Characterization of the Heparin-Mediated Activation of PKR, the Interferon-Inducible RNA-Dependent Protein Kinase

VIROLOGY

221, 180–188 (1996) 0364

ARTICLE NO.

Characterization of the Heparin-Mediated Activation of PKR, the Interferon-Inducible RNA-Dependent Protein Kinase CYRIL X. GEORGE,* DANIEL C. THOMIS,‡,1 STEPHEN J. MCCORMACK,*,2 CARL M. SVAHN,† and CHARLES E. SAMUEL*,‡,3 *Department of Molecular, Cellular and Developmental Biology, and ‡Interdepartmental Graduate Program of Biochemistry and Molecular Biology, University of California, Santa Barbara, California 93106; and †Pharmacia AB, Therapeutics Stockholm, S-112 87 Stockholm, Sweden Received March 11, 1996; accepted April 30, 1996 Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent eIF-2a protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirus VA RNA, was competed by poly(rI):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, eIF-2a phosphorylation was catalyzed by both heparinand dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules. q 1996 Academic Press, Inc.

INTRODUCTION

the best characterized of the PKR substrates, both in structural and functional terms. PKR catalyzes the phosphorylation of eIF-2a at serine residue 51 (Lasky et al., 1982; Pathak et al., 1988). Phosphorylation of eIF-2 leads to an inhibition of translation (Hershey, 1989; Samuel, 1993; Clemens, 1996). A variety of physiologic conditions, including IFN treatment and virus infection (Samuel, 1991; Schneider and Shenk, 1987), cause the phosphorylation state of eIF-2a to increase and mRNA translation to subsequently decrease. Several viruses encode RNAs that are bound by PKR and either activate or antagonize the autophosphorylation of PKR (Mathews, 1993; Samuel, 1993). The RNAbinding domain of PKR has been localized by mutational analysis to the N-terminal region of the kinase (Feng et al., 1992; Green and Mathews, 1992; Katze et al., 1991; McCormack et al., 1992). Both activator and inhibitor RNAs bind to the same PKR domain (McCormack et al., 1992; McCormack and Samuel, 1995). The RNA-binding domain of PKR includes a 20 amino acid core motif, designated R, that is conserved and often repeated in dsRNA-binding proteins from both prokaryotic and eukaryotic cells and their viruses (McCormack et al., 1992; St. Johnston et al., 1992). Curiously, in addition to certain natural and synthetic RNAs with double-stranded character, heparin has also been reported to activate PKR auto-

Among the enzymes induced by interferon (IFN)4 is the RNA-dependent protein kinase (PKR) (Samuel, 1991). PKR is a central component of the IFN-induced antiviral response (Lee and Esteban, 1993; Mathews, 1993; Meurs et al., 1992; Samuel, 1991). The PKR protein acquires protein serine/threonine kinase activity following autophosphorylation, a process mediated by RNA with double-stranded character (Clemens, 1996; Samuel, 1993). Following activation, PKR catalyzes the intermolecular phosphorylation of at least three protein substrates: the alpha subunit of protein synthesis initiation factor 2 (Samuel, 1979); the PKR protein itself (Thomis and Samuel, 1993); and the transcription factor inhibitor I-kappa B (Kumar et al., 1994). Protein synthesis factor eIF-2 is so far

1 Present address: Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138. 2 Present address: Lombardi Cancer Center, Georgetown University School of Medicine, Washington, DC 20007. 3 To whom correspondence and reprint requests should be addressed at Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, CA 93106. Fax: (805) 8934724. 4 Abbreviations used: IFN, interferon; PKR, IFN-inducible RNA-dependent protein kinase; dsRNA, double-stranded RNA; Hep, heparin.

