trans-Autophosphorylation by the isolated kinase domain is not sufficient for dimerization or activation of the dsRNA-activated protein kinase PKR

The double-stranded (ds) RNA-activated protein kinase PKR phosphorylates the alpha-subunit of the eukaryotic initiation factor 2 (eIF2alpha) and inhibits translation initiation. PKR contains two dsRNA binding domains in its amino terminus and a kinase domain in its carboxy terminus. dsRNA binding activates PKR from a latent state by inducing dimerization and trans-autophosphorylation. Recent studies show that PKR is also activated by caspase cleavage to remove the inhibitory dsRNA binding domains. In this report, we show that the isolated kinase domain of PKR is a constitutively active monomeric kinase that has an activity similar to that of wild-type PKR. We used a solid-phase kinase assay system to show that PKR does not transfer its own phosphate to either PKR or eIF2alpha but rather uses the gamma-phosphate from ATP. In addition, the isolated autophosphorylated kinase domain of PKR phosphorylated intact monomeric PKR in trans in a reaction that did not require dsRNA binding. However, this trans-phosphorylation did not occur at the critical Thr446/451 sites and was not sufficient to induce dimerization and/or activation of PKR. The results show that dsRNA binding domains of PKR are not only required for dimerization of PKR but also required for phosphorylation of Thr446/451 sites of PKR. The results imply that even though the isolated kinase domain of PKR phosphorylates intact PKR and eIF2alpha, it is unable to activate PKR.     

Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.     

Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKR: role of dimerization and cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2).

An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the alpha subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein-protein interactions and that trans-autophosphorylation is the final step in the mechanism of activation of PKR.     

Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop

Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.     

Activation of Protein Kinase PKR Requires Dimerization-induced cis-Phosphorylation within the Activation Loop

Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKR^T446∼P-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKR^T446∼P-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization.     

Higher-order substrate recognition of eIF2alpha by the RNA-dependent protein kinase PKR

In response to binding viral double-stranded RNA byproducts within a cell, the RNA-dependent protein kinase PKR phosphorylates the alpha subunit of the translation initiation factor eIF2 on a regulatory site, Ser51. This triggers the general shutdown of protein synthesis and inhibition of viral propagation. To understand the basis for substrate recognition by and the regulation of PKR, we determined X-ray crystal structures of the catalytic domain of PKR in complex with eIF2alpha. The structures reveal that eIF2alpha binds to the C-terminal catalytic lobe while catalytic-domain dimerization is mediated by the N-terminal lobe. In addition to inducing a local unfolding of the Ser51 acceptor site in eIF2alpha, its mode of binding to PKR affords the Ser51 site full access to the catalytic cleft of PKR. The generality and implications of the structural mechanisms uncovered for PKR to the larger family of four human eIF2alpha protein kinases are discussed.     

Conserved intermolecular salt bridge required for activation of protein kinases PKR, GCN2, and PERK

The protein kinases PKR, GCN2, and PERK phosphorylate translation initiation factor eIF2alpha to regulate general and genespecific protein synthesis under various cellular stress conditions. Recent x-ray crystallographic structures of PKR and GCN2 revealed distinct dimeric configurations of the kinase domains. Whereas PKR kinase domains dimerized in a back-to-back and parallel orientation, the GCN2 kinase domains displayed an antiparallel orientation. The dimerization interfaces on PKR and GCN2 were localized to overlapping surfaces on the N-terminal lobes of the kinase domains but utilized different intermolecular contacts. A key feature of the PKR dimerization interface is a salt bridge interaction between Arg(262) from one protomer and Asp(266) from the second protomer. Interestingly, these two residues are conserved in all eIF2alpha kinases, although in the GCN2 structure, the two residues are too remote to interact. To test the importance of this potential salt bridge interaction in PKR, GCN2, and PERK, the residues constituting the salt bridge were mutated either independently or together to residues with the opposite charge. Single mutations of the Asp (or Glu) and Arg residues blocked kinase function both in yeast cells and in vitro. However, for all three kinases, the double mutation designed to restore the salt bridge interaction with opposite polarity resulted in a functional kinase. Thus, the salt bridge interaction and dimer interface observed in the PKR structure is critical for the activity of all three eIF2alpha kinases. These results are consistent with the notion that the PKR structure represents the active state of the eIF2alpha kinase domain, whereas the GCN2 structure may represent an inactive state of the kinase.     

