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.     

Inhibition of the protein kinase PKR by the internal ribosome entry site of hepatitis C virus genomic RNA

Translation of the hepatitis C genome is mediated by internal ribosome entry on the structurally complex 5' untranslated region of the large viral RNA. Initiation of protein synthesis by this mechanism is independent of the cap-binding factor eIF4E, but activity of the initiator Met-tRNA(f)-binding factor eIF2 is still required. HCV protein synthesis is thus potentially sensitive to the inhibition of eIF2 activity that can result from the phosphorylation of the latter by the interferon-inducible, double-stranded RNA-activated protein kinase PKR. Two virally encoded proteins, NS5A and E2, have been shown to reduce this inhibitory effect of PKR by impairing the activation of the kinase. Here we present evidence for a third viral strategy for PKR inhibition. A region of the viral RNA comprising part of the internal ribosome entry site (IRES) is able to bind to PKR in competition with double-stranded RNA and can prevent autophosphorylation and activation of the kinase in vitro. The HCV IRES itself has no PKR-activating ability. Consistent with these findings, cotransfection experiments employing a bicistronic reporter construct and wild-type PKR indicate that expression of the protein kinase is less inhibitory towards HCV IRES-driven protein synthesis than towards cap-dependent protein synthesis. These data suggest a dual function for the viral IRES, with both a structural role in promoting initiation complex formation and a regulatory role in preventing inhibition of initiation by PKR.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

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.     

Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase.

The interferon-inducible, RNA-dependent protein kinase (PKR) is activated by autophosphorylation, a process mediated by double-stranded RNA. A catalytically deficient, histidine-tagged mutant PKR protein [His-PKR(K296R)] was used as the substrate for characterization of the intermolecular phosphorylation catalyzed by purified wild-type PKR [PKR(Wt)]. The intermolecular autophosphorylation of His-PKR(K296R) by PKR(Wt) was RNA dependent. Excess His-PKR(K296R) substrate inhibited both the auto- and the trans-phosphorylation activities of PKR(Wt). Inhibition of PKR(Wt) by His-PKR(K296R) was relieved by higher concentrations of activator double-stranded RNA. Phosphopeptide analysis revealed that the sites of intermolecular autophosphorylation in His-PKR(K296R) were very similar, if not identical, to the sites that were autophosphorylated in PKR(Wt) and suggest a multiple of four major phosphorylation sites per PKR molecule.     

Autophosphorylation of carboxy‐terminal residues inhibits the activity of protein kinase CK1α

CK1 constitutes a protein kinase subfamily that is involved in many important physiological processes. However, there is limited knowledge about mechanisms that regulate their activity. Isoforms CK1δ and CK1ε were previously shown to autophosphorylate carboxy‐terminal sites, a process which effectively inhibits their catalytic activity. Mass spectrometry of CK1α and splice variant CK1αL has identified the autophosphorylation of the last four carboxyl‐end serines and threonines and also for CK1αS, the same four residues plus threonine‐327 and serine‐332 of the S insert. Autophosphorylation occurs while the recombinant proteins are expressed in Escherichia coli. Mutation of four carboxy‐terminal phosphorylation sites of CK1α to alanine demonstrates that these residues are the principal but not unique sites of autophosphorylation. Treatment of autophosphorylated CK1α and CK1αS with λ phosphatase causes an activation of 80–100% and 300%, respectively. Similar treatment fails to stimulate the CK1α mutants lacking autophosphorylation sites. Incubation of dephosphorylated enzymes with ATP to allow renewed autophosphorylation causes significant inhibition of CK1α and CK1αS. The substrate for these studies was a synthetic canonical peptide for CK1 (RRKDLHDDEEDEAMS*ITA). The stimulation of activity seen upon dephosphorylation of CK1α and CK1αS was also observed using the known CK1 protein substrates DARPP‐32, β‐catenin, and CK2β, which have different CK1 recognition sequences. Autophosphorylation effects on CK1α activity are not due to changes in Km_app for ATP or for peptide substrate but rather to the catalytic efficiency per pmol of enzyme. This work demonstrates that CK1α and its splice variants can be regulated by their autophosphorylation status. J. Cell. Biochem. 106: 399–408, 2009. © 2008 Wiley‐Liss, Inc.     