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Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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phosphorylation (Hovanessian and Galabru, 1987; Patel et al., 1994). Heparin is a heterogeneously sized glycosaminoglycan, consisting of repeating disaccharide subunits of hexuronic acid and D-glucosamine with a high degree of sulfation (Casu, 1985; Jackson et al., 1991). Heparin is bound by a variety of different proteins and subsequently affects an array of physiologic processes. Among them are growth factor-induced cell growth and differentiation modulated by heparin-binding polypeptide growth factors (Ruoslahti and Yamaguchi, 1991) and thrombin-regulated hemostasis modulated by heparin-binding antithrombin III (Hirsch, 1991). Although it has been described that PKR binds to heparin-Sepharose and that commercially obtained heparin of heterogeneous sulfation and size activates PKR autophosphorylation (Hovanessian and Galabru, 1987; Patel et al., 1994), little additional information is available concerning the effect of heparin on PKR activity. As an extension of our studies on the structure and function of the PKR kinase, we examined small heparin oligosaccharides of defined lengths for their ability to activate PKR autophosphorylation and subsequent eIF2a kinase activity. Comparison of results obtained for purified human PKR and heparin with those obtained for PKR and dsRNA suggest unique structural and functional bases of PKR interaction with these two classes of polyanionic biomolecules. MATERIALS AND METHODS Materials Heparin oligosaccharides of defined sizes and high sulfate content were prepared by nitrous acid depolymerization of heparin and fractionation as described by Sudhalter et al. (1989); size homogeneity of the heparin oligosaccharides was determined by high-performance gel permeation chromatography. Sulfur content was determined by elemental analysis and was 11.7% for the hexasaccharide, 12.6% for the octasaccharide, and 12.6% for the hexadecasaccharide. Unfractionated heparin of 18to 25-residue average chain length was from Sigma. Poly(rI):poly(rC) of 200-residue average chain length was generously provided by the Antiviral Substances Program, National Institute of Allergy and Infectious Diseases, NIH. Purified eIF-2 was generously provided by W. C. Merrick, Case Western Reserve University (Cleveland, OH). Other materials were as previously described (Thomis and Samuel, 1993; McCormack et al., 1994).

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al., 1985), except that the procedure was modified to include a MonoQ ion-exchange fast liquid chromatography fractionation step. For production of the HisPKR(K296R) protein, the cDNA encoding the full-length PKR(K296R) was subcloned into pBlueBacHisC (InVitroGen) and a recombinant baculovirus was produced (Thomis and Samuel, 1993). The His-PKR(K296R) protein was purified 45 hr after infection of Sf21 cells. The washed cell pellet was resuspended in lysis buffer (20 mM HEPES, pH 7.5; 0.5 M KCl; 0.65% Nonidet P-40; 1 mM PMSF, 10 mg/ml aprotinin; 2 mg/ml leupeptin), and a 100,000 g supernatant fraction (S100) was prepared. His-PKR(K296R) was purified from the S100 by Ni2/-chelating Sepharose column chromatography (fast flow metal-chelating Sepharose, Pharmacia). The 100 mM imidazole eluate containing His-PKR(K296R) was dialyzed against MonoS buffer (20 mM HEPES, pH 7.5; 0.1 mM EDTA; 100 mM KCl; 0.01% Triton X-100; 10% glycerol; 5 mM b-mercaptoethanol; 0.5 mM PMSF) and then further fractionated by MonoS FPLC ion exchange chromatography, using a linear salt gradient from 100 to 500 mM KCl in MonoS buffer. MonoS fractions were dialyzed against MonoQ buffer (20 mM HEPES, pH 7.5; 50 mM KCl; 2.5 mM MgCl2 ; 2.5 mM MnCl2 ; 0.01% Triton X-100; 10% glycerol; 5 mM b-mercaptoethanol; 0.5 mM PMSF). PKR protein kinase assay [g-32P]ATP-mediated PKR autophosphorylation catalyzed by PKR(Wt) was carried out in the absence of added activator or in the presence of double-stranded RNA or heparin, or both, as indicated. For measurement of the eIF-2a kinase activity of PKR, 0.25 mg of purified eIF-2 was added to the standard reaction mixture. For measurement of the intermolecular phosphorylation of HisPKR(K296R), the purified His-PKR(K296R) substrate was present at a twofold-molar excess over PKR(Wt). The 32Plabeled polypeptide products were analyzed by SDS– polyacrylamide gel electrophoresis and autoradiography. Quantitation was by scanning autoradiograms that included internal dilution standards, using an LKB Ultroscan XL laser densitometer. The procedures have been described in detail (Samuel et al., 1986). Western immunoblot analysis Western immunoblots were prepared as previously described using antiserum prepared against recombinant human PKR expressed in Escherichia coli (Thomis et al., 1992). Gel mobility-shift assay

