J R Cook

Robert Wood Johnson University Hospital, New Brunswick, New Jersey, United States

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Publications (13)92.79 Total impact

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    ABSTRACT: The Jak family of protein-tyrosine kinases are crucial for the signaling of a large number of different polypeptide ligands, including the interferons, many cytokines, erythropoietin, and growth factors. Through their interaction with receptors, the Jaks initiate a signaling cascade resulting in the activation of gene transcription and ultimately a cellular response to various ligands. In addition to their role in cellular signaling, alteration of Jak activity has been implicated in several disease states. In identifying Jak2-interacting proteins with the yeast two-hybrid system, we cloned the human homologue of the Drosophila melanogaster tumor suppressor gene lethal () tumorous imaginal discs, which encodes the protein Tid56. Drosophila Tid56 and its human homologue hTid-1 represent members of the DnaJ family of molecular chaperones. The TID1 gene encodes two splice variants hTid-1(S) and hTid-1(L). We confirmed the interaction between Jak2 and hTid-1(S) or hTid-1(L) by immunoprecipitation from COS-1 cells expressing these proteins. The interaction between endogenous hTid-1 and Jak2 was shown in HEp2 cells. We further showed that hTid-1 interacts with the human interferon-gamma (Hu-IFN-gamma) receptor subunit IFN-gamma R2. In addition, using a chimeric construct where the extracellular domain of IFN-gamma R2 was fused to the kinase domain of Jak2, we showed that hTid-1 binds more efficiently to the chimera with an active kinase domain than to a similar construct with an inactive kinase domain. Additionally, the data demonstrate that hTid-1 isoforms as well as Jak2 interact with Hsp70/Hsc70 in vivo, and the interaction between Hsp70/Hsc70 and hTid-1 is reduced after IFN-gamma treatment. Furthermore, both hTid-1(S) and hTid-1(L) can modulate IFN-gamma-mediated transcriptional activity.
    Journal of Biological Chemistry 01/2002; 276(52):49034-42. · 4.65 Impact Factor
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    ABSTRACT: We have identified a new mammalian protein arginine N-methyltransferase, PRMT5, formerly designated Janus kinase-binding protein 1, that can catalyze the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine in a variety of proteins. A hemagglutinin peptide-tagged PRMT5 complex purified from human HeLa cells catalyzes the S-adenosyl-l-[methyl-(3)H]methionine-dependent in vitro methylation of myelin basic protein. When the radiolabeled myelin basic protein was acid-hydrolyzed to free amino acids, and the products were separated by high-resolution cation exchange chromatography, we were able to detect two tritiated species. One species co-migrated with a omega-N(G)-monomethylarginine standard, and the other co-chromatographed with a symmetric omega-N(G),N(G')-dimethylarginine standard. Upon base treatment, this second species formed methylamine, a breakdown product characteristic of symmetric omega-N(G),N(G')-dimethylarginine. Further analysis of these two species by thin layer chromatography confirmed their identification as omega-N(G)-monomethylarginine and symmetric omega-N(G),N(G')-dimethylarginine. The hemagglutinin-PRMT5 complex was also able to monomethylate and symmetrically dimethylate bovine histone H2A and a glutathione S-transferase-fibrillarin (amino acids 1-148) fusion protein (glutathione S-transferase-GAR). A mutation introduced into the S-adenosyl-l-methionine-binding motif I of a myc-tagged PRMT5 construct in COS-1 cells led to a near complete loss of observed enzymatic activity. PRMT5 is the first example of a catalytic chain for a type II protein arginine N-methyltransferase that can result in the formation of symmetric dimethylarginine residues as observed previously in myelin basic protein, Sm small nuclear ribonucleoproteins, and other polypeptides.
    Journal of Biological Chemistry 09/2001; 276(35):32971-6. · 4.65 Impact Factor
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    ABSTRACT: The yeast protein Hsl7p is a homologue of Janus kinase binding protein 1, JBP1, a newly characterized protein methyltransferase. In this report, Hsl7p also is shown to be a methyltransferase. It can be crosslinked to [(3)H]S-adenosylmethionine and exhibits in vitro protein methylation activity. Calf histones H2A and H4 and bovine myelin basic protein were methylated by Hsl7p, whereas histones H1, H2B, and H3 and bovine cytochrome c were not. We demonstrated that JBP1 can complement Saccharomyces cerevisiae with a disrupted HSL7 gene as judged by a reduction of the elongated bud phenotype, and a point mutation in the JBP1 S-adenosylmethionine consensus binding sequence eliminated all complementation by JBP1. Therefore, we conclude the yeast protein Hsl7p is a sequence and functional homologue of JBP1. These data provide evidence for an intricate link between protein methylation and macroscopic changes in yeast morphology.
