Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.3.23 (GAS)
957 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Prolactin (PRL) induces transcriptional activation of milk protein genes, such as the whey acidic protein (WAP), beta-casein, and beta-lactoglobulin genes, through a signaling cascade encompassing the Janus kinase Jak2 and the mammary gland factor (MGF; also called Stat5), which belongs to the family of proteins of signal transducers and activators of transcription (STAT). We isolated and sequenced from mouse mammary tissue Stat5 mRNA and a previously unreported member, which we named Stat5b (Stat5 is renamed to Stat5a). On the protein level Stat5a and Stat5b show a 96% sequence similarity. The 5' and 3' untranslated regions of the two mRNAs are not conserved. Stat5a comprises 793 amino acids and is encoded by a mRNA of 4.2 kb. The Stat5b mRNA has a size of 5.6 kb and encodes a protein of 786 amino acids. Both Stat5a and Stat5b recognized the GAS site (gamma-interferon-activating sequence; TTCNNNGAA) in vitro and mediated PRL-induced transcription in COS cells transfected with a PRL receptor. Stat5b also induced basal transcription in the absence of PRL. Similar levels of Stat5a and Stat5b mRNAs were found in most tissues of virgin and lactating mice, but a differential accumulation of the Stat5 mRNAs was found in muscle and mammary tissue. The two RNAs are present in mammary tissue of immature virgin mice, and their levels increase up to day 16 of pregnancy, followed by a decline during lactation. The increase of Stat5 expression during pregnancy coincides with the activation of the WAP gene.
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PMID:Cloning and expression of Stat5 and an additional homologue (Stat5b) involved in prolactin signal transduction in mouse mammary tissue. 756 26

Interleukin-3 (IL-3) is an important regulator of hemopoiesis and considerable effort has been directed towards the study of its mechanism of signal transduction. In this paper, we describe the first molecular identification of a STAT transcription factor that is activated by IL-3. STATs exist in a cytoplasmic, transcriptionally inactive form which, in response to extracellular signals, become tyrosine phosphorylated and translocate to the nucleus where they bind to specific DNA elements. Several of these DNA elements were found which bind proteins in an IL-3-responsive manner. Analysis of these bandshift complexes with available antibodies to the known STATs suggests that IL-3 activates the DNA-binding ability of STAT5, a protein which was originally characterized as a prolactin-responsive transcription factor in sheep. IL-5 and granulocyte-macrophage colony stimulating factor (GM-CSF), which share a common signaling receptor subunit with IL-3, also activate STAT5. Unexpectedly, two murine STAT5 homologs, 96% identical to each other at the amino acid level, were isolated and IL-3-dependent GAS binding could be reconstituted in COS cells transfected with IL-3 receptor and either STAT5 cDNA. In IL-3-dependent hemopoietic cells, both forms of STAT5 are expressed and activated in response to IL-3.
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PMID:Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. 772 Jul 7

The rat homolog of sheep mammary gland factor (MGF)/Stat5 has been isolated and used to study the regulation of Stat5 during mammary gland development and PRL regulation in COS cells transfected with Stat5a and the PRL receptor. Two alternatively spliced isoforms, designated Stat5a1 and Stat5a2, were identified, the latter encoding a carboxy-terminal truncated protein. A polyclonal antibody to a carboxy-terminal peptide of Stat5a1 was generated and used to measure the level of this isoform during mammary gland development and after PRL induction in COS cells transiently transfected with Stat5a and the long form of the PRL receptor. Surprisingly, Stat5a mRNA and protein were readily detected both in virgin rats and after mammary gland involution. The levels of Stat5a increased during pregnancy, were highest in late pregnancy, and then, unexpectedly, decreased during lactation, the time at which the highest levels of milk protein gene expression are observed. Electrophoretic mobility shift assays using the specific anti-Stat5a1 antisera demonstrated that Stat5a1 comprises part of the heterogeneous, PRL-inducible, protein-DNA complex associated with the beta-casein GAS site. Immunocytochemical analysis detected considerable cytoplasmic and some nuclear staining for Stat5a1 during late pregnancy and predominantly nuclear staining during early lactation. The lack of correspondence of Stat5a gene expression and beta-casein gene expression suggests that Stat5 activation may facilitate the interaction of other factors binding within composite response elements identified recently in the milk protein gene promoters that are then responsible for the stable expression of milk protein genes in terminally differentiated mammary epithelial cells.
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PMID:Regulation of mammary gland factor/Stat5a during mammary gland development. 858 36