Purification of PKR(Wt) and His-PKR(K296R) The wild-type PKR enzyme was purified from ribosomal salt-wash fractions prepared from IFN-treated human amnion U cells as previously described for the phosphorylation of PKR(Wt) from mouse fibroblast cells (Berry et

The gel mobility-shift analysis was carried out following the procedure of Gatignol et al. (1993) with the following slight modifications (McCormack and Samuel, 1995). The final reaction mixture (10 ml) contained 15 mM Tris– HCl, pH 7.8, 70 mM NaCl, 10 mM KCl, 1 mM EDTA, 6%

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(v/v) glycerol, 0.01% (v/v) Triton X-100, 25 ng ovalbumin, 2 units of RNasin (Promega), 10 ng yeast RNA (5*–3* oligonucleotides), recombinant PKR (5 ng protein), and 32 P-labeled adenovirus VAI probe (2.3 nM) as indicated. Incubation was for 20 min at room temperature before loading onto a 10% native acrylamide gel (80:1 acrylamide:bisacrylamide) made in 0.5X Tris-borate-EDTA buffer. Gels were prerun at 47 for ú1 hr at 200 V before addition of the sample; electrophoresis was then at 280 V for 5 hr or until the xylene cyanol was approximately 3 cm from the bottom of the gel. The gels were then dried and subjected to autoradiography. In competition experiments the competitor, either poly(rI):poly(rC) or heparin-16, was added at the same time as the 32P-labeled adenovirus VAI probe. RESULTS Minimum-size requirement of heparin for PKR activation PKR(Wt) purified from IFN-treated cells is dependent upon dsRNA for activation of autophosphorylation, a wellestablished observation (Clemens, 1996; Samuel, 1993). For the PKR(Wt) used in this study, which was purified from human U cells, readily detectable autophosphorylation was obtained with 0.01 mg/ml poly(rI):poly(rC) and saturated by 0.1 mg/ml (data not shown, Fig. 4). Confirming the original finding of Hovanessian and Galabru (1987), commercially available heparin likewise activated PKR autophosphorylation, displaying a relatively sharp

FIG. 1. Activation of PKR autophosphorylation by heparin and doublestranded RNA. [g-32P]ATP-mediated autophosphorylation of purified PKR was measured as described under Materials and Methods. The standard reaction mixture contained homogeneously sized heparin oligosaccharide at the indicated concentration (mg/ml) as follows: heparin hexasaccharide (Hep-6, lanes b-g); octasaccharide (Hep-8, lanes h-m); hexadecasaccharide (Hep-16, lanes n-r); or, no heparin added (lane a). The autoradiogram is of the complete gel area.

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FIG. 2. Activation of PKR(Wt) kinase by either heparin or doublestranded RNA leads to eIF-2a phosphorylation. [g-32P]ATP-mediated autophosphorylation of the a subunit of protein synthesis initiation factor eIF-2 catalyzed by purified PKR was measured as described under Materials and Methods. The standard reaction mixture contained dsRNA (poly(rI):poly(rC), 0.5 mg/ml), heparin (Hep-16, 1.0 mg/ml), and eIF-2 (0.25 mg) as indicated. The autoradiogram is of the complete gel area. PKR, position of the autophosphorylated protein PKR; a, position of the alpha subunit of eIF-2.

