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Michael G. Katze - One of the best experts on this subject based on the ideXlab platform.
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pancreatic β cell failure and diabetes in mice with a deletion mutation of the endoplasmic reticulum molecular chaperone gene P58ipk
Diabetes, 2005Co-Authors: Warren C Ladiges, Marcus J. Korth, Sue E Knoblaugh, John Morton, Bryce L Sopher, Carole R Baskin, Alasdair Macauley, Alan G Goodman, Renee C Leboeuf, Michael G. KatzeAbstract:The endoplasmic reticulum (ER) transmits apoptotic signals in the pancreas during ER stress, implicating ER stress-mediated apoptosis in the development of diabetes. P58(IPK) (DNAJC3) is induced during ER stress and functions as a negative feedback component to inhibit eIF-2alpha signaling and attenuate the later phases of the ER stress response. To gain insight into a more comprehensive role of P58(IPK) function, we generated deletion mutant mice that showed a gradual onset of glucosuria and hyperglycemia associated with increasing apoptosis of pancreatic islet cells. Lack of P58(IPK) had no apparent effect on the functional integrity of viable beta-cells. A set of genes associated with apoptosis showed altered expression in pancreatic islets from P58(IPK)-null mice, further substantiating the apoptosis phenotype. The data provide in vivo evidence to support the concept that P58(IPK) functions as a signal for the downregulation of ER-associated proteins involved in the initial ER stress response, thus preventing excessive cell loss by degradation pathways. Insulin deficiency associated with the absence of P58(IPK) mimics beta-cell failure associated with type 1 and late-stage type 2 diabetes. P58(IPK) function and activity may therefore provide a novel area of investigation into ER-mediated mechanistic and therapeutic approaches for diabetes.
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Inhibition of Double-Stranded RNA- and Tumor Necrosis Factor Alpha-Mediated Apoptosis by Tetratricopeptide Repeat Protein and Cochaperone P58IPK
Molecular and cellular biology, 1999Co-Authors: Norina M. Tang, Bryan R.g. Williams, Marcus J. Korth, Michael Gale, Marlene Wambach, Sandy D. Der, Sudip K. Bandyopadhyay, Michael G. KatzeAbstract:P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.
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The 58-kDa Cellular Inhibitor of the Double Stranded RNA-dependent Protein Kinase Requires the Tetratricopeptide Repeat 6 and DnaJ Motifs to Stimulate Protein Synthesis in Vivo
The Journal of biological chemistry, 1996Co-Authors: Norina M. Tang, Michael G. KatzeAbstract:Double stranded RNA-dependent protein kinase (PKR) is a double stranded RNA-activated, interferon-induced serine-threonine kinase that participates in both the antiviral and antiproliferative properties of interferon. We previously found that influenza virus inhibited PKR function by recruiting or activating a cellular inhibitor termed P58(IPK). The present study was undertaken to complement our earlier analyses, which demonstrated that P58(IPK) efficiently inhibited PKR autophosphorylation and activity in vitro. We now report that P58(IPK) down-regulates PKR and, in turn, stimulates protein synthetic rates inside the cell. Using transfection analysis, we show that P58(IPK) stimulates translation of secreted embryonic alkaline phosphatase reporter gene mRNA. Furthermore, we found that at least two regions of the P58(IPK) molecule were required for PKR inhibitory activity in COS-1 cells: (i) the DnaJ similarity region at the carboxyl terminus (amino acids 391-504); and (ii) the tetratricopeptide repeat 6 (TPR6) domain (amino acids 222-255) located in the middle of the P58(IPK) protein and within the eukaryotic protein synthesis initiation factor 2alpha homology region. P58(IPK) variants lacking either one of these regions were unable to stimulate secreted embryonic alkaline phosphatase protein synthetic rates. Consistent with this data is the observation that the DeltaTPR6 mutant (the P58(IPK) variant lacking the TPR6 motif) failed to block PKR activity in vitro. Based on these data and our earlier in vitro functional and PKR-P58(IPK) binding analyses, a revised model of PKR regulation by P58(IPK) is presented.
