Showing papers on "Transcription factor published in 1994"
TL;DR: The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, severe phase response, and radiation damage.
Abstract: NF-kappa B is a ubiquitous transcription factor. Nevertheless, its properties seem to be most extensively exploited in cells of the immune system. Among these properties are NF-kappa B's rapid posttranslational activation in response to many pathogenic signals, its direct participation in cytoplasmic/nuclear signaling, and its potency to activate transcription of a great variety of genes encoding immunologically relevant proteins. In vertebrates, five distinct DNA binding subunits are currently known which might extensively heterodimerize, thereby forming complexes with distinct transcriptional activity, DNA sequence specificity, and cell type- and cell stage-specific distribution. The activity of DNA binding NF-kappa B dimers is tightly controlled by accessory proteins called I kappa B subunits of which there are also five different species currently known in vertebrates. I kappa B proteins inhibit DNA binding and prevent nuclear uptake of NF-kappa B complexes. An exception is the Bcl-3 protein which in addition can function as a transcription activating subunit in th nucleus. Other I kappa B proteins are rather involved in terminating NF-kappa B's activity in the nucleus. The intracellular events that lead to the inactivation of I kappa B, i.e. the activation of NF-kappa B, are complex. They involve phosphorylation and proteolytic reactions and seem to be controlled by the cells' redox status. Interference with the activation or activity of NF-kappa B may be beneficial in suppressing toxic/septic shock, graft-vs-host reactions, acute inflammatory reactions, acute phase response, and radiation damage. The inhibition of NF-kappa B activation by antioxidants and specific protease inhibitors may provide a pharmacological basis for interfering with these acute processes.
4,708 citations
TL;DR: The results suggest that the physiologic role of PPAR gamma 2 is to regulate development of the adipose lineage in response to endogenous lipid activators and that this factor may serve to link the process of adipocyte differentiation to systemic lipid metabolism.
3,420 citations
TL;DR: In this paper, an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice was identified.
Abstract: Previously, we have isolated and characterized an enhancer from the 5'-flanking region of the adipocyte P2 (aP2) gene that directs high-level adipocyte-specific gene expression in both cultured cells and transgenic mice. The key regulator of this enhancer is a cell type-restricted nuclear factor termed ARF6. Target sequences for ARF6 in the aP2 enhancer exhibit homology to a direct repeat of hormone response elements (HREs) spaced by one nucleotide; this motif (DR-1) has been demonstrated previously to be the preferred binding site for heterodimers of the retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR). We have cloned a novel member of the peroxisome proliferator-activated receptor family designated mPPAR gamma 2, and we demonstrate that a heterodimeric complex of mPPAR gamma 2 and RXR alpha constitute a functional ARF6 complex. Expression of mPPAR gamma 2 is induced very early during the differentiation of several cultured adipocyte cell lines and is strikingly adipose-specific in vivo. mPPAR gamma 2 and RXR alpha form heterodimers on ARF6-binding sites in vitro, and antiserum to RXR alpha specifically inhibits ARF6 activity in adipocyte nuclear extracts. Moreover, forced expression of mPPAR gamma 2 and RXR alpha activates the adipocyte-specific aP2 enhancer in cultured fibroblasts, and this activation is potentiated by peroxisome proliferators, fatty acids, and 9-cis retinoic acid. These results identify mPPAR gamma 2 as the first adipocyte-specific transcription factor and suggest mechanisms whereby fatty acids, peroxisome proliferators, 9-cis retinoic acid, and other lipids may regulate adipocyte gene expression and differentiation.
2,237 citations
TL;DR: Reporter constructs containing truncated promoter regions revealed that activation of NF-kappa B/Rel is critical in the induction of iNOS by LPS, however, additional, newly synthesized proteins contribute to the NF- kappa Bd-dependent transcription factor complex on the iN OS promoter in LPS-treated mouse macrophages.
2,195 citations
TL;DR: The ubiquitin-proteasome pathway has been shown to play an essential role in two proteolytic processes required for activation of the transcription factor NF-κB as discussed by the authors.
2,059 citations
TL;DR: The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs.
