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Kenneth M. Murphy - One of the best experts on this subject based on the ideXlab platform.
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Role of the STAT4 N domain in receptor proximal tyrosine phosphorylation.
Molecular and cellular biology, 2000Co-Authors: Theresa L. Murphy, E. D. Geissal, J. D. Farrar, Kenneth M. MurphyAbstract:The STAT (signal transducers and activators of transcription) family of transcription factors reside as latent cytoplasmic monomers which are phosphorylated on a conserved tyrosine residue in response to ligand-induced receptor activation (5, 15, 38). Following tyrosine phosphorylation, STAT proteins undergo homo- and heterodimerization via reciprocal interactions involving their conserved SH2 domains, followed by STAT dimer nuclear translocation and participation in transcriptional regulation of various cytokine responsive genes. STAT dimers bind the palindromic gamma interferon (IFN-γ)-activated sequence (GAS) TTCNmGAA, where m equals 3 for all STAT proteins except Stat6 (6, 7, 14, 19). STAT-specific binding site preferences have been identified involving both the central nucleotide core and sequences flanking the core palindrome (10, 25, 33, 34). Furthermore, higher-order interactions between STAT dimers, in which a tetrameric complex of two STAT dimers cooperatively binds two adjacent GAS elements, have been described for Stat1, STAT4, and Stat5 (42, 43, 47). It has been suggested that such tetramer formation is facilitated through interactions between the highly conserved N-terminal domain (N domain), facilitating STAT dimer binding to low-affinity, nonconsensus STAT binding sites (47). Other functions for the N domain have also been suggested, including roles in nuclear localization and binding to CREB-binding protein, or p300 (39, 48). The specificity of STAT activation by cytokines is in part mediated by the selective interaction of their SH2 domains with distinct tyrosine-containing motifs located within the cytoplasmic domains of specific cytokine receptors. In addition, each STAT protein has private physiologic functions exerted presumably by selective activation of distinct target genes. One important STAT-dependent biologic function involves T helper differentiation. In particular, STAT4 activation plays a significant role in directing development of T helper type 1 (Th1) T cells from naive CD4+ precursors (16, 18, 41). Control of Th1 differentiation is exerted both by the tissue-restricted expression of STAT4 and by the limited activation of STAT4 by only certain cytokines. STAT4 is activated by interleukin 12 (IL-12) (1, 16), a cytokine which potently induces Th1 development (13, 22) through recruitment to a tyrosine-based motif in the IL-12 receptor β2 (IL-12Rβ2) subunit via the STAT4 SH2 domain (26). IL-12 activates STAT4 in all species examined, and the requirement for STAT4 in Th1 development has been confirmed by targeted deletion of STAT4 in mice (18, 41). IFN-α also activates STAT4 and induces Th1 development in human T cells (1, 32), but not mouse T cells (32, 46). STAT4 activation by IFN-α, however, does not involve direct binding to the cytoplasmic domain of the IFN-α receptor (IFN-αR), but instead occurs through an intermediate step (8). First, IFN-α signaling leads to phosphorylation of a conserved tyrosine in the receptor cytoplasmic domain that acts to recruit Stat2, which is subsequently phosphorylated on a conserved tyrosine 690. Stat2 serves as an adapter that binds the SH2 domains of both Stat1 and STAT4. Stat1 binds to tyrosine 690 of Stat2; however, STAT4 binds to a distinct region of Stat2, specifically to the most carboxy-terminal regions of Stat2. In summary, IL-12 and IFN-α each induce Th1 development and activate STAT4, but the STAT4 SH2 domain interaction differs between these receptor pathways. How STAT4 promotes Th1 development is unclear. STAT4 could directly regulate activity of the IFN-γ gene. Recombinant STAT4 produces a footprint on specific sites within the IFN-γ gene promoter and first intron, sites which are low affinity and nonconsensus. The cooperative interaction of adjacent sites with STAT4 dimers binding as a tetramer via adjacent amino termini was suggested as a mechanism for augmenting IFN-γ gene expression (47), although the requirement for these sites in regulation of the native IFN-γ gene has not been established. Alternately, STAT4 may regulate expression of other signaling molecules or transcription factors that act in Th1 development. For example, STAT4 is required for the Th1-specific expression of the Ets transcription family member ERM (28), although a role for ERM in IFN-γ gene expression has not