concentration dependence between 0.1 and 1.0 mg/ml (data not shown). Commercially available heparin is a heterogeneously sized and sulfated polymer, typically ranging from Mr 5,000 to 30,000 (Casu, 1985). To define the minimal structural requirements of heparin required to activate PKR, heparin oligosaccharides of defined sizes and high sulfate content were prepared by nitrous acid depolymerization of commercially obtained heparin. These small heparin oligosaccharides were tested for their ability to activate purified PKR autophosphorylation (Fig. 1). Heparin hexasaccharide (Hep-6) was a poor PKR activator (Fig. 1, lanes b–g), whereas the heparin hexadecasaccharide (Hep-16) was an efficient activator of PKR autophosphorylation (Fig. 1, lanes n–r). Heparin octasaccharide (Hep8) likewise activated PKR autophosphorylation to a comparable extent as Hep-16, but higher concentrations were required of Hep-8 (Fig. 1). As shown in Fig. 2, PKR activated by Hep-16 was able to catalyze the phosphorylation of the alpha subunit of eIF-2 (lane d), a well-established property of PKR activated by dsRNA (Clemens, 1996; Samuel, 1993). Intra- and intermolecular autophosphorylation of PKR Two models have been proposed for the activation of PKR; one postulates that intramolecular autophosphory-

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mixtures that contained His-PKR(K296R) (Fig. 3, lanes 8 and 9). As controls, when the catalytic-deficient HisPKR(K296R) mutant protein was examined alone, it was not phosphorylated either in the absence or presence of added dsRNA (Fig. 3, lanes 4 and 5) as previously reported (Thomis and Samuel, 1993) or in the presence of heparin (lane 6). Also, consistent with the results obtained earlier in Fig. 1, when the PKR(Wt) protein was examined alone, both Hep-16 and dsRNA activated autophosphorylation (Fig. 3, lanes 2 and 3), but PKR(Wt) lacked kinase activity in the absence of added activator (Fig. 3, lane 1). Heparin and dsRNA act independently of each other in the activation of PKR

FIG. 3. Intermolecular phosphorylation of His-PKR(K296R) substrate by PKR(Wt) kinase is double-stranded RNA-dependent. In vitro phosphorylation reaction mixtures contained no activator (lanes 1, 4, and 7); 0.1 mg/ml poly[rI]:poly[rC] (ds, lanes 2, 5, and 8); or 2.0 mg/ml heparin as Hep-16 (H, lanes 3, 6, and 9). Lanes 1–3 contained PKR(Wt), lanes 4–6 contained a molar amount of His-PKR(K296R) similar to the amount of PKR(Wt) in lanes 1–3, and lanes 7–9 contained equimolar amounts of PKR(Wt) and His-PKR(K296R) (Wt / K296R). Reaction mixtures were incubated at 307 for 5 min; 32P-labeled phosphoproteins were fractionated on a SDS–PAGE (10% polyacrylamide) gel and visualized by autoradiography. The positions of the 67-kDa PKR(Wt) and 71-kDa HisPKR(K296R) protein bands are indicated.

lation is involved in the activation of PKR (Berry et al., 1985; Galabru et al., 1989), whereas the other proposes the involvement of intermolecular PKR phosphorylation (Kostura and Mathews, 1989). Recent studies definitively established that autophosphorylation of PKR may indeed include intermolecular phosphorylation events, because the histidine-tagged PKR(K296R) mutant is a substrate for PKR(Wt) kinase activated by dsRNA (Thomis and Samuel, 1993). The K296R mutation, which substitutes an arginine for lysine at position 296 of kinase catalytic subdomain II of PKR, destroys PKR kinase activity (Katze et al., 1991; Thomis and Samuel, 1992). The engineered N-terminal histidine tag adds about 4 kDa to the size of PKR(K296R) and thus allows electrophoretic separation of PKR(Wt) and His-PKR(K296R) from each other (Thomis and Samuel, 1993). As shown in Fig. 3, efficient intermolecular phosphorylation of His-PKR(K296R) in trans by PKR(Wt) was observed in the presence of dsRNA (Fig. 3, lane 8), but was completely absent in the absence of activator RNA (Fig. 3, lane 7). Surprisingly, when heparin was used to activate PKR(Wt), the intermolecular phosphorylation of HisPKR(K296R) was marginally detectable (Fig. 3, lane 9) compared to when dsRNA was used as the activator (Fig. 3, lane 8). However, the intramolecular phosphorylation of PKR(Wt) was readily detected, both with Hep-16 (lane 9) and with dsRNA (lane 8) as the activator, in the reaction