GH is known to activate JAK2 tyrosine kinase and members of the Stat family of transcription factors, including Stats 1, 3, and 5. The recent observation that at least two Stat5 proteins (Stat5A and Stat5B) exist in mouse and human, raises the question of whether GH activates both Stat5A and Stat5B and, if so, whether the requirements for activation are the same. An initial report investigating this issue demonstrated GH-dependent activation of Stat5A but not Stat5B. In this paper, we demonstrate (in COS cells expressing rat GH receptor (rGHR) and either Stat5A or Stat5B, 3T3-F442A fibroblasts, and CHO cells expressing rGHR) that GH induces tyrosyl phosphorylation of both Stat5A and Stat5B. Similar time courses of phosphorylation were observed for the two proteins. Interestingly, the pattern of observed bands differs for the two forms of Stat5. Two closely migrating Stat5A bands can be detected in cells treated with or without GH. Both of these bands become tyrosyl phosphorylated in response to GH. Three species of Stat5B are observed in untreated cells. An additional, more slowly migrating Stat5B band, appears upon treatment with GH. The three more slower migrating Stat5B bands observed in response to GH contain phosphorylated tyrosyl residues. We further demonstrate that GH induces binding of Stat5A and Stat5B, as well as Stat1, to the GAS-like element in the beta-casein promoter. We and others have demonstrated previously that specific regions of GHR are required for GH-dependent activation of what is here identified as Stat5B. To gain insight into the mechanism by which GH promotes tyrosyl phosphorylation of Stat5A, GH-dependent tyrosyl phosphorylation of Stat5A was examined in CHO cells expressing truncated and mutated rGHR. The results indicate that Stat5A and Stat5B require the same regions of rGHR for maximal activation by GH: the C-terminal half of the cytoplasmic domain; tyrosines 333 and/or 338 in the N-terminal half of the cytoplasmic domain; and the regions required for JAK2 activation. To dissect further the mechanism by which GH activates Stat5A and B, the requirement for JAK2 in GH-dependent Stat5 tyrosyl phosphorylation was assessed using JAK2-deficient cells expressing GHR (gamma2A-GHR) and the wild-type parental cell line expressing GHR (2C4-GHR). GH-induced tyrosyl phosphorylation of Stat5B in 2C4-GHR cells but not in the JAK2 deficient, gamma2A-GHR cells, indicating that JAK2 is required for GH-dependent tyrosyl phosphorylation of Stat5B. Western blotting revealed that Stat5A is not expressed in this cell type. Taken together, these findings suggest that: 1) GH activates both Stat5A and Stat5B in several cell types; 2) the pattern of bands observed differs for Stat5A and Stat5B; 3) GH-dependent tyrosyl phosphorylation of Stat5A requires specific regions of GHR, and these requirements are the same as for Stat5B; and 4) JAK2 kinase is required for GH-dependent tyrosyl phosphorylation of Stat5B and, most likely, Stat5A.
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PMID:Growth hormone-induced tyrosyl phosphorylation and deoxyribonucleic acid binding activity of Stat5A and Stat5B. 923 97

The Nb2 PRL receptor (PRL-R) is known to mediate PRL signaling to the interferon (IFN) regulatory factor-1 (IRF-1) gene via the family of signal transducers and activators of transcription or Stats. To analyze the components of the PRL-R/Stat/IRF-1 signaling pathway, various PRL-R, Stat, and IRF-1-CAT reporter constructs were transiently cotransfected into COS cells. First, mutations in the IFNgamma-activated sequence (GAS), either multimerized or in the context of the 1.7-kb IRF-1 promoter, failed to mediate a PRL response, showing that the IRF-1 GAS is a target of PRL signaling. Next, pairwise alanine substitutions into conserved residues in the proline-rich motif or Box 1 region and two tyrosine mutations, Y308F and Y382F, in the PRL-R intracellular domain all impaired PRL signaling to multimerized GAS or to the 1.7-kb IRF-1 promoter. Furthermore, these PRL-R mutants mediated reduced Stat1 binding to the IRF-1 GAS. Transfection of Stat1 further enhanced PRL signaling to the IRF-1 promoter, suggesting that Stat1 is a positive mediator of PRL action. These studies show that both membrane proximal and distal residues of the PRL-R are involved in signaling to the IRF-1 gene. Further, Stat1 and the GAS element are important for PRL activation of the IRF-1 gene.
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PMID:Multiple prolactin (PRL) receptor cytoplasmic residues and Stat1 mediate PRL signaling to the interferon regulatory factor-1 promoter. 925 25

