The regulation of the protein kinase PKR by RNA

Biochimie (1996) 78, 909-914 © Soci6t6 franqaise de biochimie el biologic mol6culaire / Elsevier, Paris

The regulation of the protein kinase P K R by RNA HD Robertson a, MB Mathews b aDepartment of BiochemistD, Cornell Universi~. Medical College, 1300 York Avenue, New York, NY 10021; bUMDNJ-New Jersey Medical School, Department of Biochemisto, and Molecular Biology, 185 South Orange Avenue, Universi~ Heights, Newark, NJ 07103, USA

(Received 10 November 1996; accepted 4 December 1996) Summary m A model is presented for the regulation of the double-stranded RNA (dsRNA)-activated mammalian protein kinase PKR, which is involved in protein synthesis inhibition and the antiviral response in cells. A series of previous findings abut PKROs behavior are reviewed, including its effects on translation; the activation of its protein kinase activity; binding sites for PKR on RNA; PKR0s protein domains, which include two double-stranded RNA binding motifs (dsRBMs); and the likelihood of PKR dimer form,tion. The model which emerges to account for many of these observations includes the suggestion that PKR dimers form which are stabilized and rearranged upon binding to dsRNA regions 60 bp or longer. The hypothesis includes protein conformational changes within each member of a PKR dimer bound to dsRNA which re-position an inhibitory polypeptide domain and thus allow kinase activation. Also considered are ways in which PKR interacts with imperfectly duplexed, highly structured RNA molecules. dsRNA / dsRBM / protein dimerization / protein synthesis inhibition Introduction

One of the most enduring mysteries in the sphere of regulatory RNA:protein interactions concerns the mammalian protein kinase PKR (also called DAI, p68 kinase and PIelF2 kinase). This activity was originally detected as the enzyme responsible for double-stranded (ds) RNA-dependent protein ~ynthesis inhibition in poliovirus-infected HeLa cells [ II and in cell-free translation systems made from rabbit reticulocytes [2]. While the target of dsRNA's mysterious ability to inhibit protein synthesis initiation was eventually shown to be the protein kinase PKR, which autophosphorylates in the presence of dsRNA and then blocks protein synthesis by phosphorylating initiation factor elF2 [3-5], some properties of this reaction remain unexplained to this day. For example, as first demonstrated by Hunt and Ehrenfeld [I, 2] protein synthesis inhibition in vitro (now known to correlate with PKR activation) begins at very low concentrations of dsRNA (below 1 ng/mL), but this inhibition disappears at high dsRNA concentrations as if some component had been 'diluted out'. Any model for PKR action must take this effect (fig 1) into account. Furthermore, in the time since these initial discoveries, many additional properties of PKR have been described but no overall working model explaining its behavior has emerged. It is the purpose of the present communication first to present an up-to-date account including relevant aspects of PKR's behavior and that of a group of other proteins containing a similar RNA-binding motif, the 'double-stranded RNA-binding motif', or dsRBM. We then describe studies on the size and specificity of RNA binding

by PKR, and discuss how this relates to kinase activation and, in a number of viral systems, to the inhibition of kinase activation by specialized viral RNAs. Studies of both RNA:protein and protein:protein interactions will be reviewed. Finally, we propose a hypothesis which accounts for a number of seemingly contradictory observations about PKR and other dsRRM-contai,ing proteins, and outline some ways in which this model can be tested.