Abstract: The transcription factor nuclear factor-kappa B (NF-kappa B) is critical for the inducible expression of multiple cellular and viral genes involved in inflammation and infection including interleukin-1 (IL-1), IL-6, and adhesion molecules. The anti-inflammatory drugs sodium salicylate and aspirin inhibited the activation of NF-kappa B, which further explains the mechanism of action of these drugs. This inhibition prevented the degradation of the NF-kappa B inhibitor, I kappa B, and therefore NF-kappa B was retained in the cytosol. Sodium salicylate and aspirin also inhibited NF-kappa B-dependent transcription from the Ig kappa enhancer and the human immunodeficiency virus (HIV) long terminal repeat (LTR) in transfected T cells.
1,771 citations
TL;DR: It is demonstrated that the transcription factor GATA-2 plays a critical role in haematopoiesis, particularly of an adult type, and proposed that it regulates genes controlling growth factor responsiveness or the proliferative capacity of early haem atopoietic cells.
Abstract: Blood cell development relies on the expansion and maintenance of haematopoietic stem and progenitor cells in the embryo. By gene targeting in mouse embryonic stem cells, we demonstrate that the transcription factor GATA-2 plays a critical role in haematopoiesis, particularly of an adult type. We propose that GATA-2 regulates genes controlling growth factor responsiveness or the proliferative capacity of early haematopoietic cells.
1,414 citations
TL;DR: Investigating whether one of the mechanisms by which glucocorticoids exert their antiinflammatory activities is through inhibition of gene activation mediated by NF-kappa B suggests that direct interactions between NF- kappa B and glucoc Corticoid receptor may partly account for the antiinflammatory properties of glucocORTicoids in vivo.
Abstract: Glucocorticoids, which are widely used as antiinflammatory agents, downregulate the expression of the interleukin 6 gene and of additional cytokine genes involved in inflammatory responses. Conversely, the transcription factor NF-kappa B, a member of the Rel family of transcription factors, has been implicated in the induction of multiple genes involved in the early processes of immune and inflammatory responses. This prompted us to investigate whether one of the mechanisms by which glucocorticoids exert their antiinflammatory activities is through inhibition of gene activation mediated by NF-kappa B. We report that, in intact cells, activation of the interleukin 6 promoter by a combination of the factor NF-IL6 and the p65 subunit of NF-kappa B is inhibited by dexamethasone (ligand)-activated glucocorticoid receptor. Conversely, activation of the mouse mammary tumor virus promoter by a combination of dexamethasone and glucocorticoid receptor is inhibited by overexpression of p65. Furthermore, we provide evidence for physical association between glucocorticoid receptor and p65 in protein crosslinking and coimmunoprecipitation experiments, using either in vitro translated proteins or those present in cell extracts. These studies suggest that direct interactions between NF-kappa B and glucocorticoid receptor may partly account for the antiinflammatory properties of glucocorticoids in vivo.
1,046 citations
TL;DR: The purification and cloning of APRF are reported and it is observed that p91 is not tyrosine phosphorylated in response to IL-6, and that selective activation of p91-related factors may explain the diversity of cellular responses to different cytokines.
997 citations
TL;DR: Sterol-regulated proteolysis of a membrane-bound transcription factor provides a novel mechanism by which transcription can be regulated by membrane lipids.
985 citations
TL;DR: The cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53, a protein partner of the retinoblastoma-susceptibility gene product, RB, which appears to function as a positive regulator or signal for entry into S phase.
Abstract: The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. The E2F-1 transcription factor is a protein partner of the retinoblastoma-susceptibility gene product, RB. E2F-1 appears to function as a positive regulator or signal for entry into S phase. To explore possible interactions of p53 and E2F-1 in the cell cycle, a human E2F-1 expression plasmid was introduced into a murine cell line containing a temperature-sensitive p53 allele which produces a p53 protein in the wild-type conformation at 32 degrees C and the mutant form at 37.5 degrees C. Coexpression of the wild-type p53 protein and E2F-1 in these cells resulted in a rapid loss of cell viability through a process of apoptosis. Thus, the cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53.
TL;DR: Data from a PCR-based technique and in situ hybridization revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis, and these data may outline a genetic program responsible for neuronal programmed cell death.