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As an approach to assess the mechanism of activation of PKR autophosphorylation by heparin as compared to dsRNA, and whether the two classes of anionic activators can act synergistically, heparin and dsRNA were examined alone and in combination with each other (Fig. 4). Hep-16, at a low concentration (0.1 mg/ml) that activated autophosphorylation of PKR only to a marginally detectable extent (Fig. 1B, lane o, and Fig. 4, lane n), did not significantly affect the concentration of dsRNA required to activate autophosphorylation (Fig. 4, lanes a–d compared to lanes e–h). Interestingly, a concentration of Hep-16 saturating for activation of autophosphorylation, 10 mg/ml (Fig. 1B, lane r and Fig. 4, lane p), yielded a lower level of phosphorylation than did a saturating level of dsRNA but did not adversely affect the enhanced phosphorylation observed with dsRNA (Fig. 4, lanes a–d compared to lanes i–l). The RNA-binding site of PKR has been localized to a novel R motif within the N-terminal region of the protein, a motif now recognized as part of numerous cellular and viral dsRNA-binding proteins (St. Johnston et al., 1992; McCormack et al., 1992; Samuel, 1993). Purified recombinant PKR mutant K296R(1–551) efficiently bound 32P-labeled adenovirus VA RNA, both by Northwestern analysis

FIG. 4. Activation of PKR autophosphorylation by heparin and doublestranded RNA, either alone or in combination with each other. [g-32P]ATP-mediated autophosphorylation of purified PKR was measured as described under Materials and Methods. The standard reaction mixture contained dsRNA and/or heparin at the indicated concentrations (mg/ml). The region of the autoradiogram corresponding to the 67-kDa 32P-labeled PKR is shown.

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added to the reaction mixture (Fig. 6A, lane C), consistent with the prior observation that dsRNA is required for the binding of ATP to PKR (Bischoff and Samuel, 1985; Galabru and Hovanessian, 1987) and the finding described herein that dsRNA and Hep-16 in combination activated PKR autophosphorylation (Fig. 5). By contrast, preincubation of PKR with 1 mg/ml of Hep-16 for 20 min at 307 in the absence of ATP surprisingly prevented subsequent autophosphorylation when [g-32P]ATP and dsRNA were added to the reaction mixture (Fig. 6A, lane d). Preincubation on ice under the same conditions had no adverse effect (data not shown).

FIG. 5. Electrophoretic mobility-shift competition analysis of PKR RNA binding activity. Gel mobility shift assays were carried out as described under Materials and Methods, with constant concentrations of human PKR protein and adenovirus VAI RNA probe and competitor as indicated. Lane a, probe alone; lanes b and g, no competitor; lane c, Hep16 at 1 mg/ml, lane d, Hep-16 at 10 mg/ml, lane e, poly(rI):poly(rC) at 0.1 mg/ml; and lane f, poly(rI):poly(rC) at 1 mg/ml.

as previously reported (McCormack et al., 1994) and by gel mobility-shift analysis as shown in Fig. 5 (lanes b and g). The binding of VA RNA was effectively competed by dsRNA (lanes e and f), but not by heparin (lanes c and d), suggesting that the two activators bind to independent sites on PKR (Fig. 5). Hovanessian and Galabru (1987) originally showed that natural PKR(Wt) protein binds to heparin-Sepharose. This binding to heparin is independent of a functional dsRNA-binding activity, because the full-length recombinant PKR proteins PKR(K64E), PKR(K296R), and PKR(Wt) expressed in COS cells (McCormack et al., 1994) all bound to heparin-agarose (data not shown).

