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Purification and Activation of the Double-Stranded RNA-Dependent Eif-2 Kinase DAI MATTHEW Kosturat and MICHAEL B

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Purification and Activation of the Double-Stranded RNA-Dependent Eif-2 Kinase DAI MATTHEW Kosturat and MICHAEL B MOLECULAR AND CELLULAR BIOLOGY, Apr. 1989, p. 1576-1586 Vol. 9, No. 4 0270-7306/89/041576-11$02.00/0 Copyright C) 1989, American Society for Microbiology Purification and Activation of the Double-Stranded RNA-Dependent eIF-2 Kinase DAI MATTHEW KOSTURAt AND MICHAEL B. MATHEWS* Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, New York 11724 Received 14 September 1988/Accepted 3 January 1989 The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsl and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of -70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNA,. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNA, blocked autophosphory- lation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNA, also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNA, would block activation by preventing the interaction between DAI and dsIINA. Phosphorylation of the a subunit of eucaryotic protein state, and activation is accompanied by p68 phosphoryla- synthesis initiation factor eIF-2 (eIF-2oa) prevents catalytic tion; on the assumption that p68 is a single polypeptide recycling of the protein in polypeptide chain initiation and species and that no other protein is involved, the activation results in the inhibition of protein synthesis (reviewed in event would constitute an autophosphorylation. Detailed reference 30). Control of translational initiation by this study of the enzyme has been hampered by difficulties in mechanism has been demonstrated, or at least inferred, in a purifying it without either eliciting its activation or losing its variety of systems, including hemin-deprived rabbit reticu- dsRNA dependence, and most attempts to isolate the en- locytes, virus-infected mammalian cells, and cells subjected zyme have met with only moderate success. Nevertheless, to heat shock or changes in redox state (see reference 21 for considerably enriched preparations have been obtained from reviews). Although many effectors and environmental con- the ribosomal salt wash fraction of both rabbit reticulocytes ditions influence the level of eIF-2 phosphorylation, only (32-34) and interferon-induced tissue culture cells (3, 13, 38, two protein kinases that are capable of catalyzing this 41, 44, 45). Highly purified preparations of DAI contain a reaction have been identified (26): the hemin-controlled single major polypeptide subunit of -70 kDa, and the native regulator HCR, found chiefly in reticulocytes, and the dou- enzyme has a molecular mass of approximately 70,000 Da, ble-stranded RNA (dsRNA)-activated inhibitor (DAI, also as determined by sedimentation velocity and gel filtration. known as P1, dsl, and PKdS). DAI, also found in reticulo- These observations suggested that the p68 phosphoprotein cytes, is present at low levels in most mammalian cells and and DAI are identical, but the hypothesis remained un- tissue culture cell lines and at greatly increased levels after proven since complete purity was not achieved. Taking an interferon treatment (reviewed in references 1, 10, and 19). alternative approach, Hovanessian et al. generated a mono- The enzyme plays a role in establishment of the antiviral clonal antibody against the phosphorylated p68 protein (17). state, and several viruses have elaborated defense measures The antibody, coupled to a resin matrix, was able to immo- to counter this form of interference in their replication (12, bilize DAI, which confirmed that the p68 protein was in- 14, 15, 29, 35, 36, 40, 43). Viruses may also exploit the volved in the activity of DAI, but elution of the immunoad- enzyme to achieve selective production oftheir own proteins sorbent yielded a polypeptide of -48 kDa (p48) in addition to (12, 26a). The ubiquity of the enzyme suggests that it may p68 (6). Further study led to the suggestion that DAI is a function in normal, uninfected cells (for example, during dimer of p68 and p48 apd that these two species have growth and differentiation events [31]), but this idea remains dissimilar kinase activities; p48 was thought to phosphory- to be fully substantiated. late p68 in the presence of dsRNA, and phosphorylated p68 Exposure of cell extracts containing DAI to dsRNA re- was held to possess the eIF-2a kinase activity. More recent sults in phosphorylation of a protein of -70 kilodaltons