Prolactin (PRL) induces transcriptional activation of not only growth-related genes such as interferon regulatory factor-1 (IRF-1) but also differentiation-specific genes such as beta-casein through a signaling cascade consisting of Janus kinases and Stat (signal transducer and activator of transcription) factors. To understand better the role of Stats in PRL signaling, we cloned rat Stat5b from a PRL-responsive T cell line Nb2. A Stat5b-specific peptide antibody was generated. In PRL receptor reconstituted COS cells cotransfected with Stat5b or Stat5a, both Stat5 proteins become tyrosine phosphorylated and bind to the IRF-1 GAS (interferon-gamma activation sequence) element in a PRL-inducible manner. Unexpectedly, both Stat5b and Stat5a inhibit PRL induction of the IRF-1 promoter, but they mediate PRL stimulation of the beta-casein promoter. Stat5-mediated inhibition was observed only at the native IRF-1 promoter and not at the isolated IRF-1 GAS element linked to a heterologous thymidine kinase promoter. Mutational analyses showed that the DNA binding activity of Stat5b is not required, but the carboxyl-terminal transactivation domain is essential for Stat5b to inhibit PRL induction of the IRF-1 promoter. These results suggest that Stat5b mediates inhibition via protein-protein interactions. In contrast, both DNA binding and transactivation domains of Stat5b are required to mediate PRL induction of the beta-casein promoter. Furthermore, a carboxyl-terminal truncated dominant negative Stat5b can reverse Stat5b inhibition at the IRF-1 promoter. These studies suggest that Stat proteins can act as not only positive but also negative regulators of gene transcription. Further, Stat5 can modulate gene expression without binding to DNA but via protein-protein interactions.
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PMID:Transcriptional inhibition by Stat5. Differential activities at growth-related versus differentiation-specific promoters. 934 Nov 15

The PRL receptor (PRL-R) signals through the Janus tyrosine kinases (JAK) and other non-JAK tyrosine kinases, some of which are preassociated with the PRL-R. To clone PRL-R interacting proteins, the intracellular domain (ICD) of the long form of the PRL-R was used in a yeast two-hybrid screen of a human B cell cDNA library. One PRL-R interacting protein was identified as the 42-kDa form of the enzyme 2',5'-oligoadenylate synthetase (OAS). The in vivo interactions in yeast were further confirmed by an in vitro interaction assay and by coimmunoprecipitation in transfected mammalian cells. Functionally, OAS reduced the basal activity of two types of promoters in transiently transfected COS-1 cells. In the presence of PRL, OAS inhibited PRL induction of the immediate early IRF-1 (interferon-regulatory factor 1) promoter, but not PRL induction of the differentiation-specific beta-casein promoter, suggesting that OAS exerts specific effects on immediate early gene promoters. The inhibitory effects of OAS were accompanied by a reduction in PRL-inducible Stat1 (signal transducer and activator of transcription 1) DNA binding activity at the IRF-1 GAS (interferon-gamma-activated sequence) element. These results demonstrate a novel interaction of OAS with the PRL-R and suggest a role for OAS in modulating Stat1-mediated signaling to an immediate early gene promoter. Although previously characterized as a regulator of ribonuclease (RNase) L antiviral responses, OAS may have additional effects on cytokine receptor signal transduction pathways.
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PMID:Association of 2',5'-oligoadenylate synthetase with the prolactin (PRL) receptor: alteration in PRL-inducible stat1 (signal transducer and activator of transcription 1) signaling to the IRF-1 (interferon-regulatory factor 1) promoter. 1067 1

