 9, 10979-10983 47 Beattie-McGrath E. Tartaglia J. Chang H-W, Jacobs BL. Paoletti E 0992) Assessment of E3L and K3L deletion mutants to grow in interferon treated cells. Pox Virus Workshop, Abstract # 155 48 Carroll K. Elroy-Stein O. Moss B, Jagus R (1993) Recombinant vaccinia vires K3L gene product prevents activation of PKR. J Biol Chem 268. 1283712842 49 Davies MV, Elroy-Stein O. Jagus R, Moss B. Kaufman RI (1992) Vaccinia virus K3L gene product potentiates translation by inhibiting PKR. J Viro166, 1943-1950 50 Katze MG. Wambach M, Wong ML, Garfinkel M. Meurs E. Williams B, Hovanessian AG. Barber GN (1991) Functional expression and RNA binding analysis of dsRNA dependent PKR in a ceil-free system. Mol Cel Bioi ! !. 5497-5505 51 Meilor H, Proud CG (1990) A synthetic peptide substrate for elF-2a-specific protein kinases. Biochem Biophys Res Conmmn 178, 430-437 52 Baringa M (1992) Viruses launch their own "Star Wars'. Science 258. 17301731 53 Smith GL, Chan YS (199 I) Two vaccinia virus proteins structurally related to the IL-I receptor and the immunoglobulin superfamily. J Gen Viro172, 511518 54 Ray CA, Black TA. Kronheim SR, Greenstreet TA. Sleath PR, Salveson GS. Pickup DJ (1992) Viral inhibition of inflammation: cow-pox virus encodes an inhibitor of the IL- I ~ converting enzyme. Cell 69. 597--604 55 Hershey JWB (1993) Introduction to translational initiation factors and their regulation by phosphorylation. Semin Virol 4. 201-208 56 Rhoads RE (1993) Regulation of eukaryotic protein synthesis by initiation factors. J Biol Chem 268, 3017-3020 57 Duncan R. Hershey JWB (1985) Regulation of initiation factors during translational repression caused by serum deprivation. J Biol Chem 260. 5493-5497 58 Rowlands AG, Montine KS. Henshaw EC, Panniers R (1988) Physiological stresses inhibit elF-2B in Ehrlich cells. Fur J Biochem ! 75, 93-99 59 Montine KS, Henshaw EC (1989) Serum growth thctors cause rapid stimulation of protein synthesis and dephosphorylation of eiF-2ct in serum deprived Ehrlich cells. Biochim Biophys Acta 1014. 282-288 60 Chert J J. Throop MS. Gehrke L, Kuo l. Pal JY, Brodsky M, London IM (1991) Cloning of cDNA of eiF-2~-PK of rabbit reticulocytes: homology to yeast GCN2 and PKR. Pro¢ Nail Acad Sci USA 88. 7729-7733 61 Chong KL, Feng L, Schappert K, Meurs E. Donohue TF. Friesen JD. Hovahessian AG. Williams BRG (1992) Human p68 kinase (PKR) exhibits growth suppression in yeast and homology to the translational regulator, GCN2. E M B O J I I, 1553-1562 62 Dever T. Chen JJ. Barber GN. Cigan AM. Feng L, Donohue TF, London IM. Kat:,e MG. Hinnebusch AG (1993) Mammalian elF-2cx-PKs functionally substitute for GCN2 and stimulate GCN4 translation in yeast. Proc Nail Acad Sci USA 9(I, 4616--4620 63 Zinn K, Keller A, Whitemore LA. Maniatis T (1988) 2-Aminopurine selectively inhibits the induction of ~.bm, rferon, c-fi~s, and c-myc. Science 240, 210-213 64 Tiwari RK, Kusari J, Kumar R. Sen G (1988) Gene induction by interferons and dsRNA: selective inhibition by 2-aminopurine. Mol Cei Biol 8. 42894294 65 Hall DJ. Jones SD. Kaplan DR, Whitman M, Rollins BJ. Stiles CD (1989) Evidence for a novel signal transduction pathway activated by PDGF and dsRNA. Mol (?el Biol 9. ! 705-1713 66 Mundschau LJ, Failer DV (1992) Oncogenic ras induces inhibition of PKR. J Biol Chem 267. 23092-23098 67 Zullo JN, Cochran BH, Huang AS, Stiles C (1985) PDGF and dsRNA stimulate expression of the same genes in 3T3 cells. Cell 43,793-800 68 Zullo JN. Failer DV (1988) p21 v-ras inhibits induction of c-myc and c-los expression by PDGE Mol Cei Biol 8, 5080-5085 69 Clemens M! (1992) Suppression with a difference. Nature 360, 210--21 i 70 Hovanessian AG (1993) dsRNA-activated protein kinase (PKR): antiproliferative antiviral and antitumoral functions. Semin Virol 4, 237-245 71 Lengyel P (1993) Tumor suppressor genes: News about the interferon connection. Proc Nad Acad Sci USA 90, 5893-5895 72 Koromilas AE, Roy S. Barber GN, Katze MG, Sonenberg N (1992) Malignant transformation by a mutant of the INF-induced PKR. Science 257, 1685-1689

