Characterization and regulation of the 58,000-dalton cellular inhibitor of the interferon-induced, dsRNA-activated protein kinase.

The P68 protein kinase is a serine/threonine kinase induced by interferon treatment and activated by double-stranded RNAs (dsRNAs). Once activated, the kinase phosphorylates its natural substrate, the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently purified from influenza virus-infected cells a P68 kinase inhibitor, found to be a 58-kDa cellular protein. We have now investigated the mechanisms by which the 58-kDa inhibitor regulates P68 kinase activity and how the inhibitor itself is controlled. The 58-kDa inhibitor did not function by degrading or sequestering the dsRNA activator of P68 but could repress phosphorylation of eIF-2 alpha by an already activated protein kinase. Utilizing antibody prepared against a 58-kDa-specific peptide, we showed that the 58-kDa proteins from infected and uninfected cells were present in equivalent amounts. Although kinase inhibitory activity could not be detected in crude uninfected cell extracts, ammonium sulfate treatment unmasked this activity and allowed purification of the cellular inhibitor with identical chromatographic properties as that from influenza virus-infected cells. Finally, we have identified and partially purified a specific inhibitor of the 58-kDa protein which we refer to as an "anti-inhibitor." Based on these data, we present a model depicting the complex regulation of the interferon-induced protein kinase in eukaryotic cells.     

Differential effects of two interferon-induced translational inhibitors on initiation of protein synthesis.

At least two different mechanisms for the inhibition of mRNA translation operate in extracts of interferon-treated L cells. One is mediated by an interferon-induced protein kinase which, when activated by double-stranded RNA and ATP, phosphorylates the small subunit of initiation factor eIF-2. Addition of the purified interferon-induced protein kinase to L cell extracts, strongly reduces the amount of methionyl-tRNA bound to 40-S ribosomal subunits. The second translational inhibition is due to the synthesis of (2'-5')oligo(adenylate) by interferon-induced enzyme E. The oligonucleotide in turn activates a ribonuclease F constitutively present in L cells. Addition of the purified nuclease with its oligonucleotide activator to L cell extracts produces a strong decrease in polyribosome formation and an accumulation of initiation complex. These experiments differentiate the effects of the two interferon-induced inhibitors on mRNA translation.     

Characterization of a novel serine/threonine kinase associated with nuclear bodies

A novel protein kinase, Mx-interacting protein kinase (PKM), has been identified in a yeast two-hybrid screen for interaction partners of human MxA, an interferon-induced GTPase with antiviral activity against several RNA viruses. A highly conserved protein kinase domain is present in the N-terminal moiety of PKM, whereas an Mx interaction domain overlaps with C-terminal PEST sequences. PKM has a molecular weight of about 127,000 and exhibits high sequence homology to members of a recently described family of homeodomain-interacting protein kinases. Recombinant PKM has serine/threonine kinase activity that is abolished by a single amino acid substitution in the ATP binding domain (K221W). PKM catalyzes autophosphorylation and phosphorylation of various cellular and viral proteins. PKM is expressed constitutively and colocalizes with the interferon-inducible Sp100 protein and murine Mx1 in discrete nuclear structures known as nuclear bodies.     

Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2 alpha kinase

The VAI RNA of adenovirus is a small, RNA polymerase III-transcribed species required for efficient translation of host cell and viral mRNAs late after infection. The growth of a viral mutant that is unable to produce the RNA is inhibited by interferon, while wild-type virus is not affected. VAI RNA prevents activation of the interferon-induced P1/eIF-2 alpha kinase. This inhibition can be reproduced in extracts of interferon-treated cells where purified VAI RNA prevents activation of latent kinase by double-stranded RNA.     

Heat-shock-induced denaturation of proteins. Characterization of the insolubilization of the interferon-induced p68 kinase.