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the P58 cellular inhibitor complexes with the interferon induced double stranded rna dependent protein kinase pkr to regulate its autophosphorylation and activity
Journal of Biological Chemistry, 1996Co-Authors: Stephen J Polyak, Marlene Wambach, Norina Tang, Glen N Barber, Michael G. KatzeAbstract: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 α 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 α 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.
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the 58 kilodalton inhibitor of the interferon induced double stranded rna activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Glen N Barber, Samantha Thompson, Tae Gyu Lee, Ted S Strom, Rosemary Jagus, Andre Darveau, Michael G. KatzeAbstract: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.
Bingdong Sha - One of the best experts on this subject based on the ideXlab platform.
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structural insight into the protective role of P58 ipk during unfolded protein response
Methods in Enzymology, 2011Co-Authors: Jiahui Tao, Bingdong ShaAbstract:Abstract P58(IPK) has been identified as an ER molecular chaperone to maintain protein-folding homeostasis. P58(IPK) expression can be significantly upregulated during unfolded protein responses (UPR), and it may play important roles in suppressing the ER protein aggregations. To investigate the mechanism how P58(IPK) functions to promote protein folding within ER, we have determined the crystal structure of P58(IPK) TPR domain at 2.5 A resolution. P58(IPK) contains nine TPR motifs and a C-terminal J domain within its primary sequence. The crystal structure of P58(IPK) revealed three subdomains (I, II, and III) with similar folds and each domain contains three TPR motifs. Our data also showed that P58(IPK) acts as a molecular chaperone by interacting with the unfolded proteins such as luciferase, rhodanese, and insulin. The P58(IPK) structure reveals a conserved hydrophobic patch located in subdomain I that may be involved in binding the misfolded polypeptides. We have proposed a working model for P58(IPK) to act together with Bip to prevent protein aggregations and promote protein foldings within ER.
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crystal structure of P58 ipk tpr fragment reveals the mechanism for its molecular chaperone activity in upr
Journal of Molecular Biology, 2010Co-Authors: Jiahui Tao, Kseniya Petrova, David Ron, Bingdong ShaAbstract:P58(IPK) might function as an endoplasmic reticulum molecular chaperone to maintain protein folding homeostasis during unfolded protein responses. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the endoplasmic reticulum, we have determined the crystal structure of P58(IPK) TPR fragment to 2.5 A resolution by the SAD method. The crystal structure of P58(IPK) revealed three domains (I-III) with similar folds and each domain contains three TPR motifs. An ELISA assay indicated that P58(IPK) acts as a molecular chaperone by interacting with misfolded proteins such as luciferase and rhodanese. The P58(IPK) structure reveals a conserved hydrophobic patch located in domain I that might be involved in binding the misfolded polypeptides. Structure-based mutagenesis for the conserved hydrophobic residues located in domain I significantly reduced the molecular chaperone activity of P58(IPK).
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preliminary x ray crystallographic studies of mouse upr responsive protein P58 ipk tpr fragment
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2008Co-Authors: Jiahui Tao, David Ron, Bingdong ShaAbstract:Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), which can promote protein folding and misfolded protein degradation and attenuate protein translation and protein translocation into the ER. P58(IPK) has been proposed to function as a molecular chaperone to maintain protein-folding homeostasis in the ER under normal and stressed conditions. P58(IPK) contains nine TPR motifs and a C-terminal J-domain within its primary sequence. To investigate the mechanism by which P58(IPK) functions to promote protein folding within the ER, a P58(IPK) TPR fragment without the C-terminal J-domain was crystallized. The crystals diffract to 2.5 A resolution using a synchrotron X-ray source. The crystals belong to space group P21, with unit-cell parameters a = 83.53, b = 92.75, c = 84.32 A, α = 90.00, β = 119.36, γ = 90.00°. There are two P58(IPK) molecules in the asymmetric unit, which corresponds to a solvent content of approximately 60%. Structure determination by MAD methods is under way.
Glen N Barber - One of the best experts on this subject based on the ideXlab platform.
