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Biomedical subjects

M G Katze

Publications and source records attributed to M G Katze.

At least 55 records · Page 3Linked to original sources
PubMed1996

The P58 cellular inhibitor complexes with the interferon-induced, double-stranded RNA-dependent protein kinase, PKR, to regulate its autophosphorylation and activity.

The 58-kDa protein, referred to as P58, is a cellular inhibitor of the interferon-induced, double-stranded RNA-activated protein kinase, PKR. The P58 protein inhibits both the autophosphorylation of PKR and the phosphorylation of the PKR natural substrate, the alpha subunit of eukaryotic initiation factor eIF-2. Sequence analysis revealed that P58 is a member of the tetratricopeptide family of proteins. Utilizing experimental approaches, which included coprecipitation or coselection of native and recombinant wild-type and mutant proteins, we found that P58 can efficiently complex with the PKR protein kinase. Attempts to map the P58 interactive sites revealed a correlation between the ability of P58 to inhibit PKR in vitro and bind to PKR. The DnaJ sequences, present at the carboxyl terminus of P58, were dispensable for binding in vitro, while sequences containing the eIF-2 alpha similarity region were essential for efficient complex formation. Furthermore, not all tetratricopeptide motifs were necessary for PKR-P58 interactions. Initial experiments to map the binding domains present in PKR showed that P58 complexed with PKR molecules that lacked the first RNA binding domain but did not bind to a PKR mutant containing only the amino terminus. These data, taken together, demonstrate that P58 inhibits PKR through a direct interaction, which is likely independent of the binding of double-stranded RNA to the protein kinase.

3T3 Cells↗
PubMed1996

Chromosomal assignment of the gene encoding the human 58-kDa inhibitor (PRKRI) of the interferon-induced dsRNA-activated protein kinase to chromosome 13q32.

The 58-kDa inhibitor (p58) of the interferon-induced dsRNA-activated protein kinase (PKR) is a cellular protein recruited by the influenza virus to down-regulate the activity of PKR during virus infection. The inhibitor also appears to play a role in the regulation of cellular gene expression in the absence of viral infection and has oncogenic properties when overexpressed. Using fluorescence in situ hybridization, we have mapped the p58 gene (PRKRI) to human chromosome 13 band q32. Aberrations in the structure or number of chromosome 13 have been identified in a variety of human cancers, particularly in acute leukemia.

Chromosome Mapping↗
PubMed1996

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.

Base Sequence↗
PubMed1995

Interferon treatment inhibits virus replication in HIV-1- and SIV-infected CD4+ T-cell lines by distinct mechanisms: evidence for decreased stability and aberrant processing of HIV-1 proteins.

We have examined the effects of interferon (IFN)-alpha/beta on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse-chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.

Animals↗
PubMed1995

Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor.

The NS1 protein of influenza A virus binds not only to poly(A) and a stem-bulge region in U6 small nuclear RNA (snRNA), but also to double-stranded (ds) RNA. Binding assays with NS1 protein mutants established that the previously identified RNA-binding domain of the NS1 protein is required for binding to ds RNA as well as for binding to poly(A) and U6 snRNA. In addition, dsRNA competed with U6 snRNA for binding to the NS1 protein, consistent with both RNAs sharing the same binding site on the protein. As a consequence of its binding to dsRNA, the NS1 protein blocks the activation of the dsRNA-activated protein kinase (PKR) in vitro. This kinase phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (elF-2 alpha), leading to a decrease in the rate of initiation of translation. Assays using purified PKR and purified elF2 demonstrated that the NS1 protein blocks the dsRNA activation of PKR, and experiments using reticulocyte extracts showed that the NS1 protein blocks the inhibition of translation caused by dsRNA activation of PKR. The implications of these results for control mechanisms occurring in influenza virus-infected cells are discussed.

Animals↗
PubMed1995

Translational control by influenza virus. Identification of cis-acting sequences and trans-acting factors which may regulate selective viral mRNA translation.