been demonstrated. In addressing potential STAT4 tetramer interactions for IFN-γ regulation, we became interested in the role of the STAT4 N domain in mediating STAT dimer-dimer interactions. The structure of the STAT N domain was determined from the isolated N domain from STAT4, which was found to naturally pack as a dimer in the crystal (43). In the STAT4 N domain, composed of eight alpha helices which form a hook-like structure, a conserved tryptophan residue, W37, was shown to be engaged in critical internal polar interactions between interacting helices of reciprocal N domain subunits (43). For functional analysis, the role of this tryptophan was evaluated in Stat1 rather than STAT4; however, mutation of this tryptophan prevented tetramer formation of recombinant Stat1 protein and caused the loss of an IFN-γ augmentation of a synthetic promoter composed of multimerized GAS elements. Furthermore, mutation of this conserved tryptophan in Stat5 was recently shown to prevent the ability of Stat5 to undergo tetramer formation on the adjacent STAT sites present in the IL-2Rα chain promoter (17). So far, the role of this tryptophan-mediated N domain dimerization had not been evaluated for STAT4 nor evaluated in a system where native physiologic responses to STAT4 activation could be observed. To this end, we have carried out a mutational analysis of the STAT4 N domain for IFN-α- and IL-12-induced STAT4 activation using cell lines that lack STAT4 expression and primary T cells derived from STAT4-deficient mice. Surprisingly, our results point to additional roles of the STAT4 N domain beyond mediating tetramer formation on DNA. Our results indicate that the STAT4 N domain also can influence the ability of STAT proteins to undergo successful interactions with cytokine receptor complexes. Importantly, the W37A mutation within the N domain of STAT4 interferes with IFN-α-induced tyrosine phosphorylation of the STAT4 monomer, interrupting STAT4 activation before formation of the STAT4 dimer. This result precludes any conclusions regarding functional tetramer activity based on this mutation for STAT4. Finally, the data suggest that N-domains may be involved in targeting certain STATs to receptors, influencing their suitability as substrates for receptor-dependent kinases.
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selective loss of type i interferon induced STAT4 activation caused by a minisatellite insertion in mouse stat2
Nature Immunology, 2000Co-Authors: David J Farrar, Theresa L. Murphy, Janice D. Smith, Stewart Leung, George R Stark, Kenneth M. MurphyAbstract:The use of murine systems to model pathogen-induced human diseases presumes that general immune mechanisms between these species are conserved. One important immunoregulatory mechanism involves linkage of innate and adaptive immunity to direct the development of T helper subsets, for example toward subset 1 (TH1) development through STAT4 activation. In analyzing type I interferon signaling, we uncovered a difference between murine and human cells which may affect how these two species control linkage between innate and adaptive immunity. We show that in humans, type I interferons induce TH1 development and can activate STAT4 by recruitment to the IFN-alpha receptor complex specifically via the carboxy-terminus of STAT2. However, the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its capacity to activate STAT4, but not other STATs. This defect in murine Stat2 suggests that the signals leading to STAT4 activation and TH1 development in CD4+ T cells are different between mice and humans.
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Recruitment of STAT4 to the Human Interferon-α/β Receptor Requires Activated Stat2
The Journal of biological chemistry, 2000Co-Authors: J. David Farrar, Theresa L. Murphy, Janice D. Smith, Kenneth M. MurphyAbstract:Abstract STAT4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although STAT4 is activated by interleukin (IL)-12 in both human and murine T cells, STAT4 is activated by interferon (IFN)-α only in human, but not murine, CD4+ T cells. This species-specific difference in cytokine activation of STAT4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of STAT4 recruitment and activation by the human IFN-α receptor. Analysis of phosphopeptide binding analysis suggests that STAT4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-α receptor complex. Expression of murine STAT4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-α-induced STAT4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of STAT4 by the IL-12 receptor, STAT4 activation by the human IFN-α receptor occurs through indirect recruitment by intermediates involving Stat2.
