Double-stranded RNA-activated protein kinase (PKR) is negatively regulated by 60S ribosomal subunit protein L18

The double-stranded RNA (dsRNA)-activated protein kinase (PKR) provides a fundamental control step in the regulation of protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2alpha), a process that prevents polypeptide chain initiation. In such a manner, activated PKR inhibits cell growth and induces apoptosis, whereas disruption of normal PKR signaling results in unregulated cell growth. Therefore, tight control of PKR activity is essential for regulated cell growth. PKR is activated by dsRNA binding to two conserved dsRNA binding domains within its amino terminus. We isolated a ribosomal protein L18 by interaction with PKR. L18 is a 22-kDa protein that is overexpressed in colorectal cancer tissue. L18 competed with dsRNA for binding to PKR, reversed dsRNA binding to PKR, and did not directly bind dsRNA. Mutation of K64E within the first dsRNA binding domain of PKR destroyed both dsRNA binding and L18 interaction, suggesting that the two interactive sites overlap. L18 inhibited both PKR autophosphorylation and PKR-mediated phosphorylation of eIF-2alpha in vitro. Overexpression of L18 by transient DNA transfection reduced eIF-2alpha phosphorylation and stimulated translation of a reporter gene in vivo. These results demonstrate that L18 is a novel regulator of PKR activity, and we propose that L18 prevents PKR activation by dsRNA while PKR is associated with the ribosome. Overexpression of L18 may promote protein synthesis and cell growth in certain cancerous tissue through inhibition of PKR activity.     

Regulation of the interferon-inducible protein kinase PKR and (2''-5'')oligo(adenylate) synthetase by a catalytically inactive PKR mutant through competition for double-stranded RNA binding

This study evaluated the long-term effect of an ultrasonic toothbrush used as part of a daily oral hygiene regimen on supragingival plaque, gingivitis, and gingival bleeding. Compared with a conventional toothbrush, the ultrasonic toothbrush was significantly more effective in reducing plaque formation (p < 0.05), removing plaque (p < 0.05), and reducing gingivitis (p < 0.05) during the 6-month study period.     

Cell cycle regulation of the double stranded RNA activated protein kinase, PKR

The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells. The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase. PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter. In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase. Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity. In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis. Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase. Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells. Taken together these results suggest that PKR may play a role in cell cycle progression.     

Gene V protein dimerization and cooperativity of binding of poly(dA)

Gene V protein of bacteriophage f1 is a dimeric protein that binds cooperatively to single-stranded nucleic acids. In order to determine whether a monomer-dimer equilibrium has an appreciable effect upon the thermodynamics of gene V protein binding to nucleic acids, the dissociation constant for the protein dimer was investigated using size-exclusion chromatography. At concentrations ranging from 5 x 10(-10) to 1.2 x 10(-5) M, the Stokes radius of the protein was that expected of the dimer of the gene V protein. The Stokes radius of the protein was also independent of salt concentration from 0.2 to 1.0 M NaCl in a buffer containing 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA. The binding of the dimeric gene V protein to poly(dA) was studied using a simplified lattice model for protein-protein interactions adapted for use with a dimeric protein that binds simultaneously to two strands of nucleic acid. Interpretation of the salt dependence, C = [d log(Kint omega)]/[d log(NaCl)], of binding of such a dimeric protein to nucleic acid using the theory of Record et al. (Record, M. T., et al. (1976) J. Mol. Biol. 107, 145-158) indicates that C is a function of the numbers of cations and anions released from protein and nucleic acid upon binding of the dimer, not of the monomer. Cooperativity of gene V protein binding to poly(dA) was studied with titration experiments that are sensitive to the degree of cooperativity of binding. The cooperativity factor omega, defined as the ratio of the binding constant for a site adjacent to a previously bound dimer to that for an isolated site, was found to be relatively insensitive to salt, with a value in the range of 2000-7000 for binding to poly(dA) at 3 degrees C and at 23 degrees C. This high cooperativity factor supports the suggestion that protein-protein contacts play a major role in the formation of the superhelical gene V protein-single-stranded nucleic acid complex.     

Requirement of PKR dimerization mediated by specific hydrophobic residues for its activation by double-stranded RNA and its antigrowth effects in yeast

The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic alpha-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.     

Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport

A wide variety of biological activities including the major metabolic actions of insulin is regulated by phosphatidylinositol (PI) 3-kinase. However, the downstream effectors of the various signaling pathways that emanate from PI 3-kinase remain unclear. Akt (protein kinase B), a serine-threonine kinase with a pleckstrin homology domain, is thought to be one such downstream effector. A mutant Akt (Akt-AA) in which the phosphorylation sites (Thr308 and Ser473) targeted by growth factors are replaced by alanine has now been shown to lack protein kinase activity and, when overexpressed in CHO cells or 3T3-L1 adipocytes with the use of an adenovirus vector, to inhibit insulin-induced activation of endogenous Akt. Akt-AA thus acts in a dominant negative manner in intact cells. Insulin-stimulated protein synthesis, which is sensitive to wortmannin, a pharmacological inhibitor of PI 3-kinase, was abolished by overexpression of Akt-AA without an effect on amino acid transport into the cells, suggesting that Akt is required for insulin-stimulated protein synthesis. Insulin activation of p70 S6 kinase was inhibited by approximately 75% in CHO cells and approximately 30% in 3T3-L1 adipocytes, whereas insulin-induced activation of endogenous Akt was inhibited by 80 to 95%, by expression of Akt-AA. Thus, Akt activity appears to be required, at least in part, for insulin stimulation of p70 S6 kinase. However, insulin-stimulated glucose uptake in both CHO cells and 3T3-L1 adipocytes was not affected by overexpression of Akt-AA, suggesting that Akt is not required for this effect of insulin. These data indicate that Akt acts as a downstream effector in some, but not all, of the signaling pathways downstream of PI 3-kinase.     

Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation

Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.     

Substance P-induced mitogenesis in human astrocytoma cells correlates with activation of the mitogen-activated protein kinase signaling pathway

The neuropeptide substance P (SP) regulates many biological processes through binding to and activating the SP receptor (NK-1 subtype). Activation of the SP receptor induces mitogenesis in several cell types. In this study, we characterized the mitogenic response induced by SP peptide in the U-373MG astrocytoma cell line and showed that activation of the SP receptor induces [3H]thymidine incorporation into DNA. We also found that SP potently induces c-myc mRNA and protein in the U-373MG cells. Tyrphostin A25, which blocks activity of tyrosine kinases, significantly inhibited SP-induced mitogenesis, suggesting that the mitogenic response induced by SP peptide involves phosphorylation by tyrosine kinases. Furthermore, stimulation of the SP receptor activates tyrosine phosphorylation and enzymatic activity of extracellular signal-regulated kinases (Erk1 and Erk2), also called the mitogen-activated protein kinases (MAPKs). This result suggests that MAPKs participate in the SP peptide-induced signaling pathway. The addition of CP 96,345 ([(2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1 -azabicyclo[2.2.2]octan-3-amine]; an NK-1 receptor antagonist) or PD 098059 (MEK1 inhibitor) inhibited both DNA synthesis and activation of the MAPK pathway, substantiating that SP stimulates mitogenesis by activating the MAPK pathway through receptors of the NK-1 subtype. Our results demonstrate that SP peptide is a strong mitogen in the U-373MG astrocytoma cell line and establish a clear correlation between SP-induced mitogenesis and activation of MAPK signaling pathway.     

Regulated expression of the interferon-induced protein kinase p68 (PKR) by vaccinia virus recombinants inhibits the replication of vesicular stomatitis virus but not that of poliovirus

A direct antiviral role of the interferon-induced human protein kinase p68 has been shown only against encephalomyocarditis virus (EMCV) and vaccinia virus (VV). To determine if p68 kinase (PKR) has a broad antiviral effect, we have used coinfections between VV recombinants expressing p68 kinase under regulation of the lac I operator/repressor elements of Escherichia coli and two RNA viruses, vesicular stomatitis virus (VSV) and poliovirus. In cells coinfected with VV recombinants and VSV, induction with isopropyl-B-D-thiogalactoside (IPTG) of wild-type p68 kinase or a mutant lacking the dsRNA binding domain resulted in inhibition of both VV and VSV protein synthesis. This inhibition is not observed in cells infected with a catalytically inactive point mutant lys-arg296 of p68 kinase. When cells are coinfected with VV recombinants and poliovirus, induction of active p68 kinase resulted in a decrease in VV proteins but not in poliovirus proteins or poliovirus yields. Immunoblot analysis revealed that p68 kinase was expressed during mixed infections. Our results demonstrate a differential effect of p68 kinase on the replication of VV, VSV, and poliovirus. We suggest that in a particular virus-cell system, the different sensitivity of a virus to p68 kinase is probably due to levels of active enzyme.     