Heat-shock stress causes inactivation and aggregation of various cellular proteins which become further insoluble. Previous studies have shown that the interferon-induced p68 kinase activity was greatly reduced in extracts of heat-shocked HeLa cells, and that the loss of activity was due to a decreased solubility of the enzyme. Here we show that the p68 kinase which is normally evenly distributed in the cytoplasm, aggregates as a thick ring around the nucleus in heat-shocked cells. The 70-kDa constitutive heat-shock proteins are major insolubilized proteins during stress and we find them to colocalize with the p68 kinase after stress. Treatments of cells with drugs which disrupt the cytoskeleton, such as colcemid and cytochalasin E, do not hinder the enzyme insolubilization during heat-shock. On the contrary, heat-protectors such as glycerol and deuterium oxide (D2O) keep the p68 kinase under a soluble and active form during heat-shock stress. Similarly, an attenuation of the insolubilization of this enzyme is observed in cells rendered thermo-tolerant by a previous heat-shock, suggesting that heat-shock proteins may also contribute to the protection. During the recovery period at normal temperature after heat-shock, resolubilization occurs and most of the enzyme is again recovered under an active soluble form.     

Activation of double-stranded RNA-activated protein kinase in HeLa cells after poliovirus infection does not result in increased phosphorylation of eucaryotic initiation factor-2.

Protein kinase activity in general is stimulated at least 5- to 10-fold in ribosomal salt wash preparations from poliovirus-infected HeLa cells compared with those from mock-infected cells. The stimulation of kinase activity is manifested by increased phosphorylation of ribosome-associated polypeptides having approximate molecular weights of 135,000, 120,000, 85,000, 68,000, 65,000, 40,000, 28,000, 25,000, and 21,000. The Mr 68,000 phosphoprotein is structurally identical to the interferon-induced, double-stranded RNA-activated protein kinase (P1) which phosphorylates the alpha subunit of eucaryotic initiation factor-2 (eIF-2). A similar protein of Mr 68,000 is more phosphorylated in poliovirus-infected cells than in mock-infected cells. Increased phosphorylation of P1 protein in poliovirus-infected cells, however, does not result in an increased phosphorylation of the alpha subunit of endogenous or exogenously added eIF-2, both in vitro and in vivo. These results suggest that a mechanism must exist in poliovirus-infected HeLa cells which prevents further phosphorylation of eIF-2 by the activated kinase.     

Activation of the purified protein tyrosine kinase domain of the epidermal growth factor receptor

Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.     

Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.     

Ethoxylated alcohol (Neodol-12) and other surfactants in the assay of protein kinase C

Most commonly used surfactants were found to be inhibitors of partially purified rat brain protein kinase C at or above their critical micellar concentrations (CMC). These include sodium lauryl sulfate, deoxycholate, octyl glucoside, dodecyl trimethylammonium bromide, linear alkylbenzene sulfonate and Triton X-100. Several detergents, including the nonionic surfactants digitonin and Neodol-12 (ethoxylated alcohol), did not inhibit protein kinase C activity, even at concentrations greater than their CMC, while the anionic surfactant, AEOS-12 (ethoxylated alcohol sulfate), inhibited enzyme activity only slightly (less than 8%). Since these latter surfactants have little or no inhibitory effect on protein kinase C, they may be of value in solubilizing cells and tissues for the determination of enzyme activity in crude extracts. Among the detergents tested, sodium lauryl sulfate and linear alkylbenzene sulfonate significantly stimulated protein kinase C activity in the absence of phosphatidylserine and calcium. This was found to be dependent on the presence of histone in the protein kinase C assay. These detergents failed to stimulate protein kinase C activity when endogenous proteins in the partially purified rat brain extracts were used as the substrate. Our results indicate that activity of protein kinase C can be modified by the conditions of the assay and by the detergents used to extract the enzyme.     

Constitutive expression of human double-stranded RNA-activated p68 kinase in murine cells mediates phosphorylation of eukaryotic initiation factor 2 and partial resistance to encephalomyocarditis virus growth.