We have shown that sequences contained within the viral mRNA 5'-untranslated region (UTR) played a critical role in directing selective influenza viral mRNA translation. We therefore attempted to identify transacting factors that may regulate viral mRNA translation through interactions with the 5'-UTR and at the same time map the precise sequences to which these factors bind. We can now demonstrate that multiple cellular proteins interact with influenza viral but not cellular 5'-UTRs using gel mobility shift and UV cross-linking analyses. Gel supershift studies revealed that the La autoantigen was one of the cellular proteins that interacted with the viral 5'-UTR. Utilizing mutants of the viral mRNA 5' UTR, we have determined that sequences within the very 5'-conserved region and nucleotides immediately 3' are necessary but not always sufficient for binding certain cellular proteins. Northwestern analysis showed the binding of a distinct subset of cellular proteins to the viral 5'-UTR, but also demonstrated interactions of the viral nonstructural protein NS1. Gel shift analysis with purified recombinant NS1 confirmed the binding of the viral protein to a specific region of the viral 5'-UTRs. A model describing the possible role of these cellular and viral RNA-binding proteins in regulating influenza virus mRNA translation will be discussed.

Base Sequence↗
PubMed1995

Molecular mechanisms responsible for malignant transformation by regulatory and catalytic domain variants of the interferon-induced enzyme RNA-dependent protein kinase.

Double-stranded RNA-dependent protein kinase (PKR) is suggested to play an important role in both the antiviral and antiproliferative arms of the interferon response. To gain insights into the molecular mechanisms underlying PKR's growth regulatory properties, we examined the biological and biochemical properties of PKR variants containing either a mutation in catalytic domain II (PKR-M1) or a deletion of RNA binding domain I (PKR-M7) in both reticulocyte translation extracts and in vitro kinase assays with purified reagents and compared these results with those using the same mutants stably expressed in vivo. While wild-type PKR (PKR-WT) efficiently inhibited mRNA translation in a reticulocyte extract, the inactive PKR-M1 had no effect. The PKR-M7 mutant was modestly inhibitory in this assay. The PKR-M1 variant was able to reverse the translational inhibitory effects and increased eukaryotic initiation factor (eIF)-2 alpha phosphorylation levels caused by addition of double-stranded RNA to reticulocyte extract, whereas PKR-M7 could not. Both PKR-M1 and PKR-M7 functioned as transdominant inhibitors of PKR-WT in our in vitro kinase assays. While the inhibition by PKR-M1 required a vast excess of mutant to shut down PKR function, PKR-M7 inhibited PKR-WT at approximately stoichiometric levels. To complement these experiments, we compared growth rates and alpha phosphorylation levels in transformed cell lines overexpressing either PKR-M1 or PKR-M7. Levels of endogenous eIF-2 alpha phosphorylation were significantly more diminished in PKR-M7 overexpressing cells compared with PKR-M1. These paradoxical data will be discussed in terms of the potential molecular mechanisms underlying malignant transformation caused by the PKR variants.

3T3 Cells↗
PubMed1995

Regulation of the interferon-induced PKR: can viruses cope?

Viruses that fail to block the lethal effects of the double-stranded-RNA-activated protein kinase (PKR) may be doomed; why do so many viruses go to so much trouble to downregulate this interferon-induced protein kinase? PKR may regulate cell growth and proliferation in uninfected cells, suggesting that it also participates in the antiproliferative arm of the interferon response.

Animals↗
PubMed1995

Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation.

Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.

3T3 Cells↗
PubMed1994

The 58-kilodalton inhibitor of the interferon-induced double-stranded RNA-activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties.