Activation of PKR enzymic function by dsRNA-binding at the N-terminal region of the protein presumably results from an RNA-mediated conformational change affecting the C-terminal kinase catalytic subdomains, because ATP can be bound by PKR only in the presence of dsRNA activator (Bischoff and Samuel, 1985; Galabru and Hovanessian, 1987). Because of these earlier observations obtained with dsRNA, the effect of preincubation with heparin was also examined. Preincubation of PKR with dsRNA in the absence of ATP did not impair subsequent autophosphorylation when [g-32P]ATP and Hep-16 were

FIG. 6. Effect of preincubation on activation of PKR autophosphorylation by heparin and double-stranded RNA. (A) Purified PKR was preincubated (/) or not (0) for 20 min at 307 under standard conditions, except no ATP was present and either Hep-16 (1 mg/ml) or poly(rI):poly(rC) (0.5 mg/ml) was added as indicated. Following preincubation, 100 mM ATP containing [g-32P]ATP and dsRNA were added and the standard reaction mixture was incubated for an additional 20 min at 307 before analysis. (B) Preincubation was as for (A), except Hep-16 at the indicated concentration (mg/ml) or dsRNA at 0.5 mg/ml was present. Following preincubation, 100 mM ATP containing [g-32P]ATP and either dsRNA (0.5 mg/ml) or Hep-16 at the indicated concentration was added and the standard reaction was incubated for an additional 20 min at 307 before analysis. (C) Preincubation was as for (A), except Hep-16 (1 mg/ml) and ATP (100 mM unlabeled) were present as indicated and preincubation was at 307 for the specified period of time. Following preincubation, [g-32P]ATP and dsRNA (0.5 mg/ml) were added and the standard reaction was incubated for an additional 20 min at 307 before analysis. For (A), the autoradiogram is of the complete gel area; for (B) and (C) the autoradiograms correspond to the region of the 67-kDa 32 P-labeled PKR protein.

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with [32P]ATP (Fig. 7A) consistent with the results shown in Fig. 6, parallel reactions carried out with unlabeled ATP and then analyzed by Western immunoblot analysis did not reveal detectable degradation of PKR (Fig. 7B). The slightly retarded mobility of PKR observed in lane b* of the Western blot (Fig. 7B) corresponds to the autophosphorylated form of PKR (Fig. 7A, lane b); it is well-established that the phosphorylated form of PKR often displays a retarded mobility on SDS–PAGE as compared to the unphosphorylated form of PKR (Lasky et al., 1982; Thomis and Samuel, 1993). DISCUSSION

We considered the possibility that preincubation with Hep-16 led to degradation of PKR and thus loss of autophosphorylation activity. However, the results shown in Fig. 7 suggest that this is unlikely. Although preincubation with Hep-16 in the absence of ATP impaired subsequent dsRNA-dependent autophosphorylation measured