The cDNA encoding interferon-induced human double-stranded RNA-activated p68 kinase was expressed in murine NIH 3T3 cells by using the pcDNA1/neo vector. Several stable clones were selected which expressed either the wild-type kinase or an inactive mutant possessing a single amino acid substitution in the invariant lysine 296 in the catalytic domain II. The transfected wild-type kinase showed properties similar to those of the natural kinase, such as subcellular ribosomal localization and dependence on double-stranded RNA for autophosphorylation. Upon infection with encephalomyocarditis virus (EMCV), wild-type- but not mutant-expressing clones were found to partially resist virus growth. Such natural antiviral activity was virus specific, since no inhibition was observed in the case of vesicular stomatitis virus infection. In accord with EMCV inhibition, the wild-type p68 kinase was found to be highly phosphorylated during infection. Furthermore, its natural substrate, the small subunit of protein synthesis initiation factor eIF2, was phosphorylated. These results demonstrate that p68 kinase is activated during EMCV infection, leading to reduced virus production.     

Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR.

The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R) was a substrate for the purified wild-type human PKR kinase. This intermolecular phosphorylation of mutant PKR(K296R) by wild-type PKR was dependent on double-stranded RNA and was inhibited by 2-aminopurine. Finally, PKR mRNA was capable of mediating an autoactivation of wild-type PKR kinase autophosphorylation in vitro.     

Inhibitory sequences in the N-terminus of the double-stranded-RNA-dependent protein kinase, PKR, are important for regulating phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha).

During viral infection, phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) by the interferon-induced RNA-dependent protein kinase, PKR, leads to inhibition of translation initiation and viral proliferation. Activation of PKR is mediated by association of virally encoded double-stranded RNAs (dsRNAs) with two dsRNA binding domains (dsRBDs) located in the N-terminus of PKR. To better understand the molecular mechanisms regulating PKR, we characterized the activities of wild-type and mutant versions of human PKR expressed and purified from yeast. The catalytic rate of eIF2alpha phosphorylation by our purified PKR was increased in response to dsRNA, but not single-stranded RNA or DNA, consistent with the properties previously described for PKR purified from mammalian sources. While both dsRBD1 and dsRBD2 were required for activation of PKR by dsRNA, only deletion of dsRBD1 severely reduced the basal eIF2alpha kinase activity. Removal of as few as 25 residues at the C-terminal junction of dsRBD2 dramatically increased eIF2alpha kinase activity and characterization of larger deletions that included dsRBD1 demonstrated that removal of these negative-acting sequences could bypass the dsRBD1 requirement for in vitro phosphorylation of eIF2alpha. Heparin, a known in vitro activator of PKR, enhanced eIF2alpha phosphorylation by PKR mutants lacking their entire N-terminal sequences, including the dsRBDs. The results indicate that induction of PKR activity is mediated by multiple mechanisms, one of which involves release of inhibition by negative-acting sequences in PKR.     

Oligomeric structure and catalytic activity of G type casein kinase. Isolation of the two subunits and renaturation experiments

Casein kinase G purified from bovine tissue is an oligomeric cyclic nucleotide-independent protein kinase made of two different monomers, namely an alpha (Mr = 38 kilodaltons) and a self-phosphorylatable beta (Mr = 27 kilodaltons) subunit. Treatment of the native enzyme under denaturing conditions (0.5 M NaCl, 4 M LiCl, and 20 to 35% formamide) resulted in a progressive selective removal of the beta subunit following gel filtration and a correlated loss of activity of the corresponding renatured enzyme. Mild digestion with papain resulted in a proteolytic alteration limited to the beta monomer with a concomitant partial loss of the enzyme activity. Isolation of the alpha and beta casein kinase G subunits was achieved by preparative reversed polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Renaturation of the proteins following sodium dodecyl sulfate removal by acetone and/or Triton X-100 treatment allowed reconstitution of a functional casein kinase G. Whereas the isolated alpha subunit was found to exhibit a weak catalytic activity, addition of the beta subunit was required for recovery of a maximal casein kinase activity. The process was dose-dependent and reached a plateau for an alpha:beta subunit molar ratio of approximately 1 to 1. These data suggest that while the casein kinase G alpha subunit bears the catalytic site, stoichiometric combination with the beta subunit is required for optimal enzymatic activity. A possible role of the beta subunit as a regulatory component of casein kinase G activity in the intact cell remains to be examined.     