The interferon-induced RNA-dependent protein kinase (PKR) is considered to play an important role in the cellular defense against viral infection and, in addition, has been suggested to be a tumor suppressor gene because of its growth-suppressive properties. Activation of PKR by double-stranded RNAs leads to the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) and a resultant block to protein synthesis initiation. To avoid the consequences of kinase activation, many viruses have developed strategies to down-regulate PKR. Recently, we reported on the purification and characterization of a cellular inhibitor of PKR (referred to as p58), which is activated during influenza virus infection. Subsequent cloning and sequencing has revealed that p58 is a member of the tetratricopeptide repeat (TPR) family of proteins. To further examine the physiological role of this PKR inhibitor, we stably transfected NIH 3T3 cells with a eukaryotic expression plasmid containing p58 cDNA under control of the cytomegalovirus early promoter. By taking advantage of a recently characterized p58 species-specific monoclonal antibody, we isolated cell lines that overexpressed p58. These cells exhibited a transformed phenotype, growing at faster rates and higher saturation densities and exhibiting anchorage-independent growth. Most importantly, inoculation of nude mice with p58-overexpressing cells gave rise to the production of tumors. Finally, murine PKR activity and endogenous levels of eIF-2 alpha phosphorylation were reduced in the p58-expressing cell lines compared with control cells. These data, taken together, suggest that p58 functions as an oncogene and that one mechanism by which the protein induces malignant transformation is through the down-regulation of PKR and subsequent deregulation of protein synthesis.

3T3 Cells↗
PubMed1994

Intrarectal inoculation of macaques by the simian immunodeficiency virus, SIVmne E11S: CD4+ depletion and AIDS.

Macaca nemestrina and Macaca fascicularis were inoculated with various doses of a single-cell clone of SIVmne-infected HuT 78 cells (E11S) by both the intravenous and intrarectal routes. Animals inoculated intravenously at each dose seroconverted and virus was isolated from peripheral blood mononuclear cells, but only the high-dose intrarectally exposed macaques became viremic and seroconverted. However, some seronegative, virus isolation negative intrarectally inoculated macaques showed evidence of infection and disease.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1993

Translational control by influenza virus. Selective translation is mediated by sequences within the viral mRNA 5'-untranslated region.

In cells infected by influenza virus type A, host cell protein synthesis declines rapidly and dramatically, while influenza viral protein synthesis occurs efficiently throughout infection. Previously, we had shown that the selective translation of influenza viral mRNAs in infected cells occurred in a cap-dependent manner and was due at least in part to structures inherent in the mRNAs. Using chimeras containing the noncoding and coding regions of cellular and viral mRNAs, we can now report that the selective translation is mediated by sequences within the 5'-untranslated regions (UTR) of the viral mRNAs. Polysome analysis confirmed that a 45-nucleotide sequence contained in the 5'-UTR of the influenza viral nucleocapsid protein was necessary and sufficient to allow the host cell translational machinery to discriminate between viral and cellular mRNAs. In reciprocal experiments in which the 5'-UTR of the cellular mRNA-secreted embryonic alkaline phosphatase replaced the nucleocapsid protein 5'-UTR, viral protein synthesis was inhibited in virus-infected cells, resembling host protein synthesis. Finally, we demonstrated that the 5'-UTR of another influenza viral mRNA, that encoding the nonstructural protein, also conferred resistance to the shutoff of protein synthesis in influenza virus-infected cells.

Animals↗
PubMed1993

Mammalian eukaryotic initiation factor 2 alpha kinases functionally substitute for GCN2 protein kinase in the GCN4 translational control mechanism of yeast.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) in Saccharomyces cerevisiae by the GCN2 protein kinase stimulates the translation of GCN4 mRNA. The protein kinases heme-regulated inhibitor of translation (HRI) and double-stranded RNA-dependent eIF-2 alpha protein kinase (dsRNA-PK) inhibit initiation of translation in mammalian cells by phosphorylating Ser-51 of eIF-2 alpha. We show that HRI and dsRNA-PK phosphorylate yeast eIF-2 alpha in vitro and in vivo and functionally substitute for GCN2 protein to stimulate GCN4 translation in yeast. In addition, high-level expression of either mammalian kinase in yeast decreases the growth rate, a finding analogous to the inhibition of total protein synthesis by these kinases in mammalian cells. Phosphorylation of eIF-2 alpha inhibits initiation in mammalian cells by sequestering eIF-2B, the factor required for exchange of GTP for GDP on eIF-2. Mutations in the GCN3 gene, encoding a subunit of the yeast eIF-2B complex, eliminate the effects of HRI and dsRNA-PK on global and GCN4-specific translation in yeast. These results provide further in vivo evidence that phosphorylation of eIF-2 alpha inhibits translation by impairing eIF-2B function and identify GCN3 as a regulatory subunit of eIF-2B. These results also suggest that GCN4 translational control will be a good model system to study how mammalian eIF-2 alpha kinases are modulated by environmental signals and viral regulatory factors.