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recruitment of STAT4 to the human interferon α β receptor requires activated stat2
Journal of Biological Chemistry, 2000Co-Authors: David J Farrar, Theresa L. Murphy, Janice D. Smith, Kenneth M. MurphyAbstract:Abstract STAT4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although STAT4 is activated by interleukin (IL)-12 in both human and murine T cells, STAT4 is activated by interferon (IFN)-α only in human, but not murine, CD4+ T cells. This species-specific difference in cytokine activation of STAT4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of STAT4 recruitment and activation by the human IFN-α receptor. Analysis of phosphopeptide binding analysis suggests that STAT4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-α receptor complex. Expression of murine STAT4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-α-induced STAT4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of STAT4 by the IL-12 receptor, STAT4 activation by the human IFN-α receptor occurs through indirect recruitment by intermediates involving Stat2.
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interleukin 12 signaling in t helper type 1 th1 cells involves tyrosine phosphorylation of signal transducer and activator of transcription stat 3 and STAT4
Journal of Experimental Medicine, 1995Co-Authors: N G Jacobson, Zhong Zhong, James E Darnell, Susanne J Szabo, R M Webernordt, Robert D Schreiber, Kenneth M. MurphyAbstract:Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and STAT4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, STAT4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of STAT4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of STAT4.
Fabrice Gouilleux - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological Inhibition of Oncogenic STAT3 and STAT5 Signaling in Hematopoietic Cancers
Cancers, 2020Co-Authors: Marie Brachet-botineau, Marion Polomski, Heidi Neubauer, Ludovic Juen, Damien Hédou, Marie-claude Viaud-massuard, Gildas Prié, Fabrice GouilleuxAbstract:Signal Transducer and Activator of Transcription (STAT) 3 and 5 are important effectors of cellular transformation, and aberrant STAT3 and STAT5 signaling have been demonstrated in hematopoietic cancers. STAT3 and STAT5 are common targets for different tyrosine kinase oncogenes (TKOs). In addition, STAT3 and STAT5 proteins were shown to contain activating mutations in some rare but aggressive leukemias/lymphomas. Both proteins also contribute to drug resistance in hematopoietic malignancies and are now well recognized as major targets in cancer treatment. The development of inhibitors targeting STAT3 and STAT5 has been the subject of intense investigations during the last decade. This review summarizes the current knowledge of oncogenic STAT3 and STAT5 functions in hematopoietic cancers as well as advances in preclinical and clinical development of pharmacological inhibitors.
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Transforming properties of chimeric TEL-JAK proteins in Ba/F3 cells.