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the P58 cellular inhibitor complexes with the interferon induced double stranded rna dependent protein kinase pkr to regulate its autophosphorylation and activity
Journal of Biological Chemistry, 1996Co-Authors: Stephen J Polyak, Marlene Wambach, Norina Tang, Glen N Barber, Michael G. KatzeAbstract: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 α 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 α 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.
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the 58 kilodalton inhibitor of the interferon induced double stranded rna activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Glen N Barber, Samantha Thompson, Tae Gyu Lee, Ted S Strom, Rosemary Jagus, Andre Darveau, Michael G. KatzeAbstract: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.
Lorenzo Moretta - One of the best experts on this subject based on the ideXlab platform.
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the human leukocyte antigen hla c specific activatory or inhibitory natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions
Journal of Experimental Medicine, 1996Co-Authors: Roberto Biassoni, Claudia Cantoni, Michela Falco, Simonetta Verdiani, Cristina Bottino, Massimo Vitale, Romana Conte, Alessandro Poggi, Alessandro Moretta, Lorenzo MorettaAbstract:Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (P58) but also as activatory (p50) receptors. Here we show that p50 and P58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas P58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.
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existence of both inhibitory P58 and activatory p50 receptors for hla c molecules in human natural killer cells
Journal of Experimental Medicine, 1995Co-Authors: Alessandro Moretta, Cristina Bottino, Massimo Vitale, Simona Sivori, Daniela Pende, Luigia Morelli, Raffaella Augugliaro, Lorenzo MorettaAbstract:The natural killer (NK) cell-specific P58 molecules EB6 and GL183 have been shown to represent the putative surface receptors for two distinct groups of human histocompatibility leukocyte antigen (HLA) C alleles. Interaction between P58 receptors and class I molecules expressed on target cells results in inhibition of the NK-mediated cytolytic activity and thus in target cell protection. In the present study, we show that EB6 molecules may also act as receptors mediating NK cell triggering. Activatory EB6 molecules were found to be confined only to certain donors. Moreover, in these donors, only a fraction of EB6+ NK clones expressed the activatory form of EB6 molecules, while the remaining clones expressed the conventional inhibitory form. Biochemical analysis of the activatory EB6 molecules revealed a molecular mass of approximately 50 kD (p50), thus differing from the 58-kD inhibitory form. This difference was not due to differential glycosylation of the same protein, as revealed by deglycosylation experiments of isolated EB6 molecules. Treatment of purified P58 or p50/EB6 molecules with proteolytic enzymes, including V8-protease, chymotrypsin, and papain, showed only minor differences in the resulting peptides. Treatment with pepsin followed by two-dimensional peptide mapping demonstrated that, although the majority of peptides migrated in identical positions, differences between the two forms could be detected for at least one major peptide. Anti-EB6 monoclonal antibody (mAb)-mediated cross-linking of p50 molecules was required to trigger the cytolytic activity and the intracellular calcium ([Ca+2]i) increases in appropriate NK clones. Likewise, mAb-mediated cross linking of the P58 EB6 molecules was needed to inhibit the cytolytic activity; however, in this case, no [Ca+2]i increases could be detected. In NK clones expressing the inhibitory P58 EB6 receptors, soluble anti-EB6 mAb prevented recognition of protective Cw4 molecules and reconstituted target cell lysis. In contrast, in clones expressing the activatory p50/EB6 receptor, EB6 masking frequently resulted in partial inhibition of the cytolytic activity against Cw4+ target cells. Therefore, it appears that NK clones expressing the p50/EB6 receptors are induced to lyse Cw4+ target cells upon specific interaction with Cw4 molecules. This concept was further substantiated by experiments in which target cells were represented by the HLA-negative LCL721.221 cell line transfected with the Cw4 allele. Phenotypic and functional analysis of a large number of NK clones showed that clones expressing the activatory p50/EB6 molecules consistently coexpressed inhibitory receptors for other HLA class I alleles.(ABSTRACT TRUNCATED AT 400 WORDS)
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molecular clones of the P58 nk cell receptor reveal immunoglobulin related molecules with diversity in both the extra and intracellular domains
Immunity, 1995Co-Authors: Nicolai Wagtmann, Roberto Biassoni, Claudia Cantoni, Simonetta Verdiani, Cristina Bottino, Massimo Vitale, Alessandro Moretta, Lorenzo Moretta, Mauro S Malnati, Eric O LongAbstract:Recognition of major histocompatibility class I molecules on target cells by natural killer (NK) cells confers selective protection from NK-mediated lysis. Cross-linking of the P58 NK receptor, involved in the recognition of HLA-C alleles, delivers a negative signal that prevents target cell lysis. Molecular cloning of the P58 NK receptor reported here revealed a new member of the immunoglobulin superfamily. Five distinct P58 receptors, with sequence diversity in the immunoglobulin-related domains, were identified in a single individual. All NK clones tested expressed at least one P58 member. Three different types of transmembrane and cytoplasmic domains exist, even among receptors with closely related extracellular domains. These data revealed a repertoire of NK cells with clonally distributed P58 receptors exhibiting diversity in both extracellular and intracellular domains.