Animals↗
PubMed1993

Translational regulation by the interferon-induced double-stranded-RNA-activated 68-kDa protein kinase.

Activation of the interferon-inducible 68-kDa protein kinase (referred to as P68) by double-stranded RNA catalyzes phosphorylation of the alpha subunit of eukaryotic protein synthesis initiation factor 2. We have analyzed the transient expression of mutant and wild-type kinase molecules in transfected COS cells to examine the effects of the kinase on gene expression in the absence of other interferon-induced gene products. The wild-type P68 kinase was expressed inefficiently whereas a catalytically inactive P68 was expressed at 30- to 40-fold higher levels. Protein stability measurements and primer-extension analysis of human kinase-specific mRNA levels provided evidence that kinase expression was regulated at the level of mRNA translation. Further, cotransfection experiments revealed that the domain II catalytically inactive mutant could stimulate reporter gene protein synthesis in a transdominant manner. We also examined the expression of mutants with deletions in the N-terminal double-stranded RNA binding domains and found that a kinase construct lacking aa 156-243 was expressed at levels comparable to the wild type whereas a P68 construct lacking aa 91-243 was expressed at levels 70-fold higher. Both the inactive domain II P68 mutant and the deletion mutant lacking aa 91-243 were less inhibitory to growth in yeast due to the reduced ability to phosphorylate initiation factor 2 alpha in vivo. In conclusion we have demonstrated that the P68 kinase can regulate mRNA translation primarily of its own mRNA and to a lesser extent of a heterologous mRNA and that this regulation is notably affected by mutations in either the catalytic or N-terminal regulatory domains.

Animals↗
PubMed1993

Tumor suppressor function of the interferon-induced double-stranded RNA-activated protein kinase.

RNA-dependent protein kinase is a M(r) 68,000 protein in human cells (p68 kinase) or a M(r) 65,000 protein in murine cells (p65 kinase). p65/p68 is a serine/threonine kinase induced by interferon treatment and generally activated by double-stranded RNAs. Once activated, the known function of this kinase is inhibition of protein synthesis through phosphorylation of the eukaryotic initiation factor 2. Here we have investigated the potential for tumorigenicity in mice of murine NIH 3T3 clones expressing human p68 kinase, either the wild-type or a mutant inactive kinase with a single amino acid substitution in the invariant lysine-296 in the catalytic domain II. Expression of the mutant p68 kinase was correlated with a malignant transformation phenotype, giving rise to the production of large tumors of at least 1 cm in diameter within 7-12 days in all inoculated mice. In contrast, no tumor growth was observed for several weeks in mice inoculated with NIH 3T3 cell clones expressing either the wild-type recombinant p68 kinase or only the endogenous p65 kinase, the murine analogue of the p68 kinase. These results suggest that functional p65/p68 kinase (recently called PKR), by a still undefined mechanism, may also act as a tumor suppressor. Consequently, one of the pathways by which interferon inhibits tumor growth might be through its capacity to induce the enhanced expression of this kinase.

3T3 Cells↗
PubMed1993

Chromosomal assignment of the interferon-inducible double-stranded RNA-dependent protein kinase (PRKR) to human chromosome 2p21-p22 and mouse chromosome 17 E2.

The genes encoding P68 and P65 (PRKR), the human and mouse interferon-inducible dsRNA-dependent protein kinases, respectively, have been mapped to a single locus on human chromosome 2 (band p21) and on mouse chromosome 17 (band E2). These kinases have been implicated in the antiviral response mediated by interferon since their activation by virus-specific dsRNAs can lead to the inhibition of protein synthesis. Recently we have shown that the dsRNA-dependent kinase also may function as a tumor suppressor gene since defective mutant proteins induced malignant transformation. Identification of the chromosomal location of human PRKR permitted a survey of translocations, deletions, or other rearrangement events involving this segment of chromosome 2 in a variety of human malignancies. Finally, our results define a new region of conservation between the distal part of the short arm of chromosome 2 (band p21) and band E2 of mouse chromosome 17.

Animals↗