Several important points emerge from the results reported herein concerning the activation of PKR autophosphorylation by heparin. First, the results not only confirm that heterogeneously sized heparin activates PKR autophosphorylation but they further demonstrate the minimum size requirement for activation of PKR autophosphorylation using homogeneously sized heparin oligosaccharides. Second, heparin-activated PKR(Wt) catalyzes the phosphorylation of eIF-2a as does dsRNAactivated PKR, whereas only dsRNA-activated PKR(Wt) and not heparin-activated PKR(Wt) catalyzes the intermolecular phosphorylation of His-PKR(K296R). Third, the structural and functional basis of PKR interaction with heparin differs from that of dsRNA as revealed from competition RNA binding data and from the effects of preincubation with the polymers in the absence of ATP on subsequent PKR autophosphorylation activation. Fourth, neither heparin nor dsRNA when incubated with purified PKR mediates a detectable degradation of PKR. The minimum saccharide size of heparin required to efficiently activate PKR autophosphorylation was the heparin octasaccharide. The heparin hexasaccharide was a very poor activator of PKR autophosphorylation. These results obtained for PKR are comparable to those reported for basic fibroblast growth factor-mediated biochemical and biological activities (Ishihara et al., 1993; Ornitz et al., 1992). For example, heparin oligosaccharide with as few as 8 to 10 sugar residues was sufficient to mediate bFGF-receptor binding and induce mitogenesis. Heparin, although able to mediate the autophosphorylation of PKR(Wt) and the intermolecular phosphorylation of eIF-2a, could not mediate the intermolecular transphosphorylation of His-PKR(K296R). The finding that heparin-activated PKR(Wt) could not catalyze the intermolecular phosphorylation of His-PKR(K296R) in the manner observed for dsRNA-activated PKR(Wt) was unexpected (Thomis and Samuel, 1993, 1995). This result suggests that the intermolecular phosphorylation of His-PKR (K296R) may require the binding of either the PKR(Wt) enzyme or the His-PKR(K296R) substrate, or both, to RNA in order for subsequent intermolecular phosphorylation to occur. Alternatively, the conformational change of PKR

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FIG. 7. Effect of preincubation with heparin on activation of PKR autophosphorylation and stability of PKR. Preincubation was as described in the legend of Fig. 6A, except that the mixture contained Hep16 (1 mg/ml) as indicated. Following preincubation, dsRNA (0.5 mg/ml) and ATP, either [g-32P]ATP (A) or unlabeled ATP (B) were added as indicated. The standard reaction mixtures were then incubated for an additional 20 min at 307 before analysis. (A) The 32P-labeled samples were analyzed for [g-32P]ATP-mediated autophosphorylation of PKR. (B) The unlabeled samples were analyzed for PKR protein by Western immunoblot using 125I-protein A as described under Materials and Methods. The autoradiograms are of the complete gel areas.

The concentration of Hep-16 required during preincubation to mediate the subsequent impairment of autophosphorylation in the presence of dsRNA was between 0.1 and 1.0 mg/ml (Fig. 6B, lanes a–d), similar to the concentration of Hep-16 required for activation of PKR autophosphorylation (Fig. 1B). But preincubation of PKR with dsRNA did not impair subsequent autophosphorylation in the presence of Hep-16, even when examined at 10 mg/ml Hep-16 (Fig. 6B, lanes e–h). Inhibition of PKR autophosphorylation by preincubation at 307 with Hep16 was rapid in the absence of ATP (Fig. 6C, lanes d– f). However, the presence of 100 mM ATP during the preincubation with Hep-16 completely prevented the inhibition (Fig. 6C, lanes g and h). PKR remains intact during preincubation and subsequent autophosphorylation