PKR and eIF2alpha: integration of kinase dimerization, activation, and substrate docking

The antiviral RNA-dependent protein kinase, PKR, binds to viral double-stranded RNA in the cell and halts protein synthesis by phosphorylating the alpha subunit of the translation initiation factor eIF2. In this issue of Cell, two complementary papers Dar et al. (2005) and Dey et al. (2005) address the interaction between PKR and eIF2alpha. The structures of eIF2alpha bound to PKR reveal that PKR forms a dimer, the interface of which is essential for kinase activation, and demonstrate how this protein substrate docks to its kinase. The structures, coupled with mutagenesis analysis, also demonstrate how phosphorylation of the activation loop can allosterically couple two distal regions, the dimerization and substrate recognition interfaces.     

Heterologous dimerization domains functionally substitute for the double-stranded RNA binding domains of the kinase PKR.

The protein kinase PKR (dsRNA-dependent protein kinase) phosphorylates the eukaryotic translation initiation factor eIF2alpha to downregulate protein synthesis in virus-infected cells. Two double-stranded RNA binding domains (dsRBDs) in the N-terminal half of PKR are thought to bind the activator double-stranded RNA, mediate dimerization of the protein and target PKR to the ribosome. To investigate further the importance of dimerization for PKR activity, fusion proteins were generated linking the PKR kinase domain to heterologous dimerization domains. Whereas the isolated PKR kinase domain (KD) was non-functional in vivo, expression of a glutathione S-transferase-KD fusion, or co-expression of KD fusions containing the heterodimerization domains of the Xlim-1 and Ldb1 proteins, restored PKR activity in yeast cells. Finally, coumermycin-mediated dimerization of a GyrB-KD fusion protein increased eIF2alpha phosphorylation and inhibited reporter gene translation in mammalian cells. These results demonstrate the critical importance of dimerization for PKR activity in vivo, and suggest that a primary function of double-stranded RNA binding to the dsRBDs of native PKR is to promote dimerization and activation of the kinase domain.     

Mechanism of PKR activation: dimerization and kinase activation in the absence of double-stranded RNA

The kinase PKR is a central component of the interferon antiviral pathway. PKR is activated upon binding double-stranded (ds) RNA to undergo autophosphorylation. Although PKR is known to dimerize, the relationship between dimerization and activation remains unclear. Here, we directly characterize dimerization of PKR in free solution using analytical ultracentrifugation and correlate self-association with autophosphorylation activity. Latent, unphosphorylated PKR exists predominantly as a monomer at protein concentrations below 2 mg/ml. A monomer sedimentation coefficient of s(20,w)(0)=3.58 S and a frictional ratio of f/f(0)=1.62 indicate an asymmetric shape. Sedimentation equilibrium measurements indicate that PKR undergoes a weak, reversible monomer-dimer equilibrium with K(d)=450 microM. This dimerization reaction serves to initiate a previously unrecognized dsRNA-independent autophosphorylation reaction. The resulting activated enzyme is phosphorylated on the two critical threonine residues present in the activation loop and is competent to phosphorylate the physiological substrate, eIF2alpha. Dimer stability is enhanced by approximately 500-fold upon autophosphorylation. We propose a chain reaction model for PKR dsRNA-independent activation where dimerization of latent enzyme followed by intermolecular phosphorylation serves as the initiation step. Subsequent propagation steps likely involve phosphorylation of latent PKR monomers by activated enzyme within high-affinity heterodimers. Our results support a model whereby dsRNA functions by bringing PKR monomers into close proximity in a manner that is analogous to the dimerization of free PKR.     

Regulation of the protein kinase PKR by the vaccinia virus pseudosubstrate inhibitor K3L is dependent on residues conserved between the K3L protein and the PKR substrate eIF2alpha.