    Biochemical and Biophysical Research Communications 08/2000; 274(1):105-11. · 2.28 Impact Factor
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    ABSTRACT: With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.
    Cytokine & Growth Factor Reviews 10/1997; 8(3):189-206. · 8.83 Impact Factor
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    ABSTRACT: Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-gamma receptor (hu-IFN-gammaR) requires the formation of a complex consisting of ligand (IFN-gamma), a ligand binding receptor chain (IFN-gammaR1), and a signal transducing receptor chain (IFN-gammaR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-gamma can exert on fibroblasts. The current report assesses the ability of hu-IFN-gammaR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-gammaR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-gammaR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-gamma with an affinity comparable to that of human cells. Binding of either human or mouse IFN-gamma to its respective receptor elicits classic IFN-gamma responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2- radicals, interleukin-6, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-gamma could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-gamma could. These results demonstrate that while co-expression of hu-IFN-gammaR1 and hu-IFN-gammaR2 is necessary and sufficient for most IFN-gamma-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-gamma receptor complex. The nature and potential role of such factors in IFN-gammaR signaling is discussed.
    Journal of Biological Chemistry 01/1997; 271(51):32659-66. · 4.65 Impact Factor
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    ABSTRACT: Chinese hamster ovary cells containing the yeast artificial chromosome F136C5 (alphaYAC) respond to all type I human interferons including IFN-alphaA, IFN-beta, and IFN-omega. The alphaYAC contains at least two genes encoding interferon-alpha receptor (IFN-alphaR) chains that are required for response to type I human interferons: Hu-IFN-alphaR1 and Hu-IFN-alphaR2. We previously isolated a splice variant of the Hu-IFN-alphaR1 chain designated Hu-IFN-alphaR1s. Chinese hamster ovary cells containing a disrupted alphaYAC, which contains a deletion in the human IFNAR1 gene, were transfected with expression vectors for the Hu-IFN-alphaR1 and Hu-IFN-alphaR1s chains. With these cells, two type I interferons have been identified which can interact with the splice variant (Hu-IFN-alphaR1s) and with the Hu-IFN-alphaR1 chains: Hu-IFN-alphaA and IFN-omega. Two other type I interferons, Hu-IFN-alphaB2 and Hu-IFN-alphaF, are capable of signaling through the Hu-IFN-alphaR1 chain only and cannot utilize the splice variant Hu-IFN-alphaR1s. Hu-IFN-alphaR1 and Hu-IFN-alphaR1s differ in that the latter is missing a single subdomain of the receptor extracellular domain encoded by exons 4 and 5 of the IFNAR1 gene. These results therefore indicate that different type I interferons require different subdomains of the Hu-IFN-alphaR1 receptor chain, and that the splice variant chain (Hu-IFN-alphaR1s) is functional.
    Journal of Biological Chemistry 07/1996; 271(23):13448-53. · 4.65 Impact Factor
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    ABSTRACT: Interferon gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different chains: the IFN-gamma receptor binding subunit (IFN-gamma R, IFN-gamma R1), and a transmembrane accessory factor (AF-1, IFN-gamma R2) necessary for signal transduction. Using cell lines expressing different cloned components of the IFN-gamma receptor complex, we examined the function of the receptor components in signal transduction upon IFN-gamma treatment. A specific IFN-gamma R2:IFN-gamma cross-linked complex was observed in cells expressing both IFN-gamma R1 and IFN-gamma R2 indicating that IFN-gamma R2 (AF-1) interacts with IFN-gamma and is closely associated with IFN-gamma R1. We show that the intracellular domain of IFN-gamma R2 is necessary for signaling. Cells coexpressing IFN-gamma R1 and truncated IFN-gamma R2, lacking the COOH-terminal 51 amino acids (residues 286-337), or cells expressing IFN-gamma R1 alone were unresponsive to IFN-gamma treatment as measured by MHC class I antigen induction. Jak1, Jak2, and Stat1 alpha were activated, and IFN-gamma R1 was phosphorylated only in cells expressing both IFN-gamma R1 and IFN-gamma R2. Jak2 kinase was shown to associate with the intracellular domain of the IFN-gamma R2.