Intramolecular binding contributes to the activation of CDPK, a protein kinase with a calmodulin-like domain

The activity of calmodulin-like domain protein kinase (CDPK) is regulated by the direct binding of Ca2+. Unmodified soybean CDPK alpha and a chimeric enzyme in which the calmodulin-like domain (CLD) was replaced by VU-1 calmodulin had similar values of Vmax(app) (3.19, 3.46, and 3.60, 3.93 mumol/ min/mg, respectively), and each was activated 30-70-fold by Ca2+. To determine if activation results from the binding of the CLD to the autoinhibitory (junction) domain of CDPK alpha in a manner analogous to the activation of calmodulin-dependent enzymes by calmodulin, recombinant CLD and truncation mutants of CDPK alpha were expressed in bacteria and highly purified. In blot overlays, biotinylated CLD bound to mutants containing residues 312-328 of the junction domain. In an electrophoretic mobility shift assay CLD bound synthetic peptides containing residues 318-332 in a calcium-dependent manner, providing direct evidence for binding of CLD to a site in the junction domain. Mutants of CDPK alpha from which all or part of the CLD had been deleted were constitutively inactive. Addition of 20 microM CLD to these mutants in the presence, but not the absence, of calcium stimulated their activities, but to various degrees. His6-CDPK alpha (1-328), which contained none of the CLD, was activated only 5-fold, but the activity of His6-CDPK alpha (1-398), which retained nearly half of the CLD in its sequence, was stimulated 64-fold. The latter activity approached that of unmodified CDPK alpha and was half maximal at a CLD concentration of 7 microM. Our results suggest that binding of CLD to the junction domain contributes to, but is not sufficient for activation. Although calmodulin supported full activity of the chimeric enzyme, its addition to His6-CDPK alpha (1-398) resulted in activity that was only 6% of that of the unmodified enzyme and which was half-maximal at 20 microM Arabidopsis calmodulin. These results support the conclusion that simple binding of the calmodulin-like domain to the junction domain is not sufficient for activation.     

Modulation of the heat-induced activation of mitogen-activated protein (MAP) kinase by quercetin

Effects of quercetin, a bioflavonoid compound, on heat-induced activation of mitogen-activated protein (MAP) kinase in rat hepatoma (H4) cells were examined. Quercetin decreased cell viability and induced DNA fragmentation in heat-shocked H4 cells. MAP kinase in heat-shocked cells was activated and reached a peak at 1 hr after the heat shock, and then gradually decreased. Quercetin inhibited the heat-induced activation of MAP kinase observed at 1 hr after heat shock, but markedly stimulated MAP kinase activity at 4 hr after heat shock. Thus, quercetin modulated the heat-induced activation of MAP kinase in a biphasic manner. Present observations indicate that quercetin modulates protein phosphorylation, especially that controled by MAP kinase, in early events of heat shock response.     

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.     

Gbp1p, a protein with RNA recognition motifs, binds single-stranded telomeric DNA and changes its binding specificity upon dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

Somatostatin activation of mitogen-activated protein kinase via somatostatin receptor 1 (SSTR1)

Hormones and growth factors regulate cell growth via the mitogen-activated protein (MAP) kinase cascade. Here we examine the actions of the hormone somatostatin on the MAP kinase cascade through one of its two major receptor subtypes, the somatostatin receptor 1 (SSTR1) stably expressed in CHO-K1 cells. Somatostatin antagonizes the proliferative effects of fibroblast growth factor in CHO-SSTR1 cells via the SSTR1 receptor. However, in these cells, somatostatin robustly activates MAP kinase (also called extracellular signal regulated kinase; ERK) and augments fibroblast growth factor-stimulated ERK activity. We show that the activation of ERK via SSTR1 is pertussis toxin sensitive and requires the small G protein Ras, phosphatidylinositol 3-kinase, the serine/threonine kinase Raf-1, and the protein tyrosine phosphatase SHP-2. The activation of ERK by SSTR1 increased the expression of the cyclin-dependent protein kinase inhibitor p21(cip1/WAF1). Previous studies have suggested that somatostatin-stimulated protein tyrosine phosphatase activity mediates the growth effects of somatostatin. Our data suggest that SHP-2 stimulation by SSTR1 may mediate some of these effects through the activation of the MAP kinase cascade and the expression of p21(cip1/WAF1).     

Mutant influenza viruses with a defective NS1 protein cannot block the activation of PKR in infected cells

A short model genome RNA and also the genome RNA of influenza A virus bearing both 5'- and 3'-terminal common sequences activated the interferon-induced double-stranded-RNA-dependent protein kinase, PKR, by stimulating autophosphorylation in vitro. The activated PKR catalyzed phosphorylation of the alpha subunit of eucaryotic translation initiation factor 2 (eIF2alpha). The NS1 protein efficiently eliminated the PKR-activating activity of these RNAs by binding to them. Two mutant NS1 proteins, each harboring a single amino acid substitution at different regions, exhibited temperature sensitivity in their RNA binding activity in the mutant virus-infected cell lysates as well as when they were prepared as fusion proteins expressed in bacteria. The virus strains carrying these mutant NS1 proteins exhibited temperature sensitivity in virus protein synthesis at the translational level, as reported previously, and could not repress the autophosphorylation of PKR developing during the virus growth, which is normally suppressed by a viral function(s). As a result, the level of eIF2alpha phosphorylation was elevated 2.5- to 3-fold. The defect in virus protein synthesis was well correlated with the level of phosphorylation of PKR and eIF2alpha.