Identification of a protein kinase activity in purified foot- and-mouth disease virus.

Purified preparations of foot-and-mouth disease virus types A, O, and C contain a protein kinase activity which can transfer the gamma phosphate of [32P]ATP to virion structural proteins VP2 and VP3 and exogenous acceptor proteins. Utilizing protamine sulfate as an acceptor, the kinase activity can be demonstrated in disrupted virus but not in intact virus. The enzyme is heat labile with optimal activity at pH 7 or greater. Serine residues of protamine sulfate were identified as the amino acid phosphorylated by the protein kinase. Treatment of purified virus with trypsin, which cleaves VP3, did not affect the protein kinase activity. The results indicate that the protein kinase activity found in FMDV is present in an internally located protein of viral or host origin.     

Control of sulfur partitioning between primary and secondary metabolism

Sulfur is an essential nutrient for all organisms. Plants take up most sulfur as inorganic sulfate, reduce it and incorporate it into cysteine during primary sulfate assimilation. However, some of the sulfate is partitioned into the secondary metabolism to synthesize a variety of sulfated compounds. The two pathways of sulfate utilization branch after activation of sulfate to adenosine 5'-phosphosulfate (APS). Recently we showed that the enzyme APS kinase limits the availability of activated sulfate for the synthesis of sulfated secondary compounds in Arabidopsis. To further dissect the control of sulfur partitioning between the primary and secondary metabolism, we analysed plants in which activities of enzymes that use APS as a substrate were increased or reduced. Reduction in APS kinase activity led to reduced levels of glucosinolates as a major class of sulfated secondary metabolites and an increased concentration of thiols, products of primary reduction. However, over-expression of this gene does not affect the levels of glucosinolates. Over-expression of APS reductase had no effect on glucosinolate levels but did increase thiol levels, but neither glucosinolate nor thiol levels were affected in mutants lacking the APR2 isoform of this enzyme. Measuring the flux through sulfate assimilation using [(35) S]sulfate confirmed the larger flow of sulfur to primary assimilation when APS kinase activity was reduced. Thus, at least in Arabidopsis, the interplay between APS reductase and APS kinase is important for sulfur partitioning between the primary and secondary metabolism.     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Double-Stranded RNA-Dependent Protein Kinase Regulates the Motility of Breast Cancer Cells

Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.     

Partial characterization of a cellular factor that regulates the double-stranded RNA-dependent eIF-2 alpha kinase in 3T3-F442A fibroblasts.

The interferon-induced double-stranded RNA-dependent eIF-2 alpha kinase (dsI) has an established role in mediating part of interferon's antiviral effects. Numerous studies have suggested that dsI also has regulatory functions in cells not infected with virus. Our previous results have indicated that the activation of this kinase may be an important regulatory signal in controlling growth arrest of mouse 3T3-F442A fibroblasts prior to their subsequent differentiation to adipocytes. Here, we report that extracts from 3T3-F442A cells cultured under conditions nonpermissive for differentiation exhibit significantly reduced dsI activity and that this reduction is due, at least in part, to the presence of elevated levels of a novel inhibitor of dsI activation (dRF). This inhibitor is also detected in reduced amounts in extracts from cells cultured under conditions which are permissive for differentiation. We have achieved a 1,000-fold purification of dRF activity, and highly purified dRF preparations were found to be greatly enriched for a 15-kDa protein that was greater than 90% pure. Our results indicate that dRF is not a protein phosphatase or protease but a reversible inhibitor of dsI autophosphorylation. In addition, our results imply that dRF is a physiologic regulator of dsI, since dRF activity correlates with the ability of 3T3-F442A cells to undergo adipose conversion.