Phosphorylation of the beta subunit of casein kinase II in human A431 cells. Identification of the autophosphorylation site and a site phosphorylated by p34cdc2

To examine the phosphorylation of casein kinase II in cells, the enzyme was isolated by immunoprecipitation from metabolically labeled human epidermal carcinoma A431 cells using polyclonal antipeptide antibodies specific for either the alpha subunit or the beta subunit of the enzyme. When isolated from 32P-labeled cells, the beta subunit was found to be significantly labeled on serine residues whereas only minimal labeling was associated with the alpha subunit. In vitro, the beta subunit of purified bovine casein kinase II was autophosphorylated, also on serine residues. Cleavage of the beta subunit, that had been autophosphorylated in vitro, at tryptophan 9 and tryptophan 12 using N-chlorosuccinimide demonstrated that the autophosphorylation site is located near the amino terminus of the protein, most likely at serine 2 and serine 3. Two-dimensional maps of phosphopeptides generated by digestion of the beta subunit with endoproteinase Glu-C indicted that the majority of the phosphate that was incorporated into the protein in cells was at sites that were indistinguishable from the sites that were autophosphorylated in vitro. In addition to phosphorylation at the autophosphorylation site, the beta subunit is also phosphorylated at an additional site, serine 209, in intact cells. This residue, which is near the carboxyl terminus of the protein, can be phosphorylated in vitro by p34cdc2.     

Regulation of Yersinia Protein Kinase A (YpkA) Kinase Activity by Multisite Autophosphorylation and Identification of an N-terminal Substrate-binding Domain in YpkA

The serine/threonine protein kinase YpkA is an essential virulence factor produced by pathogenic Yersinia species. YpkA is delivered into host mammalian cells via a type III secretion system and localizes to the inner side of the plasma membrane. We have previously shown that YpkA binds to and phosphorylates the α subunit of the heterotrimeric G protein complex, Gαq, resulting in inhibition of Gαq signaling. To identify residues in YpkA involved in substrate binding activity we generated GFP-YpkA N-terminal deletion mutants and performed coimmunoprecipitation experiments. We located a substrate-binding domain on amino acids 40–49 of YpkA, which lies within the previously identified membrane localization domain on YpkA. Deletion of amino acids 40–49 on YpkA interfered with substrate binding, substrate phosphorylation and substrate inhibition. Autophosphorylation regulates the kinase activity of YpkA. To dissect the mechanism by which YpkA transmits signals, we performed nano liquid chromatography coupled to tandem mass spectrometry to map in vivo phosphorylation sites. Multiple serine phosphorylation sites were identified in the secretion/translocation region, kinase domain, and C-terminal region of YpkA. Using site-directed mutagenesis we generated multiple YpkA constructs harboring specific serine to alanine point mutations. Our results demonstrate that multiple autophosphorylation sites within the N terminus regulate YpkA kinase activation, whereas mutation of serine to alanine within the C terminus of YpkA had no effect on kinase activity. YpkA autophosphorylation on multiple sites may be a strategy used by pathogenic Yersinia to prevent inactivation of this important virulence protein by host proteins.     

Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.     

Inhibitory autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase analyzed by site-directed mutagenesis.