Abstract: We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.
TL;DR: Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene produced little or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation, and infection with Mycobacterium bovis was more severe in IRf-1-/- mice than in wild-type mice.
Abstract: Production of nitric oxide (NO) by macrophages is important for the killing of intracellular infectious agents Interferon (IFN)-gamma and lipopolysaccharide stimulate NO production by transcriptionally up-regulating the inducible NO synthase (iNOS) Macrophages from mice with a targeted disruption of the IFN regulatory factor-1 (IRF-1) gene (IRF-1-/- mice) produced little or no NO and synthesized barely detectable iNOS messenger RNA in response to stimulation Two adjacent IRF-1 response elements were identified in the iNOS promoter Infection with Mycobacterium bovis (BCG) was more severe in IRF-1-/- mice than in wild-type mice Thus, IRF-1 is essential for iNOS activation in murine macrophages
TL;DR: Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat, and indicates that IL- 4 Stat has the same functional domain for both receptor coupling and dimerization.
Abstract: Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.
TL;DR: It is reported here that the inactive Stat91 in the cytoplasm of untreated cells is a monomer and that upon IFN-gamma-induced phosphorylation it forms a stable homodimer.
TL;DR: In this paper, E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53, while co-expression of a naturally occurring loss-of-function RB mutant or wild type p53 did not suppress the induction of apoptosis under these conditions.
Abstract: E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.
TL;DR: It is reported here that micro-injection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter, and proposed that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.
Abstract: A number of signalling pathways stimulate transcription of target genes through nuclear factors whose activities are primarily regulated by phosphorylation. Cyclic AMP regulates the expression of numerous genes, for example, through the protein kinase-A (PKA)-mediated phosphorylation of transcription factor CREB at Ser 133. Although phosphorylation may stimulate transcriptional activators by modulating their nuclear transport or DNA-binding affinity, CREB belongs to a class of proteins whose phosphorylation appears specifically to enhance their trans-activation potential. Recent work describing a phospho-CREB binding protein (CBP) which interacts specifically with the CREB trans-activation domain prompted us to examine whether CBP is necessary for cAMP regulated transcription. We report here that microinjection of an anti-CBP antiserum into fibroblasts can inhibit transcription from a cAMP responsive promoter. Surprisingly, CBP also cooperates with upstream activators such as c-Jun, which are involved in mitogen responsive transcription. We propose that CBP is recruited to the promoter through interaction with certain phosphorylated factors, and that CBP may thus play a critical role in the transmission of inductive signals from cell surface receptor to the transcriptional apparatus.
TL;DR: It is shown that a partially purified hSWI/SNF complex mediates the ATP-dependent disruption of a nucleosome, thereby enabling the activators, GAL 4–VP16 and GAL4–AH, to bind within aucleosome core.
Abstract: CHROMATIN structure can affect the transcriptional activity of eukaryotic structural genes by blocking access of sequence-specific activator proteins (activators) to their promoter-binding sites. For example, the DNA-binding domain of the yeast GAL4 protein interacts very poorly with nucleosome cores compared with naked DNA2 (and see below), and binding of other activators is even more strongly inhibited. The way in which activators bind to nucleosomal DNA is therefore a critical aspect of transcriptional activation. Genetic studies have suggested that the multi-component SWI/SNF complex of Saccharomyces cerevisiae facilitates transcription by altering the structure of the chromatin. Here we identify and partially purify a human homologue of the yeast SWI/SNF complex (hSWI/SNF complex). We show that a partially purified hSWI/SNF complex mediates the ATP-dependent disruption of a nucleosome, thereby enabling the activators, GAL4-VP16 and GAL4-AH, to bind within a nucleosome core. We conclude that the hSWI/SNF complex acts directly to reorganize chromatin structure so as to facilitate binding of transcription factors.
TL;DR: The activation of the mtTFA promoter by both NRF-1 andNRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis.
Abstract: Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis.
TL;DR: Thioredoxin plays an important role in the regulation of transcriptional processes and oppositely affects NF-kappa B and AP-1 activation, and the stimulatory effect onAP-1 activity was found to involve de novo transcription of the c-jun and c-fos components but to be independent of protein kinase C activation.