Blood, 2000Co-Authors: V Lacronique, Sylvie Gisselbrecht, Fabrice Gouilleux, A. Boureux, R Monni, S Dumon, M Mauchauffé, P. Mayeux, R. Berger, J GhysdaelAbstract:The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)
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a sequence of the cis gene promoter interacts preferentially with two associated stat5a dimers a distinct biochemical difference between stat5a and stat5b
Molecular and Cellular Biology, 1998Co-Authors: Frederique Verdier, Sylvie Gisselbrecht, Christian Beisenherzhuss, Paule Varlet, Fabrice Gouilleux, Raquel Rabionet, Catherine Lacombe, Patrick Mayeux, O. Muller, Stany ChrétienAbstract:STAT proteins are latent transcription factors containing a Src homology 2 domain (SH2 domain) that become activated by tyrosine phosphorylation. The binding of the STAT SH2 domains to the phosphorylated cytokine receptors allows their tyrosine phosphorylation by Jak kinases. After dimerization and nuclear translocation, STAT dimers bind to specific DNA sequences, thereby allowing downstream gene regulation. Seven members of the STAT family have been described (STAT1α/β, STAT2, STAT3α/β, STAT4, STAT5A, STAT5B, and STAT6), and specific combinations are involved in the signaling pathways of different cytokines. The association of STAT5 with various coactivators or repressors and the binding of additional transcription factors to the promoter region of target genes, together with various combinations of STAT dimers, could contribute to the diversity and specificity of the transcriptional regulation of target genes (for reviews, see references 4, 11, and 26). STAT5, originally named mammary gland factor (MGF), has been described as a positive regulator of the β-casein (βCAS) promoter by prolactin (29). In addition to prolactin, STAT5 proteins have been shown to be activated by erythropoietin (Epo), growth hormone, interleukin 2 (IL-2), IL-3, IL-5, IL-7, IL-9, IL-15, granulocyte-macrophage colony-stimulating factor (GM-CSF), thrombopoietin, and several other growth factors. However, only a few STAT5 target sequences have been identified, among them the βCAS promoter (8), the IL-2 receptor alpha (IL-2Rα) chain enhancer (15), p21 (WAF1) (21), oncostatin M (34), the serine protease inhibitor 2.1 (31), and the CIS gene promoter (20). Different MGF boxes located inside these promoters are implicated in transcriptional regulation following STAT5 activation. Besides positive regulatory pathways, negative regulatory pathways such as phosphatases modulate the response to cytokines. The phosphatase SHP-1 has been shown to bind to both the phosphorylated Epo receptor (Epo-R) and the Jak2 kinase and to dephosphorylate these proteins, thereby leading to inactivation of Epo-R signaling (12). However, under certain conditions, STAT5 itself or carboxy-terminally truncated isoforms of STAT5 act as negative regulators of gene transcription (1, 19, 23, 24, 30). Another negative regulatory mechanism involves the protein CIS. The CIS protein is rapidly induced in hematopoietic cells by IL-2, IL-3, GM-CSF, and Epo. CIS contains an SH2 domain in the central part of the molecule and is associated with the cytoplasmic domains of the tyrosine-phosphorylated Epo-R and the β chain of IL-3R (35). CIS overexpression reduces the activation of promoters regulated by STAT5. Moreover, STAT boxes were shown to be involved in the Epo-dependent promoter activation of the murine CIS promoter in HEK 293 cells (20). In this report, we identified different nuclear factors which bound to the human CIS gene promoter. This complex combination of transcriptional factors comprised STAT5A, STAT5B, an Sp1-related family protein, and at least three GGAA binding proteins, among which one showed an Epo-dependent DNA binding capacity. Among the four STAT5 boxes of the CIS promoter, only one bound to a STAT5 tetramer. Moreover, we showed that two STAT5A dimers can interact in cooperative binding with two STAT binding sequences of low or undetectable affinity to form a tetramer. This structure involves STAT5A but not STAT5B. The difference in the behavior of the two isoforms of STAT5 could be essential for the differential transcriptional regulation of STAT5 target genes.
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Modulation of the activation status of Stat5a during LIF-induced differentiation of M1 myeloid leukemia cells
Biochimica et Biophysica Acta - Molecular Cell Research, 1998Co-Authors: Roland Piekorz, Fabrice Gouilleux, Bernd Groner, Ralf Rinke, Brigitte Neumann, Gertrud HockeAbstract:Treatment of M1 myeloid leukemia cells with leukemia inhibitory factor (LIF) causes activation of transcription factors Stat1, Stat3 and Stat5a (signal transducers and activators of transcription). DNA-binding of Stat proteins was detectable for extended periods of time in LIF-treated M1 cells, which simultaneously underwent terminal differentiation. The relative composition of Stat factors in the protein-DNA complexes changed during time. Whereas Stat3 was activated up to 36 h during treatment with LIF, Stat5a was activated only short-termed. Similarly, high expression of the immediate early gene CIS (cytokine-inducible SH2-containing protein), a known target gene of Stat5 in hematopoietic cells, occurred only during the onset of differentiation. This suggests a role of Stat5a in the early phase of LIF-induced differentiation and growth arrest of M1 cells.
