Purification and activation of the double-stranded RNA-dependent eIF-2 kinase DAI.

Abstract

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 autophosphorylation 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 synthesis initiation factor eIF-2 (eIF-2oa) prevents catalytic recycling of the protein in polypeptide chain initiation and results in the inhibition of protein synthesis (reviewed in reference 30). Control of translational initiation by this mechanism has been demonstrated, or at least inferred, in a variety of systems, including hemin-deprived rabbit reticulocytes, virus-infected mammalian cells, and cells subjected to heat shock or changes in redox state (see reference 21 for reviews). Although many effectors and environmental conditions influence the level of eIF-2 phosphorylation, only two protein kinases that are capable of catalyzing this reaction have been identified (26): the hemin-controlled regulator HCR, found chiefly in reticulocytes, and the double-stranded RNA (dsRNA)-activated inhibitor (DAI, also known as P1, dsl, and PKdS). DAI, also found in reticulocytes, is present at low levels in most mammalian cells and tissue culture cell lines and at greatly increased levels after interferon treatment (reviewed in references 1, 10, and 19). The enzyme plays a role in establishment of the antiviral state, and several viruses have elaborated defense measures to counter this form of interference in their replication (12,14,15,29,35,36,40,43). Viruses may also exploit the enzyme to achieve selective production of their own proteins (12, 26a). The ubiquity of the enzyme suggests that it may function in normal, uninfected cells (for example, during growth and differentiation events [31]), but this idea remains to be fully substantiated.Exposure of cell extracts containing DAI to dsRNA results in phosphorylation of a protein of -70 ...

Enhancement of the Interferon‐Induced Double‐Stranded RNA‐Dependent Protein Kinase Activity by Sindbis Virus Infection and Heat‐Shock Stress

“…7. P68 was reported to be an IFN-induced protein kinase (HeLa cell counterpart of PK-I) which is autophosphorylated in the presence of dsRNA (7,13). The present results thus indicate that the IFN treatment, Sindbis virus infection, and heat-shock all lead to the dsRNA-dependent autophosphorylation of the common enzyme, PK-I.…”

PKR; a sentinel kinase for cellular stress

“…The ®nding that dsRNA binding causes PKR to elongate rather than contract (Carpick et al, 1997) suggests a mechanism, whereby the dsRBD would swing away from the rest of the protein, exposing the catalytic site. The second-order kinetics of PKR autophosphorylation suggests this reaction is intermolecular (Kostura and Mathews, 1989). In fact dimerization is important for kinase activation (Langland and Jacobs, 1992;Thomis and Samuel, 1993;Romano et al, 1995;Patel et al, 1995;Ortega et al, 1996;Kaufman, 1996, 1997;Carpick et al, 1997;Tan et al, 1998) although exact molecular mechanisms and the protein structural and dsRNA requirements for dimerization, remain to be determined.…”

Unactivated PKR Exists in an Open Conformation Capable of Binding Nucleotides

“…In addition to increasing the accessibility of the active site, dsRNA binding is believed to enhance dimerization of PKR, which in turn facilitates intermolecular autophosphorylation (21). PKR is expressed in a latent form, but upon binding dsRNA it undergoes autophosphorylation at multiple serine and threonine residues (22)(23)(24)(25)(26) as well as tyrosines (27), producing an activated enzyme form.…”

“…PKR is expressed in a latent form, but upon binding dsRNA it undergoes autophosphorylation at multiple serine and threonine residues (22)(23)(24)(25)(26) as well as tyrosines (27), producing an activated enzyme form. Following autophosphorylation, PKR is constitutively active, and is capable of phosphorylating exogenous substrates such as eIF2α in the absence of dsRNA (14,21,28). PKR exists in a weak monomer-dimer equilibrium with K d = 450 μM and dimer stability is enhanced by ~500-fold upon autophosphorylation (29).…”