Abstract: The transcription factors NF-kappa B and AP-1 have been implicated in the inducible expression of a variety of genes involved in responses to oxidative stress and cellular defense mechanisms. Here, we report that thioredoxin, an important cellular protein oxidoreductase with antioxidant activity, exerts different effects on the activation of NF-kappa B and AP-1. Transient expression or exogenous application of thioredoxin resulted in a dose-dependent inhibition of NF-kappa B activity, as demonstrated in gel shift and transactivation experiments. AP-1-dependent transactivation, in contrast was strongly enhanced by thioredoxin. A similar increase of AP-1 activity was also observed with other, structurally unrelated antioxidants such as pyrrolidine dithiocarbamate and butylated hydroxyanisole, indicating that the thioredoxin-induced increase of AP-1 activation was indeed based on an antioxidant effect. Moreover, the stimulatory effect on AP-1 activity was found to involve de novo transcription of the c-jun and c-fos components but to be independent of protein kinase C activation. These results suggest that thioredoxin plays an important role in the regulation of transcriptional processes and oppositely affects NF-kappa B and AP-1 activation.
TL;DR: It is suggested that Sp3 is an inhibitory member of the Sp family, and neither the glutamine‐rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3.
Abstract: Sp1, Sp3 (SPR-2) and Sp4 (SPR-1) are human sequence-specific DNA binding proteins with very similar structural features In this report, we have analyzed Sp3 in direct comparison with Sp1 We have raised antibodies against both Sp1 and Sp3, and show that Sp3 protein, like Sp1, is expressed in various cell lines Co-transfection experiments in different mammalian cell lines reveal that in contrast to Sp1 and Sp4, Sp3 is not able to activate several Sp1 responsive promoters In addition, Sp3 also fails to activate reporter constructs in Drosophila SL2 cells lacking endogenous Sp factors Instead, we find that Sp3 represses Sp1-mediated activation in a linear dose-dependent manner A mutant of Sp3 lacking the DNA binding domain does not affect activation by Sp1, suggesting that the inhibition is most likely due to the competition with Sp1 for their common binding sites To determine if any structurally similar domain of Sp3 is able to replace partially homologous domains of Sp1, we have generated chimeric proteins and tested their activation characteristics in gene transfer experiments It appears that neither the glutamine-rich domains A and B nor the D domain of Sp1 can be replaced by the homologous regions of Sp3 Our results suggest that Sp3 is an inhibitory member of the Sp family
TL;DR: Observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.
Abstract: We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2-independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.
TL;DR: It is demonstrated that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.
TL;DR: Findings indicate that phosphorylation of TCFs occurs in response to activation of the MAP kinase pathway, and that regulation of TCF activity is an important mechanism by which the serum response element responds to growth factor signals.
TL;DR: H hepatitis B virus X protein (HBX) may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma.
Abstract: Chronic active hepatitis caused by infection with hepatitis B virus, a DNA virus, is a major risk factor for human hepatocellular carcinoma. Since the oncogenicity of several DNA viruses is dependent on the interaction of their viral oncoproteins with cellular tumor-suppressor gene products, we investigated the interaction between hepatitis B virus X protein (HBX) and human wild-type p53 protein. HBX complexes with the wild-type p53 protein and inhibits its sequence-specific DNA binding in vitro. HBX expression also inhibits p53-mediated transcriptional activation in vivo and the in vitro association of p53 and ERCC3, a general transcription factor involved in nucleotide excision repair. Therefore, HBX may affect a wide range of p53 functions and contribute to the molecular pathogenesis of human hepatocellular carcinoma.
TL;DR: Many transcription factors contain proline- or glutamine-rich activation domains and it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor.
Abstract: Many transcription factors contain proline- or glutamine-rich activation domains. Here it is shown that simple homopolymeric stretches of these amino acids can activate transcription when fused to the DNA binding domain of GAL4 factor. In vitro, activity increased with polymer length, whereas in cell transfection assays maximal activity was achieved by 10 to 30 glutamines or about 10 prolines. Similar results were obtained when glutamine stretches were placed within a [GAL4]-VP16 chimeric protein. Because these stretches are encoded by rapidly evolving triplet repeats (microsatellites), they may be the main cause for modulation of transcription factor activity and thus result in subtle or overt genomic effects.