Initial autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) occurs at Thr286 (the "autonomy" site) and converts the kinase from a Ca(2+)-dependent to a partially Ca(2+)-independent or autonomous enzyme. After removal of Ca2+/calmodulin, the autonomous kinase undergoes a "burst" of inhibitory autophosphorylation at sites distinct from the autonomy site which may be masked in the presence of bound calmodulin. This burst of Ca(2+)-independent autophosphorylation blocks the ability of calmodulin to activate the kinase. We have used site-directed mutagenesis to replace putative inhibitory autophosphorylation sites within the calmodulin binding domain of recombinant alpha-CaM kinase with nonphosphorylatable alanines and examined the effects on autophosphorylation, kinase activity, and calmodulin binding. Although prominent Ca(2+)-independent autophosphorylation occurs within the calmodulin binding domain at Thr305, Thr306, and Ser314 in wild-type alpha-CaM kinase, the inhibitory effect on kinase activity and calmodulin binding is retained in mutants lacking any one of these three sites. However, when both Thr305 and Thr306 are converted to alanines the kinase does not display inhibition of either activity or calmodulin binding. Autophosphorylation at either Thr305 or Thr306 is therefore sufficient to block both binding and activation of the kinase by Ca2+/calmodulin. Thr306 is also slowly autophosphorylated in a basal reaction in the continuous absence of Ca2+/calmodulin. Autophosphorylation of Thr306 by the kinase in either its basal or autonomous state suggests that in the absence of bound calmodulin, the region of the autoregulatory domain surrounding Thr306, rather than the region near the autonomy site, lies nearest the peptide substrate binding site of the kinase.     

Autophosphorylation: a salient feature of protein kinases

Most protein kinases catalyze autophosphorylation, a process which is generally intramolecular and is modulated by regulatory ligands. Either serine/threonine or tyrosine serves as the phosphoacceptor, and several sites on the same kinase subunit are usually autophosphorylated. Autophosphorylation affects the functional properties of most protein kinases. Members of the protein kinase family exhibit diversity in the characteristics and functions of autophosphorylation, but certain common themes are emerging.     

Yersinia protein kinase YopO is activated by a novel G-actin binding process

Pathogenic bacteria of the genus Yersinia employ a type III secretion system to inject effector proteins (Yops) into host cells. The Yops down-regulate host cell functions through unique biochemical activities. YopO, a serine/threonine kinase required for Yersinia virulence, is activated by host cell actin via an unknown process. Here we show that YopO kinase is activated by formation of a 1:1 complex with monomeric (G) actin but is unresponsive to filamentous (F) actin. Two separate G-actin binding sites, one in the N-terminal kinase region (amino acids 89-440) and one in the C-terminal guanine nucleotide dissociation inhibitor-like region (amino acids 441-729) of YopO, were identified. Actin binding to both of these sites was necessary for effective autophosphorylation of YopO on amino acids Ser-90 and Ser-95. A S90A/S95A YopO mutant was strongly reduced in substrate phosphorylation, suggesting that autophosphorylation activates YopO kinase activity. In cells the kinase activity of YopO regulated rounding/arborization and was specifically required for inhibition of Yersinia YadA-dependent phagocytosis. Thus, YopO kinase is activated by a novel G-actin binding process, and this appears to be crucial for its anti-host cell functions.     

Substrate recognition determinants for rhodopsin kinase: studies with synthetic peptides, polyanions, and polycations

Rhodopsin kinase phosphorylates serine- and threonine-containing peptides from bovine rhodopsin's carboxyl-terminal sequence. Km's for the peptides decrease as the length of the peptide is increased over the range 12-31 amino acids, reaching 1.7 mM for peptide 318-348 from the rhodopsin sequence. The Km for phosphorylation of rhodopsin is about 10(3) lower than that for the peptides, which suggests that binding of rhodopsin kinase to its substrate, photolyzed rhodopsin, involves more than just binding to the carboxyl-terminal peptide region that is to be phosphorylated. A synthetic peptide from the rhodopsin sequence that contains both serines and threonines is improved as a substrate by substitution of serines for the threonines, suggesting that serine residues are preferred as substrates. Analogous 25 amino acid peptides from the human red or green cone visual pigment, a beta-adrenergic receptor, or M1 muscarinic acetylcholine receptors are better substrates for bovine rhodopsin kinase than is the peptide from bovine rhodopsin. An acidic serine-containing peptide from a non-receptor protein, alpha s1B-casein, is also a good substrate for rhodopsin kinase. However, many basic peptides that are substrates for other protein kinases--histone IIA, histone IIS, clupeine, salmine, and a neurofilament peptide--are not phosphorylated by rhodopsin kinase. Polycations such as spermine or spermidine are nonessential activators of phosphorylation of rhodopsin or its synthetic peptide 324-348. Polyanions such as poly(aspartic acid), dextran sulfate, or poly(adenylic acid) inhibit the kinase. Poly(L-aspartic acid) is a competitive inhibitor with respect to rhodopsin (KI = 300 microM) and shows mixed type inhibition with respect to ATP.     

Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein

Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV. To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity. Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli. Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein. The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein. In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81. Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids. Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes. In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein. These results led us to consider the development of an effective anti-HCV peptide. This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.     

双链RNA激活的蛋白激酶(PKR)的克隆表达及其对丙型肝炎病毒蛋白合成的抑制

α干扰素为治疗丙型肝炎病毒(HCV)感染的主要药物,但部分患者呈干扰素耐受而不能获得持久的病毒阴转,其可能的原因之一是病毒通过其编码的蛋白(NS5A及E2)抑制干扰素诱导的抗病毒效应分子——双链RNA激活的蛋白激酶(PKR)的活性.而关于PKR是否在IFN-α抗HCV的机理中起抑制作用目前仍有争议.为研究PKR对HCV蛋白合成环节是否有抑制作用,通过构建野生型 PKR真核表达载体（pPKRwt）及主要起负性调节作用的缺失突变PKR真核表达载体（pPKRΔ6）,并将pPKRwt /pPKRΔ6 与HCV复制子RNA同时转染Huh7细胞进行共表达, 用Western印迹检测 HCV IRES 下游的NPTⅡ蛋白表达水平,与转染空载体的对照细胞及单用IFN-α处理的细胞相比较.结果显示：表达PKRwt的细胞中NPTⅡ蛋白水平低于转染空载体的对照细胞,但高于经IFN-α单独处理的细胞;表达PKRΔ6的细胞中NPTⅡ蛋白水平与对照细胞无明显差别,但PKRΔ能部分抵消IFN-α的抑制作用,说明在IFN-α抑制HCV IRES指导的蛋白合成中,PKR有一定的抑制作用,但可能还有其它的PKR非依赖机制参与.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

The 58,000-dalton cellular inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a member of the tetratricopeptide repeat family of proteins.

PKR is a serine/threonine protein kinase induced by interferon treatment and activated by double-stranded RNAs. As a result of activation, PKR becomes autophosphorylated and catalyzes phosphorylation of the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2). While studying the regulation of PKR in virus-infected cells, we found that a cellular 58-kDa protein (P58) was recruited by influenza virus to downregulate PKR and thus avoid the kinase's deleterious effects on viral protein synthesis and replication. We now report on the cloning, sequencing, expression, and structural analysis of the P58 PKR inhibitor, a 504-amino-acid hydrophilic protein. P58, expressed as a histidine fusion protein in Escherichia coli, blocked both the autophosphorylation of PKR and phosphorylation of the alpha subunit of eIF-2. Western blot (immunoblot) analysis showed that P58 is present not only in bovine cells but also in human, monkey, and mouse cells, suggesting the protein is highly conserved. Computer analysis revealed that P58 contains regions of homology to the DnaJ family of proteins and a much lesser degree of similarity to the PKR natural substrate, eIF-2 alpha. Finally, P58 contains nine tandemly arranged 34-amino-acid repeats, demonstrating that the PKR inhibitor is a member of the tetratricopeptide repeat family of proteins, the only member identified thus far with a known biochemical function.     

A catalytically active fragment of cGMP-dependent protein kinase. Occupation of its cGMP-binding sites does not affect its phosphotransferase activity

Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.     

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 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.