STATs (signal transducers and activators of transcription) are proteins with dual functions: signal transducers in the cytoplasm and transcriptional activators in the nucleus. STAT proteins act as transcription factors activated by phosphorylation on its tyrosine residues upon stimulation by various cytokines. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interaction and translocate into the nucleus to activate the transcription of various target genes. STAT5 recognizes the interferon-gamma activated site TTCNNNGAA (GAS sequence) in the promoter region of the beta-casein gene. Except for prolactin-dependent beta-casein production in mammary gland cells, the biological consequences of STAT5a activation in various systems are not clear. Here we showed that STAT5a was phosphorylated 10 min after desferrioxamine (DFO) treatment, and reached a maximum induction at 4 h in mammary epithelial cells (HC11) and transfected COS-7 cells. Under hypoxic conditions (2% O2), a maximal phosphorylation of STAT5a was observed within 6 h. EMSA (electrophoretic mobility shift assay) showed that DFO or hypoxia enhanced the binding activities of STAT5a DNA to beta-casein gene promoter in mammary epithelial cells (HC11) and transfected COS-7 cells. These results showed that DFO or hypoxia induces tyrosine phosphorylation of STAT5a and also increases the binding activity of STAT5a DNA in mammary epithelial cells. Our data suggest that the STAT5 may act as a mediator in hypoxia-mediated gene expression.
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PMID:Hypoxia activates signal transducers and activators of transcription 5 (STAT5) and increases its binding activity to the GAS element in mammary epithelial cells. 1464 87

Hypoxia, a common consequence of solid tumor growth in breast cancer or other cancers, serves to propagate a cascade of molecular pathways which include angiogenesis, glycolysis, and various cell-cycle control proteins. As we have shown previously, hypoxia activates STAT5 (signal transducer and activator of transcription 5) and increases its binding activity to the GAS element in mammary epithelial cells. In this study we attempted to elucidate the mechanism by which cyclin D1 is regulated by the STAT5 protein under hypoxic conditions. Our data demonstrate that hypoxia (2% O(2)) or desferrioxamine (DFO) induces tyrosine and serine phosphorylation of STAT5 in human breast cancer cells (MCF-7) and mammary epithelial cells (HC11). Imunoprecipitation and subsequent Western analysis showed that Jak2 leads to the tyrosine phosphorylation and activation of STAT5a or STAT5b under hypoxic conditions. Using a transfected COS-7 cell model system, we demonstrate that the activity of a cyclin D1 promoter-luciferase construct increased under hypoxic conditions or DFO treatment. The activity of the STAT5b/cyclin D1 promoter increased significantly by 12 h of hypoxia, whereas the activity of the STAT5a/cyclin D1 promoter was unaffected under hypoxic conditions. These increases in promoter activity are predominantly mediated by the Jak2/STAT5b signaling pathway. We have shown by EMSA that hypoxia induces STAT5 to bind to the cyclin D1 promoter (GAS-1) in MCF-7 and HC11 cells. These data suggest that STAT5b may mediate the transcriptional activation of cyclin D1 after hypoxic stimulation.
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PMID:Hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. 1615 12

The sesquiterpene costunolide has a broad range of biological activities and is the parent compound for many other biologically active sesquiterpenes such as parthenolide. Two enzymes of the pathway leading to costunolide have been previously characterized: germacrene A synthase (GAS) and germacrene A oxidase (GAO), which together catalyse the biosynthesis of germacra-1(10),4,11(13)-trien-12-oic acid. However, the gene responsible for the last step toward costunolide has not been characterized until now. Here we show that chicory costunolide synthase (CiCOS), CYP71BL3, can catalyse the oxidation of germacra-1(10),4,11(13)-trien-12-oic acid to yield costunolide. Co-expression of feverfew GAS (TpGAS), chicory GAO (CiGAO), and chicory COS (CiCOS) in yeast resulted in the biosynthesis of costunolide. The catalytic activity of TpGAS, CiGAO and CiCOS was also verified in planta by transient expression in Nicotiana benthamiana. Mitochondrial targeting of TpGAS resulted in a significant increase in the production of germacrene A compared with the native cytosolic targeting. When the N. benthamiana leaves were co-infiltrated with TpGAS and CiGAO, germacrene A almost completely disappeared as a result of the presence of CiGAO. Transient expression of TpGAS, CiGAO and CiCOS in N. benthamiana leaves resulted in costunolide production of up to 60 ng.g(-1) FW. In addition, two new compounds were formed that were identified as costunolide-glutathione and costunolide-cysteine conjugates.
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PMID:Reconstitution of the costunolide biosynthetic pathway in yeast and Nicotiana benthamiana. 2185 47


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