TL;DR: It is shown that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family, suggesting that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiTFamily interactions and transcriptional activities.
Abstract: The microphthalmia (mi) gene appears essential for pigment cell development and/or survival, based on its mutation in mi mice. It has also been linked to the human disorder Waardenburg Syndrome. The mi gene was recently cloned and predicts a basic/helix-loop-helix/leucine zipper (b-HLH-ZIP) factor with tissue-restricted expression. Here, we show that Mi protein binds DNA as a homo- or heterodimer with TFEB, TFE3, or TFEC, together constituting a new MiT family. Mi can also activate transcription through recognition of the M box, a highly conserved pigmentation gene promoter element, and may thereby determine tissue-specific expression of pigmentation enzymes. Six mi mutations shown recently to cluster in the b-HLH-ZIP region produce surprising and instructive effects on DNA recognition and oligomerization. An alternatively spliced exon located outside of the b-HLH-ZIP region is shown to significantly modulate DNA recognition by the basic domain. These findings suggest that Mi's critical roles in melanocyte survival and pigmentation are mediated by MiT family interactions and transcriptional activities.
TL;DR: The mechanisms involved in the prolactin activation of Stat5 in COS cells co‐transfected with cDNA encoding Stat5 and the Prolactin receptor are studied to determine whether this activation does not require ongoing protein synthesis.
Abstract: Mammary gland factor (MGF) is a transcription factor discovered initially in the mammary epithelial cells of lactating animals. It confers the lactogenic hormone response to the milk protein genes. We reported recently the isolation of the cDNA encoding MGF. MGF is a novel member of the cytokine-regulated transcription factor gene family. Members of this gene family mediate interferon alpha/beta and interferon gamma induction of gene transcription, as well as the response to epidermal growth factor and interleukin-6, and have been named signal transducers and activators of transcription (Stat). The name Stat5 has been assigned to MGF. We studied the mechanisms involved in the prolactin activation of Stat5 in COS cells co-transfected with cDNA encoding Stat5 and the prolactin receptor. Prolactin treatment of the transfected cells caused activation of Stat5 within 5-10 min. This activation does not require ongoing protein synthesis. Tyrosine kinase inhibitors prevent Stat5 activation in transfected COS cells. Treatment of recombinant Stat5 with a tyrosine-specific protein phosphatase in vitro abolishes its DNA binding activity. Prolactin stimulation of transfected cells induces Stat5 phosphorylation on tyrosine. Phosphorylation of in vitro transcribed and translated Stat5 with the Jak2 tyrosine kinase, but not with fyn, lyn or lck, confers DNA binding activity. The prolactin response of the beta-casein milk protein gene promoter can be observed in COS cells transfected with cDNA vectors encoding Stat5 and the long form of the prolactin receptor. The short form of the prolactin receptor is unable to promote Stat5 phosphorylation and confer transcriptional induction in COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Direct evidence is presented that PKR can phosphorylate I kappa B-alpha (MAD-3) and activate NF-kappa B DNA binding activity in vitro, and it is shown that dsRNA induces an unusual phosphorylated form of I k Kappa B- alpha.
Abstract: The induction of interferon (IFN) genes by viruses or double-stranded RNA (dsRNA) requires the assembly of a complex set of transcription factors on responsive DNA elements of IFN gene promoters. One of the factors necessary for regulating IFN-beta gene transcription is nuclear factor NF-kappa B, the activation of which is triggered by dsRNA. It has previously been suggested that the dsRNA-activated p68 protein kinase (PKR) may act as an inducer-receptor, transducing the signal from dsRNA to NF-kappa B through phosphorylation of the inhibitor I kappa B. We present direct evidence that PKR can phosphorylate I kappa B-alpha (MAD-3) and activate NF-kappa B DNA binding activity in vitro. We further show that dsRNA induces an unusual phosphorylated form of I kappa B-alpha. The expression of a transdominant mutant PKR is able to perturb the dsRNA-mediated signaling pathway in vivo, suggesting a role for this kinase in IFN-beta gene induction.