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the human natural killer cell receptor for major histocompatibility complex class i molecules surface modulation of P58 molecules and their linkage to cd3 zeta chain fc epsilon ri gamma chain and the p56lck kinase
European Journal of Immunology, 1994Co-Authors: Cristina Bottino, Massimo Vitale, Lorenzo Moretta, Simona Sivori, Luigia Morelli, Raffaella Augugliaro, Lucia Olcese, Ermanno Ciccone, Alessandro MorettaAbstract:The natural killer cell (NK)-specific P58 surface molecules, recognized by the GL183 and EB6 monoclonal antibodies (mAb), have been shown to represent the putative NK receptor for HLA-C molecules. The interaction between P58 receptors and HLA-C results in inhibition of the NK-mediated target cell lysis. In this study, GL183-EB6+ clones (Cw4-specific), after mAb-induced surface modulation of EB6 molecules, acquired the ability to lyse the Cw4+ C1R cells. In NK clones co-expressing both GL183 and EB6 molecules and unable to kill Cw3-protected target cells, the mAb-induced modulation of EB6 molecules resulted both in selective co-modulation of GL183 molecules and in the lysis of Cw3-transfected P815 murine cells. In line with the co-modulation experiments we also show that the GL183 and EB6 molecules can be co-immunoprecipitated from GL183+/EB6+ clones after cell lysis in the presence of digitonin. The P58 receptor also revealed an association with molecules belonging to the zeta family (i.e. CD3 zeta and Fc epsilon RI gamma chains). Two-dimensional diagonal gel analysis of the P58 complex immunoprecipitated from polyclonally activated P58+ NK cells indicated a preferential association with CD3 zeta chains either in the form of covalently linked zeta-zeta homodimers or in the form of zeta-gamma heterodimers, while gamma-gamma homodimers were detectable in low amounts. However, P58+ clones displaying a unique association with gamma-gamma homodimers could also be isolated. Probing the immunoprecipitated P58 complex with anti-p56lck antibody also revealed an association with this member of the src family. In addition, mAb-mediated signaling of NK clones via P58 molecules induced increments of P58/p56lck association. However, under the same experimental conditions that induced optimal in vivo tyrosine phosphorylation of the CD16-associated CD3 zeta chains, no tyrosine phosphorylation was detected in the P58-associated CD3 zeta chains. In these in vivo experiments neither anti-CD16 nor anti-P58 mAb could induce tyrosine phosphorylation of the gamma chains. Finally, the anti-P58-mediated inhibition of the NK cell triggering via CD16 molecules was not accompanied by a down-regulation of the tyrosine phosphorylation of the CD16-associated CD3 zeta chains.
Tae Gyu Lee - One of the best experts on this subject based on the ideXlab platform.
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the 58 kilodalton inhibitor of the interferon induced double stranded rna activated protein kinase is a tetratricopeptide repeat protein with oncogenic properties
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Glen N Barber, Samantha Thompson, Tae Gyu Lee, Ted S Strom, Rosemary Jagus, Andre Darveau, Michael G. KatzeAbstract: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.
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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
Molecular and Cellular Biology, 1994Co-Authors: Tae Gyu Lee, Samantha Thompson, Norina Tang, James G Miller, Michael G. KatzeAbstract: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.