The facts about P K R ' s behavior Effects on translation

Once activated by contact with dsRNA, PKR inhibits translation by phosphorylating the o~-subunit of initiation factor elF2 ([3]; reviewed in [4, 5]). High concentrations of dsRNA fail to cause this effect [1, 2]. In addition, dsRNA populations below a certain size limit, at:out 40 bp, and certain highly structured viral single-stranded RNAs (ssRNAs) inhibit PKR activation [4-6]. Thus, the size of the RNA binding site for PKR determines the nature of PKR's response to binding RNA. The activation of PKR by dsRNAs above the minimum size limit has been interpreted as an antiviral response by the cell to a common product of viral infection, dsRNA, and the inhibition of PKR activation by smaller, highly structured RNAs - - most notably VA RNAI of adenovirus [71., but also TAR RNA of HIV [8] and the Epstein-Ban" virus EBER RNAs [9] - - could thus be a response by these viruses to counteract the cellular PKR antivirai pathway. This view is further supported by the fact

910 while several studies have shown that the optimal dsRNA size for kinase activation is about 70-80 bp, PKR can be activated at lower efficiency by dsRNAs as small as 40 bp [6]. Whatever binding mechanism is proposed to allow kinase activation and PKR unfolding must take this into account.

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Fig 1. Activity of reovirus dsRNA and adenovirus VA RNAI in a cell-free translation system. This figure, adapted from [26], shows globin synthesis measured in a rabbit reticulocyte lysate system as d e s c r i b e d [26] after preincubation of the lysate with various concentrations of reovirus dsRNA (:L) or VA RNA (s). Results from several experiments covering different parts of the concentration range were combined.

that PKR expression is favored by the presence of interferon (reviewed in [4, 51).

Kinase activation The activation of PKR's kinase by sizable dsRNA molecules most likely results from an induced change in protein conformation by the RNA ligand. Activation by dsRNA appears to correlate with autophosphorylation, and several s i t e s have been identified in the amino terminal half of the protein [ 10]. Some of these autophosphorylation sites, lying downstream of the RNA binding region (see below) have been shown to be important for enzyme activity, while others (lying within the RNA binding region) are of unknown significance. Evidently, RNA binding alone is not enough to activate the kinase, however, as shown by the inhibitory binding to small structured viral RNAs mentioned above. Furthermore it appears that, when parts of the 68 kDa PKR protein near the amino terminus and concerned with RNA binding are removed, the kinase activity of the remaining PKR polypeptide becomes constitutive, no longer requiring activation by dsRNA binding [ 11 ]. A final fact about kinase activation which must be explained is that,

In molecules iike VA RNAI of adenovirus which inhibit PKR activation, about 25-30 bp are a~.sociated with PKR so as to be protected from nuclease in footprinting and RNase protection studies [12, 27]. In contrast, studies using synthetic dsRNAs with pre-determined sizes ranging from less than 20 to over 100 bp revealed an initial size for PKR activation of about 40 bp, and an optimal size of 70-80 bp [6]. In delta agent RNA, which activates PKR kinase under certain conditions, RNase protection shows a size equivalent to 65-70 bp associated with activation. As discussed below, PKR has two dsRBM RNA-binding polypeptide domains near its amino terminus (fig 2). Studies on related dsRBM domains from two other RNA-binding proteins using NMR have led to the proposal that about 11 bp of dsRNA is the minimum size associated with a single isolated dsRBM [13, 14]. This proposal is in good agreement with results of gel shift experiments carried out under saturating conditions with fragments of PKR containing dsRBM sequences in which a minimum size of 11-13 bp of dsRNA was found [151. A number of structural studies on PKR have identified an amino terminal fragment about 180 amino acids long, containing both dsRBMs but lacking the kinase activity, as the minimal domain required for RNA binding. A 184amino acid fragment of PKR, p20, has been expressed in E coil, purified and extensively utilized. Although the p20 fragment can associate with about 22 bp of dsRNA [16] formation of a stable complex involving PKR or the p20 fragment apparently requires about 30 bp [6]. One of the most remarkable observations to emerge from nuclease protection studies is that the p20 fragment binds and protects the same size RNA binding site as does the intact 68 kDa PKR molecule. This is true both for the inhibitory binding

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Fig 2. Structure of the PKR amino terminal legion. The positions of the two dsRBMs in the 551 amino acid PKR protein are depicted, with their initial and terminal amino acid residues indicated at the lower left and right of each. The area between dsRBMI and dsRBM2, amino acids 78-100, has been defined here as the intramotif sequence, or IMS.