The mammalian double-stranded RNA-activated protein kinase PKR is a component of the cellular antiviral defense mechanism and phosphorylates Ser-51 on the alpha subunit of the translation factor eIF2 to inhibit protein synthesis. To identify the molecular determinants that specify substrate recognition by PKR, we performed a mutational analysis on the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. High-level expression of PKR is lethal in the yeast Saccharomyces cerevisiae because PKR phosphorylates eIF2alpha and inhibits protein synthesis. We show that coexpression of vaccinia virus K3L can suppress the growth-inhibitory effects of PKR in yeast, and using this system, we identified both loss-of-function and hyperactivating mutations in K3L. Truncation of, or point mutations within, the C-terminal portion of the K3L protein, homologous to residues 79 to 83 in eIF2alpha, abolished PKR inhibitory activity, whereas the hyperactivating mutation, K3L-H47R, increased the homology between the K3L protein and eIF2alpha adjacent to the phosphorylation site at Ser-51. Biochemical and yeast two-hybrid analyses revealed that the suppressor phenotype of the K3L mutations correlated with the affinity of the K3L protein for PKR and was inversely related to the level of eIF2alpha phosphorylation in the cell. These results support the idea that residues conserved between the pseudosubstrate K3L protein and the authentic substrate eIF2alpha play an important role in substrate recognition, and they suggest that PKR utilizes sequences both near and over 30 residues from the site of phosphorylation for substrate recognition. Finally, by reconstituting part of the mammalian antiviral defense mechanism in yeast, we have established a genetically useful system to study viral regulators of PKR.     

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.     

Expression of unphosphorylated form of human double-stranded RNA-activated protein kinase in Escherichia coli

Interferon (IFN)-inducible, double-stranded (dsRNA)-activated protein kinase (PKR) is a key mediator of the antiviral and antiproliferative effects of IFN. PKR is present within cells in a latent state. In response to binding dsRNA, the enzyme becomes activated, causing autophosphorylation and an increase in specific kinase activity. In order to study PKR and its inhibitors, a large amount of the enzyme in its latent, unphosphorylated state is required. When PKR is fused to glutathione S-transferase (GST-PKR) and the fusion protein is expressed in Escherichia coli, the PKR obtained is fully activated by autophosphorylation. Therefore, we have developed an expression plasmid in which both GST-PKR and bacteriophage lambda protein phosphatase (lambda-PPase) genes were placed downstream of a T7 promoter. After induction of expression, unphosphorylated GST-PKR was obtained in good yield, and purified to near homogeneity. The purified enzyme has dsRNA-dependent activation and phosphorylates the translation initiation factor eIF2 alpha. Using the recombinant protein, we analyzed the inhibition mechanisms of two viral inhibitors, vaccinia virus K3L protein and adenovirus virus-associated RNA I (VAI RNA). K3L inhibited both autophosphorylation of PKR and phosphorylation of eIF2 alpha, whereas VAI RNA inhibited only autophosphorylation. The separation of autophosphorylation and catalytic activity shows that the recombinant PKR is useful in analyzing the functions of PKR, its inhibitors, and its regulatory molecules. The coexpression system of protein kinase with lambda-PPase described here will be applicable to obtaining unphosphorylated and unactivated forms of other protein kinases.     

Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons

Thiamine (vitamin B1) deficiency (TD) causes mild and chronic impairment of oxidative metabolism and induces neuronal death in specific brain regions. The mechanisms underlying TD-induced cell death, however, remain unclear. The double-stranded RNA-activated protein kinase (PKR), has been well known for its anti-viral function. Upon activation by viral infection or double-stranded RNA, PKR phosphorylates its substrate, the α-subunit of eukaryotic initiation factor-2 (eIF2α), leading to inhibition of translation. In response to various cellular stresses, PKR can also be stimulated by its protein activators, or its mouse homologue, PKR activator (RAX). We demonstrated that TD in mice induced phosphorylation of PKR at Thr446 and Thr451 and phosphorylation of eIF2α at Ser51 in the cerebellum and the thalamus. TD caused phosphorylation of PKR and eIF2α, as well as nuclear translocation of PKR in primary cultures of cerebellar granule neurons. PKR phosphorylation is necessary for its nuclear translocation because TD failed to induce nuclear translocation of a T446A/T451A PKR mutant. Both PKR inhibitor and dominant-negative PKR mutant protected cerebellar granule neurons against TD-induced cell death. TD promoted the association between RAX and PKR. Antioxidant vitamin E dramatically decreased the RAX/PKR association and ameliorated TD-induced cell death. Our results indicate that TD-induced neuronal death is at least partially mediated by the activation of PKR.     

The interferon-induced double-stranded RNA-activated protein kinase PKR will phosphorylate serine, threonine, or tyrosine at residue 51 in eukaryotic initiation factor 2alpha.

The family of eukaryotic initiation factor 2alpha (eIF2alpha) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.     