    Journal of Biological Chemistry 10/1995; 270(36):20915-21. · 4.65 Impact Factor
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    ABSTRACT: A set of fragmentation vectors is described which produce a deletion series of smaller yeast artificial chromosomes (YACs) from a larger parent YAC with the insertion of a eukaryotic selectable marker. In addition, new vectors were designed to permit integration of the genes encoding neomycin (neo) or hygromycin B (hyg) resistance into YACs containing inserts of human DNA. All these vectors are compatible with the yeast host strain AB1380, in which most human genomic YAC libraries are maintained. Linearized vector DNA is used to transform yeast cells in which homologous recombination between human DNA in the YAC and the Alu sequence in the fragmentation or integrating vector produces terminal deletions from the acentromeric (URA3) end of the YAC or insertion of the vector into the YAC, respectively. A set of directional deletions of a YAC is useful for genomic mapping, restriction analysis and functional measurements of large chromosomal regions. The neo and hyg eukaryotic markers permit the study of gene function after introduction of deleted YACs into mammalian cells. Transformation of YACs with the fragmentation vectors resulted in fragmentation in 21-46% of the clones examined; transformation with the integrating vector resulted in integration in 46% of the clones examined.
    Gene 05/1995; 155(2):167-74. · 2.20 Impact Factor
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    ABSTRACT: Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by induction of class I human leukocyte antigen (HLA) and protection against encephalomyocarditis virus (EMCV) infection. Whereas human chromosome 6 encodes the Hu-IFN-gamma receptor, human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. Probes from a genomic clone were used to identity cDNA clones expressing a species-specific accessory factor. These cDNA clones are able to substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma receptor-mediated induction of class I HLA antigens. However, the factor encoded by the cDNA does not confer full antiviral protection against EMCV, confirming that an additional factor encoded on human chromosome 21 is required for reconstitution of antiviral activity against EMCV. We conclude that this accessory factor belongs to a family of such accessory factors responsible for different actions of IFN-gamma.
    Cell 04/1994; 76(5):793-802. · 31.96 Impact Factor
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    ABSTRACT: A chromosomal fragmentation procedure was employed to produce a deletion set of yeast artificial chromosomes (YACs) from a parental YAC, GART D142H8, known to map to human chromosome 21q and to encode the human interferon-gamma receptor (Hu-IFN-gamma R) accessory factor gene as well as the phosphoribosylglycinamide formyltransferase (GART) gene. When expressed in Chinese hamster ovary cells, these deleted YACs retain accessory factor activity, as judged by major histocompatibility complex class I antigen inducibility, until the deletions from the acentric end exceed 390 kilobases (kb). Therefore, the accessory factor (AF-1) gene can be localized to a 150-kb region at the left (centric) end of the parental 540-kb GART YAC. Cells containing functional YACs are also able to induce the ISGF3 gamma and gamma-activated factor (GAF) transcription factors, but were not protected against encephalomyocarditis virus (EMCV) upon treatment with Hu-IFN-gamma. Therefore, the Hu-IFN-gamma R and the AF-1 are sufficient for some, but not all, of the actions of Hu-IFN-gamma. We postulate that an additional accessory factor (AF-2) required for antiviral activity against EMCV is encoded on chromosome 21q.
    Journal of Biological Chemistry 04/1994; 269(9):7013-8. · 4.65 Impact Factor
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    ABSTRACT: Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.
    Proceedings of the National Academy of Sciences 10/1993; 90(18):8737-41. · 9.81 Impact Factor
  • J R Cook, S L Emanuel, S Pestka
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    ABSTRACT: Two fragmentation vectors, pSE1 and pSE2, were developed for targeting yeast artificial chromosomes (YACs) containing human genomic DNA. Ura- yeast cells containing YACs were selected with 5-fluoro-orotic acid. Fragmented YACs were subsequently generated by transformation to a Ura+ phenotype. Over 80% of the transformants contained YACs of reduced molecular size. These fragmented YACs will prove to be useful in mapping the region of human chromosomes covered by the parental YAC. Fragmentation utilizing URA3 transformation provides a method for producing YAC deletion sets from YACs contained in AB1380 and other ura3- yeast stains. Linkage of a neomycin resistance gene to the URA3 gene facilitates functional analysis of these YACs in eukaryotic cells.
    Genetic Analysis Biomolecular Engineering 02/1993; 10(5):109-12.
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    ABSTRACT: Mutations of the human interferon gamma (IFN-gamma) receptor intracellular domain have permitted us to define a restricted region of that domain as necessary for both induction of class I major histocompatibility complex antigen by IFN-gamma and protection against encephalomyocarditis virus. This region consists of five amino acids (YDKPH), all of which are conserved in the human and murine receptors. Tyr-457 and His-461 are essential for activity. Approximately 80% of the amino acids of the intracellular domain of the receptor is not required for major histocompatibility complex class I antigen induction or for antiviral protection against encephalomyocarditis virus. The observation that there was no protection by IFN-gamma against vesiculostomatitis virus indicates that other factors, in addition to chromosome 21 accessory factor(s), are required to generate the full complement of transduction signals from the human IFN-gamma receptor.
    Proceedings of the National Academy of Sciences 01/1993; 89(23):11317-21. · 9.81 Impact Factor