911 to small viral RNAs like VA RNA [12]; and for the binding to delta agent RNA which activates the intact kinase [27]. Thus, a 20 kDa protein fragment somehow contrives to protect 65-70 bp equivalents of RNA. Other aspects of the manner of PKR's binding to RNA emerge from a recent study in which minimal requirements for fragments like p20 were rigorously explored [ 16]. The conclusion was reached that a set of 2' hydroxyls on the exterior of a dsRNA of minimal size or greater was the signal for binding. This idea of grouped 2' hydroxyls helps to explain how some, but not all, imperfectly duplexed RNAs, like the delta and VA RNAs already mentioned, can bind to PKR: such moieties are envisaged to engage in dsRNA mimicry, wherein key 2' hydroxyls are arranged in a pattern analogous to their key counterparts in authentic dsRNA. Presumably, evolutionary selection would favor such an arrangement when the dsRNA mimicry would improve fitness, eg allowing a virus to inactivate a host kinase otherwise bent on shutting down all protein synthesis for an indefinite period. A final fact which must be accounted for is the large range of duplex sizes to which the two-dsRBM p20 fragment can bind. This fact led to the proposal of the 'hinge' model of Green and Mathews [17] for PKR binding, in which the two dsRBMs (1 and 2, as shown in figure 2) associate with RNA, but the 20 or so amino acids between them can fold at different angles to allow binding of differently sized RNAs. This hinge region, amino acids 78-100 in figure 2, can also be referred to as the 'intra-motif sequence' or IMS. Facts about PKR's protein domains

From the NMR studies of dsRBMs in other RNA-binding proteins, it is clear that the dsRBM is a compact elliptical structure which seems capable of binding (and protecting fi'om nuclease) about 11-13 bp, but not many more [13, 14]. Thus, the results of nuclease protection studies using PKR and the p20 fragment in which considerably larger RNA domains are protected must be the result of binding multiple dsRBMs. For example, when the p20 fragment of PER binds 22 bp of dsRNA, this must be the result of two p20 molecules, each binding to the dsRNA with one dsRBM (particularly at high protein:RNA ratios), or it could be the outcome of one p20 molecule binding via both of its dsRBM domains. That one dsRBM could bind 22 bp seems very unlikely from the available data. Given the observation that p20 can carry out all of the RNA binding activities and variations of the parent p68 PKR molecule, its sequence and structure must hold the key to explaining them. Another observation likely to be important in any explanation of RNA binding by PKR is the fact that dsRBMI, located near the amino terminus of the protein (amino acids 11-77) binds dsRNA or VA RNA with a much higher affinity than dsRBM2 (residues 101-167), although a protein (pl0) containing only dsRBM1 binds dsRNA about 100 times less strongly than p20 which contains both dsRBMs

[15]. Thus, in addition to RNA binding, the amino acid sequerLces in the vicinity of PKR's two dsRBMs must have additional functions, one of which, contributed by dsRBM2, seems to enhance binding stability. Finally, the amino acid sequence between the two dsRBMs, amino acids 78-100, the IMS or 'hinge' of Green and Mathews [17], must have a role in explaining variable RNA binding site sizes and efficiencies. It represents a substantial portion of the p20 sequences which are not engaged in RNA binding, and perhaps in regulating activities in the remainder of the PKR molecule. Protein dimerization

Recent studies [15, 18-22] show that PKR molecules dimerize. Furthermore, some of the contacts allowing this to happen are located within the dsRBM2 amino acid sequence [20]. Dimerization as part of kinase activation would certainly simplify explanations of the Hunt-Ehrenfeld observations at high dsRNA concentration [1, 2] (fig 1), since it would allow the postulate that such conditions prevent two PKR molecules from approaching one another closely enough to form dimers and thus become activated: they would all be distributed among a huge excess of separate dsRNA molecules. As we shall see below, the assumption that dimerization happens and involves dsRBM2 sequences can contribute much to our understanding of PKR.