Phosphorylation of the RNA-dependent protein kinase regulates its RNA-binding activity

The RNA-dependent protein kinase (PKR) is an interferon-induced, RNA-activated enzyme that phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), inhibiting the function of the eIF2 complex and continued initiation of translation. When bound to an activating RNA and ATP, PKR undergoes autophosphorylation reactions at multiple serine and threonine residues. This autophosphorylation reaction stimulates the eIF2α kinase activity of PKR. The binding of certain viral RNAs inhibits the activation of PKR. Wild-type PKR is obtained as a highly phosphorylated protein when overexpressed in Escherichia coli. We report here that treatment of the isolated phosphoprotein with the catalytic subunit of protein phosphatase 1 dephosphorylates the enzyme. The in vitro autophosphorylation and eIF2α kinase activities of the dephosphorylated enzyme are stimulated by addition of RNA. Thus, inactivation by phosphatase treatment of autophosphorylated PKR obtained from overexpression in bacteria generates PKR in a form suitable for in vitro analysis of the RNA-induced activation mechanism. Furthermore, we used gel mobility shift assays, methidiumpropyl-EDTA·Fe footprinting and affinity chromatography to demonstrate differences in the RNA-binding properties of phospho- and dephosphoPKR. We found that dephosphorylation of PKR increases binding affinity of the enzyme for both kinase activating and inhibiting RNAs. These results are consistent with an activation mechanism that includes release of the activating RNA upon autophosphorylation of PKR prior to phosphorylation of eIF2α.     

PKR and GCN2 kinases and guanine nucleotide exchange factor eukaryotic translation initiation factor 2B (eIF2B) recognize overlapping surfaces on eIF2alpha

Four stress-responsive protein kinases, including GCN2 and PKR, phosphorylate eukaryotic translation initiation factor 2alpha (eIF2alpha) on Ser51 to regulate general and gene-specific protein synthesis. Phosphorylated eIF2 is an inhibitor of its guanine nucleotide exchange factor, eIF2B. Mutations that block translational regulation were isolated throughout the N-terminal OB-fold domain in Saccharomyces cerevisiae eIF2alpha, including those at residues flanking Ser51 and around 20 A away in the conserved motif K79GYID83. Any mutation at Glu49 or Asp83 blocked translational regulation; however, only a subset of these mutations impaired Ser51 phosphorylation. Substitution of Ala for Asp83 eliminated phosphorylation by GCN2 and PKR both in vivo and in vitro, establishing the critical contributions of remote residues to kinase-substrate recognition. In contrast, mutations that blocked translational regulation but not Ser51 phosphorylation impaired the binding of eIF2B to phosphorylated eIF2alpha. Thus, two structurally distinct effectors of eIF2 function, eIF2alpha kinases and eIF2B, have evolved to recognize the same surface and overlapping determinants on eIF2alpha.     

Autophosphorylation of threonine 485 in the activation loop is essential for attaining eIF2alpha kinase activity of HRI

In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.     

Activation of a GST-tagged AKT2/PKBbeta

The protein kinase AKT is a key regulator for cell growth, cell survival and metabolic insulin action. However, the mechanism of activation of AKT in vivo, which presumably involves membrane recruitment of the kinase, oligomerization, and multiple phosphorylation events, is not fully understood. In the present study, we have expressed and purified dimeric GST-fusion proteins of human protein kinase AKT2 (DeltaPH-AKT2) in milligram quantities via the baculovirus expression system. Treatment of virus-infected insect cells with the phosphatase inhibitor okadaic acid (OA) led to phosphorylation of the two regulatory phosphorylation sites, Thr309 and Ser474, and to activation of the kinase. Likewise, phosphorylation of Thr309 in vitro by recombinant PDK1 or mutation of Thr309 and Ser474 to acidic residues rendered the kinase constitutively active. However, even though the specific activity of our AKT2 was increased 15-fold compared to previous reports, GST-mediated dimerization alone did not lead to an activation of the kinase. Whereas both mutagenesis and phosphorylation led to an increase in the turnover number of the enzyme, only the latter resulted in a marked reduction (20-fold) of the apparent Km value for the exogenous substrate Crosstide, indicating that this widely used mutagenesis only partially mimics phosphorylation. Kinetic analysis of GST-AKT2 demonstrates that phosphorylation of Thr309 in the activation loop of the kinase is largely responsible for the observed reduction in Km and for a subsequent 150-fold increase in the catalytic efficiency (k(cat)/Km) of the enzyme. Highly active AKT2 constructs were used in autophosphorylation reactions in vitro, where inactive AKT2 kinases served as substrates. As a matter of fact, we found evidence for a minor autophosphorylation activity of AKT2 but no significant autophosphorylation of any of the two regulatory sites, Thr309 or Ser474.