data, however, indicate that DAI is degraded during affinity (kDa) as well as the 36-kDa subunit of eIF-2 (4, 18, 37, 44). purification and that p48 is a proteolytic breakdown product The -70-kDa protein, often referred to as p68, is associated (7), casting doubt on the description of the enzyme as a with DAI during purification and is generally believed to heterodimer. comprise the enzyme. The enzyme exists in a latent, inactive DAI is activated by very low concentrations of dsRNA, but higher concentrations inhibit enzyme activation (9). Short * Corresponding author. (<50 base pairs) or imperfect duplexes are unable to activate t Present address: Department of Biochemical and Molecular DAI (9, 23), and adenovirus VA RNA,, a molecule with Pathology, Merck, Sharp & Dohme Research Laboratory, Rahway, extensive secondary structure (22, 24), inhibits activation NJ 07065. by competent dsRNA (14, 26a, 27, 39, 42). On the basis of 1576 VOL. 9, 1989 INTERFERON-INDUCED PROTEIN KINASE DAI 1577 these observations, we proposed a model for DAI activation monitored by dsRNA-dependent phosphorylation of p68, in which phosphorylation of p68 occurs only when two eIF-2 kinase activity, and sodium dodecyl sulfate (SDS)- molecules interact with one another on a single dsRNA polyacrylamide gel analysis of samples from column frac- molecule (28). The model also proposed that VA RNA acts tions, followed by silver staining. DAI eluted at -180 to 200 by interfering with the binding of DAI to dsRNA. To test this mM KCl. The entire DAI kinase peak was pooled and model, we have purified DAI to apparent homogeneity and concentrated by dialysis against buffer B plus 50 mM potas- studied its activation and interactions with dsRNA and VA sium phosphate (pH 7.2) and 20% sucrose. The dialyzed RNA,. The data largely corroborate the model and suggest S-Sepharose pool was loaded onto a 2.5- by 8.0-cm column that activation is accompanied by a change in the substrate of hydroxylapatite (HPHT grade; Bio-Rad Laboratories, preference of the enzyme. Richmond, Calif.) equilibrated with buffer B plus 50 mM potassium phosphate (pH 7.2). The column was washed with MATERIALS AND METHODS 1 column volume of the same buffer and then eluted with a 300-ml 50 to 500 mM potassium phosphate gradient made up Materials. Human recombinant a-interferon (IFN) was the in the start buffer. The dsRNA-dependent p68 autophospho- kind gift of Paul Trotta (Schering Corp., Bloomfield, N.J.). rylation and eIF-2 kinase activity coeluted from the column Aprotinin, leupeptin, and pepstatin were from Boehringer- with a protein of -70 kDa. DAI activity was pooled and Mannheim Biochemicals (Indianapolis, Ind.). Phenylmethyl- dialyzed against 100 volumes of buffer B (pH 6.8) with 20% sulfonyl fluoride was from Sigma Chemical Co. (St. Louis, sucrose. The dialyzed hydroxylapatite pool was loaded onto Mo.). Human 293 cells, a line of adenovirus type 5-trans- an HR 5/10 Mono S column (Pharmacia) and eluted at 1.0 formed human embryo kidney cells (8), were propagated in in B suspension cultures in F-13 medium (GIBCO Laboratories, m/min with a 40-ml 50 to 500 mM KCl gradient buffer Grand Island, N.Y.) supplemented with 10% calf serum. (pH 6.8). DAI was readily identified in silver-stained gels as Preparation of extracts from IFN-treated cells. Typically, a prominent -70-kDa protein. Individual kinase fractions 40 liters of 293 cells at 3 x 105 to 4 x 105 cells per ml was were stored at -70°C. The more highly enriched fractions treated for 18 h with 800 U of IFN per ml to increase the were subjected to 15 to 35% glycerol gradients for prepara- yields of DAI. All subsequent steps were carried out at 0 to tion of highly purified DAI. Portions (150 to 200 pI) of Mono 4°C. Cells were harvested by centrifugation at 1,000 x g, S-purified DAI were layered onto gradients of 15 to 35% washed with phosphate-buffered saline and then with buffer glycerol in buffer B (pH 7.4) and centrifuged at 49,000 rpm A (10 mM KCl, 20 mM N-2-hydroxyethylpiperazine-N'-2- for 22 h at 4°C in a Beckman SW50.1 rotor. Fractions (150 ethanesulfonic acid [HEPES] [pH 7.4], 1.5 mM MgCl2, 0.1 pI) were taken from the top by using an Auto Densiflow mM EDTA, 1 mM dithiothreitol, and protease inhibitors [1 fraction collector (Buchler Instruments Div., Nuclear-Chi- ,ug of aprotinin, leupeptin, and pepstatin per ml and 1 ,uM cago Corp., Fort Lee, N.J.), and 10 pI was analyzed by gel phenylmethylsulfonyl fluoride]). The final cell pellet was electrophoresis and silver staining. Gradient-purified kinase resuspended in 3 volumes of buffer A.
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