A model for PKR action As shown in figure 3, the data arid conc!u~i,3ns reviewed here can be brought together in a model which accounts for most observations about PKR. First, we assume that 'grouped elements' involving 2' hydroxyls form the binding sites for PKR, allowing true dsRNA and viral ssRNAs that mimic dsRNA to bind in a fashion equivalent to short dsRNA. Inhibitory binding of PKR to viral ssRNAs could then be understood in the same context as PKR's binding to small dsRNAs: If the dsRNA or its viral equivalent is 30 bp or smaller, binding of two PKR molecules via their dsRBMI elements (at high PKR:RNA ratios) could occur; whereas binding of a single PKR molecule by both its dsRBMI and dsRBM2 domains could take place at low PKR:RNA ratios. In either case, the kinase activity of the PKR remains blocked because of the folding of the 'hinge" or IMS domain between the two dsRBMs. Figure 3A depicts the PKR molecule in this inactive state, and we envisage its structure as unchanged whether or not it binds a small dsRNA or its inhibitory viral ssRNA equivalent. The small highly structured viral RNAs thus act to sequester single inactive PKR molecules, depleting and finally eliminating the pool available for the dimerization and activation steps shown in figure 3B and C.

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KINASE """"-'-~ooN Fig 3. Structures of PKR involved in RNA binding and kinase activation. A. Schematic drawing of a proposed structure for an inactive PKR monomer, dsRBMs i and 2 are depicted as boxes numbered "!' and '2' The IMS is shown folded as a hinge and interacting in an inhibitory fashion (indicated by gray hatching) with the kinase domain. Amino and carboxy termini are indicated. As explained in the text, we propose that this monomeric, kinase-inhibited form of PKR can exist in solution; bound to dsRNAs or their highly structured ssRNA equivalents less than 40 bp in size; or bound to extremely high concentrations of sizable dsRNA molecules. B. Reversible dimerization of PKR. Two PKR molecules, each having the structure depicted in A, are shown dimerizing through contacts within dsRBM2, as described in the text. The IMS sequences remain associated in an inhibitory fashion, as shown, with their kinase domains. In the absence of rapid RNA binding, we envisage this dimer as transient and easily disrupted. C. The effect of sizable (> 60 bp) dsRNA binding on PKR dimers. The schematic drawing depicts the two PKR molecules in B after they have undergone a structural rearrangement induced by binding of the reversible dimer to dsRNA. As shown, the dsRBM-containing domains of the two PKR molecules shown in B have 'opened out' at the right, and the two protein molecules have rotated 180 ° with respect to each other, so that they are now aligned end-to-end rather than in parallel. The dsRBM2 contacts which stabilize the dimer remain, as shown. The dsRNA molecule is drawn as a ~wisted ¢ouble helix, with the dsRBMs now aligned along it in a longitudinally extended manner. The IMS regions have been rearranged so that their kinase inhibitory sequences (still shown by gray hatching) are kept far from the kinase domains, which are now stably activated, as described in the text.