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associated with dsRNA-binding (Bischoff and Samuel, 1985) may facilitate the intermolecular phosphorylation. Because RNA molecules too small to accommodate two PKR molecules can mediate the activation of PKR (Manche et al., 1992), the intermolecular autophosphorylation of PKR may occur by a mechanism that is not dependent upon the physical bridging of two PKR molecules by RNA, but rather is dependent upon PKR protein–protein interaction. Our results further suggest that PKR protein–protein interaction is not sufficient for intermolecular phosphorylation events to occur. Combined biochemical and genetic evidence has established that PKR undergoes intermolecular association with itself, both in vitro and in vivo (Langland and Jacobs, 1992; Cosentino et al., 1995; Patel et al., 1995; Ortega et al., 1996). In some cases, RNA-binding was neither necessary nor sufficient for PKR multimerization (Patel et al., 1995; Ortega et al., 1996), and dimerization was neither sufficient nor essential for activation (Wu and Kaufman, 1996). The structural basis of heparin binding to PKR is not known. Our finding that Hep-16 did not compete the binding of adenovirus VA RNA by PKR in the gel mobility shift assay, whereas poly(rI):poly(rC) did compete, suggests that heparin and RNA likely bind to different sites on the PKR protein or that VA RNA has a substantially higher binding affinity for PKR than does heparin. The former explanation appears most likely, because PKR(K64E) mutant protein bound to heparin-agarose and this mutation is known to destroy RNA-binding activity (McCormack et al., 1994). The RNA binding site on PKR has been localized to a novel 20 residue motif R within the N-terminal half of the PKR protein (Mathews, 1993; McCormack et al., 1992; Samuel, 1993; St. Johnston et al., 1992). Many proteins are known to bind heparin, and a comparative analysis of structurally defined heparin binding sequences has revealed a distinct spatial distribution of basic residues (Margalit et al., 1993). When heparin binding sequences fold into either an a-helix or a b-strand fold, the alignment of two basic residues was conserved at the extremes of each of the structures, but with different linear separations (Margalit et al., 1993). The amino acid sequence of PKR deduced from the human cDNA sequence (Thomis et al., 1992) includes within the Cterminal half of the PKR protein the proposed heparin binding motifs, with correct spatial separation of basic residues and respective correct predicted a-helical or bstrand secondary structures. The notion that the heparin binding site of PKR may reside in the C-terminal region of PKR would be consistent with the observed lack of competition between Hep16 and RNA in the gel shift experiment, and also the observation that deletion mutants that fail to bind to and be activated by dsRNA can be fully activated by heparin (Patel et al., 1994). Construction and analysis of deletion and site-directed substitution mutants have led to the identification of several residues within the R-motif re-

gion which are essential for the dsRNA-binding activity of PKR (Green et al., 1995; McCormack et al., 1994; McMillan et al., 1995). So far, attempts to generate PKR deletion mutants that are activated by dsRNA but not by heparin have been unsuccessful. The molecular basis of the inhibition of PKR autophosphorylation when purified PKR was preincubated with Hep-16 in the absence of ATP, but not in the presence of ATP, is unclear. A possible explanation is that preincubation of PKR and Hep-16 in the absence of ATP leads to a conformational change of PKR that precludes the subsequent binding of ATP by PKR, either in the presence or absence of dsRNA. Because proteolysis mediated by phosphorylation can be an important regulatory mechanism of gene expression as exemplified by the process of NF-kB activation (Thanos and Maniatis, 1995), we examined the possibility that PKR undergoes proteolytic degradation during preincubation with heparin. However, this does not seem likely because Western immunoblot analysis revealed the expected size of about 67-kDa by SDS–PAGE for PKR, either preincubated or not, with either Hep-16 or dsRNA. The underlying mechanism of cell growth regulation by heparin is not well understood, but may include in part the functional modulation of various heparin-binding growth factors (Ruoslahti and Yamaguchi, 1991). Interferon is also a regulator of cell growth and differentiation, and the PKR kinase is implicated in the regulation of these cellular activities and may function as a tumor suppressor (Lengyel, 1993; Samuel, 1993). Although no evidence of tumor suppressor activity of PKR was observed in mice devoid of a functional PKR gene (Yang et al., 1995), expression of functionally defective PKR protein in mouse 3T3 cells causes malignant transformation and 3T3 cells overexpressing either inactive PKR (Koromilas et al., 1992; Meurs et al., 1993) or an inhibitor of PKR (Barber et al., 1994) are highly tumorigenic when injected into nude mice. Conceivably a physiologic role for heparin activation of PKR, if one exists, may relate to the control of cell growth and differentiation via activation of PKR, just as RNA activation of PKR is implicated in the control of virus replication in IFN-treated cells (Mathews, 1993; Samuel, 1991).

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ACKNOWLEDGMENT This work was supported in part by Research Grant AI-20611 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service.

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