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Activated Stat Related Transcription Factors in Acute Leukemia
Leukemia & lymphoma, 1997Co-Authors: V Gouilleux-gruart, Fabrice Gouilleux, Françoise Debierre-grockiego, Jean-claude Capiod, Jean-franÇois Claisse, Jacques Delobel, Lionel PrinAbstract:Cell proliferation and differentiation are under the control of cytokines and growth factors. Different signaling pathways are involved in the transmission of a specific signal through successive phosphorylation and dephosphorylation of proteins leading to gene transcription necessary for growth and differentiation. The cytokines and growth factors activate the Stat family of transcription factors. The Jak-Stat pathway is essential for cytokine signal transduction. Dysregulation of this cascade might lead to uncontrolled hematopoiesis. Studies have been carried out to examine the functionality of this pathway in cells from patients with acute leukemia. Members of the Stat protein family (Stat1, Stat3 and Stat5) are constitutively activated in cells collected from some acute leukemias suggesting dysregulation of the Jak-Stat pathway. Evidence of the existence of constitutively activated spliced variants of Stat3 and Stat5 proteins are described. The mechanisms of such activation remain to be clarified.
John J Oshea - One of the best experts on this subject based on the ideXlab platform.
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regulating type 1 ifn effects in cd8 t cells during viral infections changing STAT4 and stat1 expression for function
Blood, 2012Co-Authors: Pilar M Gil, John J Oshea, Wendy T. Watford, Xin Wang, Mickael J Y Ploquin, Seunghwan Lee, Kwangsin Kim, Yuka Kanno, Christine A BironAbstract:Type 1 IFNs can conditionally activate all of the signal transducers and activators of transcription molecules (STATs), including STAT4. The best-characterized signaling pathways use STAT1, however, and type 1 IFN inhibition of cell proliferation is STAT1 dependent. We report that type 1 IFNs can basally stimulate STAT1- and STAT4-dependent effects in CD8 T cells, but that CD8 T cells responding to infections of mice with lymphocytic choriomenigitis virus have elevated STAT4 and lower STAT1 expression with significant consequences for modifying the effects of type 1 IFN exposure. The phenotype was associated with preferential type 1 IFN activation of STAT4 compared with STAT1. Stimulation through the TCR induced elevated STAT4 expression, and STAT4 was required for peak expansion of antigen-specific CD8 T cells, low STAT1 levels, and resistance to type 1 IFN-mediated inhibition of proliferation. Thus, a mechanism is discovered for regulating the consequences of type 1 IFN exposure in CD8 T cells, with STAT4 acting as a key molecule in driving optimal antigen-specific responses and overcoming STAT1-dependent inhibition of proliferation.
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discrete roles of STAT4 and stat6 transcription factors in tuning epigenetic modifications and transcription during t helper cell differentiation
Immunity, 2010Co-Authors: Golnaz Vahedi, Hiroaki Takatori, Haydee L Ramos, Hayato Takahashi, Jonathan Liang, Gustavo Gutierrezcruz, Wendy T. Watford, Chongzhi Zang, Weiqun Peng, John J OsheaAbstract:Summary Signal transducer and activator of transcription 4 (STAT4) and STAT6 are key factors in the specification of helper T cells; however, their direct roles in driving differentiation are not well understood. Using chromatin immunoprecipitation and massive parallel sequencing, we quantitated the full complement of STAT-bound genes, concurrently assessing global STAT-dependent epigenetic modifications and gene transcription by using cells from cognate STAT-deficient mice. STAT4 and STAT6 each bound over 4000 genes with distinct binding motifs. Both played critical roles in maintaining chromatin configuration and transcription of a core subset of genes through the combination of different epigenetic patterns. Globally, STAT4 had a more dominant role in promoting active epigenetic marks, whereas STAT6 had a more prominent role in antagonizing repressive marks. Clusters of genes negatively regulated by STATs were also identified, highlighting previously unappreciated repressive roles of STATs. Therefore, STAT4 and STAT6 play wide regulatory roles in T helper cell specification.