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913 How does activation occur? Several facts cited above lead to the strong suggestion that activation requires PKR dimerization: the Hunt-Ehrenfeld observations at high dsRNA concentration [1, 2]; the binding of one dsRBM to only ! l-13 bp; and the requirement for some kind of structural rearrangement of PKR beyond RNA binding. Moreover, activation is bimolecular with respect to PKR [23]. Furthermore, the need to explain how protein domains as small as the RNA-binding regions of PKR can require such large RNA stretches for activation is especially acute in light of the fact that both the p20 fragment of PKR and the full-sized 68 kDa protein protect the same 65-70 bp equivalents in delta RNA [27]. Figure 3B and C show a dimerization model in which two PKR molecules associate reversibly through dsRBM2 contacts (fig 3B), associate with dsRNA of sufficient size to activate, and undergo a structural rearrangement which stabilizes the PKR dimer and activates the kinase (fig 3C). The postulated dimerization site in dsRBM2 brings the two PKR molecules together and stretches out the connecting polypeptide (the hinge or IMS) in each. The longitudinal extension by both bound PKR molecules which accompanies the structural rearrangement associated with dsRNA binding (fig 3C) accomplishes both of the key goals associated with PKR activation: a much more sizable region of RNA (60 bp or more) is stably bound in such a manner that it would be protected from nuclease digestion; and the dimerization has caused a structural change in the protein, freeing the kinase domain from its former inhibitory associations. A hypothesis such as this also explains why dsRNAs as short as 40 bp begin to activate the PKR kinase. As soon as the RNA domain is large enough to accommodate one longitudinally extended PKR bound to the dsRBM2 of another, the first will be activated. But only when the RNA size reaches the 67 bp or more found by Manche et a l [6] to give full kinase activity will all four dsRBM domains of both molecules be fully involved in RNA binding, structural rearrangement and kinase derepression. This imperfect activation of PKR by 40 bp fragments in fact comprises strong support for the model illustrated. There are additional facts which now fit into an overall context in light of the hypothesis shown in figure 3. First, the ability of high dsRNA concentrations to inhibit PKR activation can be understood in light of figure 3A, in which monomeric p68 molecules are inactive whether or not they are bound to any RNA. Excess dsRNA, of any size, would trap newly synthesized PKR monomers, preventing their involvement in activation. Further, if we assume as above that the PKR dimers shown in figure 3B are unstable, and that a molar excess of dsRNA molecules would encourage binding of the two monomers to separate RNA molecules, th'~s further promoting dimer destabilization, the end result of high dsRNA concentrations would be a population of stably bound, inactivated PKR monomers. Second, the model for PKR kinase activation whereby the dsRNA-related structural rearrangement overcomes the inhibitory

binding of the IMS regions to tile kinase domains (fig 3C) also allows us to explain the finding that removal of amino terminal sequences from the PKR molecule causes the kinase activity of the remaining polypeplide to become constitutive, no longer requiriag activation by dsRNA bindine [ l 1,23]. We would view this removal of dsRBM-containing amino acid sequences as analogous to their stable, longitudinally extended binding to dsRNA: b~th circumstances prevent the PKR IMS region from ~ibiting the kinase, causing its irreversible activation. ThJ,' studies in which the order of dsRBMI and dsRBM2 are reversed (so that dsRBM2 is now nearer the amino terminus of PKR) show an increase in kinase activity [24, 25]. This can now be understood by suggesting that dimerization continues to occur as shown in figure 3B and C (with the dsRBM2 dimerization sites aligned in their new, amino terminal positions) but that the newly constituted IMS domain has a lower intrinsic affinity than its wild type counterpart for the kinase domain, so that stable kinase activation is easier to achieve. Finally, what experiments come to mind which will allow tests of the activation model shown in figure 3? Since such a key inhibitory role is postulated for the IMS or 'hinge' region of PKR, it is clear that experiments involving mutations and disruptions of this amino acid sequence between the two dsRBMs will shed light on its function. For example, the data about reversal of dsRBM! and dsRBM2 [ 11, 23] cited above suggest careful studies of the interaction between IMS sequences and the PKR kinase domain. The fact that the IMS region contains phosphorylation sites as well [10] emphasizes its potential importance in regulating kinase activity in PKR molecules. A second area of experimentation would test the idea that sites in dsRBM2 of PKR are critical for dimerization, RNA protection and activation. Constructs in which the dsRBM2 dimerization sites are disrupted should allow their importance to be evaluated. Thirdly, the ability of the p20 fragment of PKR to bind and protect 60 base pairs or more should be tested with dsRNA molecules and additional highly structured ssRNAs which activate the PKR kinase. We conclude that the demonstration of RNA-mediated regulation of a protein kinase involving both induced conformational shifts and protein subunit dimerization is within reach.

Acknowledgments We thank Olivia Neel lbr helpful discussions. This work was supported by grants from NIH, AI-31067 to HDR and AI-34552 to MBM.

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