791 73 Meur~ E. Galabru J, Barber GN, Katze MG. Hovanessian AG (1993) Interferon-induced dsRNA activated protein kinase: antiviral and antitumoral functions. Proc Natl Acad Sci USA 90, 232-236 74 Barber GN. Edelhoff S, Katze MG, Distecher CM (1993) Chromosomal assignment of the interferon induced PKR to human chromosome 2p21 and mouse chromosome ! 7E2. Genomics ! 6. 765-767 75 Hanash SM, Beretta L, Barcroft CL. Sheldon S. Glarer TW, Ungar D, Sonenberg N (19931 Mapping of the gene for PKR to chromosome region 2P21-22: a site of rearrangements in myeloproliferative disorders, Genes Chromosomes Cancer 8. 34-37 76 Haines GK. Ghadge GD. Becket S, Kies M, Pelzer H. Thimmapaya B, Radosevich JA (1993) Correlation of the expression of PKR (1368) with differentialion in head and neck squamous cell carcinoma. Virchows Archiv B Cell Patho163. 289-295 77 Haines GK, Beeker S, Ghadge G, Kies M~ Pelzer H. Rados~vi~;h 3A (1993) Expression of PKR(p68) in squamous cell carcinoma of the head and neck region. Arch Otolaryngoi Head Neck Surg 119, i 142-1147 78 Barber GN, Thompson S, Lee TG. Strom T, Jagus R, Darveau A. Katze MG (1994) The 58-kDa inhibitor of PKR is a TPR protein with oncogenic properties. Proc Natl Acad Sci USA 91,4278-4282 79 Marcus Pl. Sekellick MJ (19881 2-aminopurine blocks selective and reversibly an early stage in INF induction. J Gen Viral 69, 1637-1645 80 Xanthoudakis S. Cohen L. Hiscott J (1989) Multiple protein-DNA interactions within human INF-I~ regulatory elements. J Biol Chem 264, ! i 39ii45 81 Visvanathan KV, Goodboum S (19891 dsRNA activates binding of NF-~:B to an inducible element in the human ~-interferon promoter. EMBO J 8, 11291138 82 Lenardo MI, Fan CM, Maniatis T, Baltimore D 09891 The involvement of NF-~:B in p-interferon gene regulation reveals its role as a widely inducible mediator of signal transduction. Cell 57, 287-294 83 McMillan NAJ, Kumar A, Hovanessian AG, Galabm J, Williams BRG (19931 Targets for regulation by PKR. In: Translational Control and Cancer St George's Hospital, London 84 Sharp TV, Xiao Q, Jeffrey I, Gewert DR, Clemens MJ (19931 Reversal of dsRNA-induced inhibition of protein synthesis by a catalytically inactive mutant of PKR. Eur J Biochem 214, 945-948

85 Thomis DC, Samuel CE (19931 Mechanism of interferon action: PKR: evidence for intermolecular autophosphorylation and autoactivation. J ViroI 67, 7695-7700 86 Goebl M, Yanagida M (19911 The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci 16, 173-177 87 Sikorski RS, Boguski MS, Goebl M, Hieter P (19901 A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell 60, 307-317 88 Hirano T, Kinoshita N, Morikawa M, Yanagida M (19901 Snap helix with knob and hole: essential repeats in S pombe nuclear protein nuc2 ÷. Cell 60, 319-328 89 Sikorski RS, Michaud WA, Wooton JC, Boguski MS, Connelly C, Hieter P (19901 TPR proteins as essential component~ of the yeast cell cycle. Cold Spring Harbor Syrup Quant Bioi 56, 663-673 90 Judwa~ R, Petryshyn R (19911 Partial characterization of a cellular factor that regulates dsRNA-dependent elF-2ct kinase in 3T3-F442A fibroblasts. Mol (?el Biol ! !, 3259-3267 9 ! Judware R, Petryshyn R (1992) Mechanism of action of a cellular inhibitor of the dsRNA--dependent protein kinase from 3T3-F442A cells. J Biol Chem 267, 21685-21690 92 Saito S, Kawakita M (19911 Inhibitor of interferon-induced dsRNA-dependent protein kinase. Microbial Immuno135, ! 105-1114 93 Saito S (1990) Enhancement of the interferon-induced dsRNA-dependent protein kinase by Sindbis virus infection and heat-shock stress. Microbial lmmuno134, 859--870 94 Rice AP, Kostura M. Mathews MB (1989) Identification of a 90-kDa polypeptide that associates with adenovirus VA RNA and is phosphorylated by PKR. J Biol Chem 264, 20632-20637 95 Laskey SR, Jacobs BL, Samuel CE (1982) Mechanism of interferon action: characterization of sites of phosphorylation in the interferon-induced phosphoprotein Pi from mouse fibroblasts. J Biol Chem 257, ! 1087-11093 96 Laureat AG, Krust B, Galabru J, Svab J, Hovanessian AG (1985) Monoclonal antibodies to PKR and their use for detection of PKR in human cells. P w c Nati Acad Sci USA 82, 4341-4345 97 Shagger H, van Jagow G (1987) Tdcine-SDS-PAGE for the separation of proteins in the 1-i 00 kDa range, Anal Biochem 168, 368-379 98 Yuwen H, Cox JH, Yewdell JW, Bennick JR. Moss B (1993) Nuclear localization of dsRNA-binding protein encoded by the vaccinia virus E3L gene. Virology 195, 732-744