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critical role for STAT4 activation by type 1 interferons in the interferon γ response to viral infection
Science, 2002Co-Authors: Khuong B Nguyen, John J Oshea, Wendy T. Watford, Rachelle Salomon, Sigrun R Hofmann, Gary C Pien, Akio Morinobu, Massimo Gadina, Christine A BironAbstract:Interferons (IFNs) are essential for host defense. Although the antiviral effects of the type 1 IFNs IFN-α and IFN-β (IFN-α/β) have been established, their immunoregulatory functions, especially their ability to regulate IFN-γ production, are poorly understood. Here we show that IFN-α/β activate STAT4 directly (STAT, signal transducers and activators of transcription) and that this is required for IFN-γ production during viral infections of mice, in concert with T cell receptor–derived signals. In contrast, STAT1 appears to negatively regulate IFN-α/β induction of IFN-γ. Thus, type 1 IFNs, in addition to interleukin-12, provide pathways for innate regulation of adaptive immunity, and their immunoregulatory functions are controlled by modulating the activity of individual STATs.
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activation of STAT4 by il 12 and ifn alpha evidence for the involvement of ligand induced tyrosine and serine phosphorylation
Journal of Immunology, 1996Co-Authors: Shinhyeong Cho, Chris M Bacon, D S Finbloom, R C Rees, Chitra Sudarshan, R Pine, John J OsheaAbstract:The immunoregulatory cytokine IL-12 plays a central role in cell-mediated immune responses through its effects on NK cells and T lymphocytes. While IL-12 is known to share some functions with other cytokines, such as IFN-alpha, it also maintains distinct roles, such as its ability to induce Th1 differentiation. The molecular basis for these unique and overlapping functions is not well understood. IL-12 has previously been shown to induce tyrosine phosphorylation and DNA-binding of STAT3 and STAT4, members of the signal transducers and activators of transcription (STAT) family. Because STAT4 has only been shown to be activated in response to IL-12, this specificity has been suggested to be a basis for the unique actions of IL-12. In this study, we demonstrated that STAT4 activation by IL-12 is not unique; IL-12 and IFN-alpha, but not IFN-gamma, induced tyrosine phosphorylation and DNA binding of STAT4. Since tyrosine and serine phosphorylation of STAT1 have previously been shown to be important in IFN-alpha-mediated signaling, we also investigated IL-12- and IFN-alpha-induced serine phosphorylation of STAT4. We demonstrated that both cytokines induced serine phosphorylation. This modification was not required for DNA binding, but may be important in STAT-mediated transcription. Thus, STAT4 activation was not IL-12 specific, and IL-12 and IFN-alpha activated STAT4 through both tyrosine and serine phosphorylation. These findings have significant implications for understanding the role of STAT4 in mediating biologic functions; specifically, the data argue that the unique effects of IL-12 cannot be solely explained by STAT4 activation.
Christine A Biron - One of the best experts on this subject based on the ideXlab platform.
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regulating type 1 ifn effects in cd8 t cells during viral infections changing STAT4 and stat1 expression for function
Blood, 2012Co-Authors: Pilar M Gil, John J Oshea, Wendy T. Watford, Xin Wang, Mickael J Y Ploquin, Seunghwan Lee, Kwangsin Kim, Yuka Kanno, Christine A BironAbstract:Type 1 IFNs can conditionally activate all of the signal transducers and activators of transcription molecules (STATs), including STAT4. The best-characterized signaling pathways use STAT1, however, and type 1 IFN inhibition of cell proliferation is STAT1 dependent. We report that type 1 IFNs can basally stimulate STAT1- and STAT4-dependent effects in CD8 T cells, but that CD8 T cells responding to infections of mice with lymphocytic choriomenigitis virus have elevated STAT4 and lower STAT1 expression with significant consequences for modifying the effects of type 1 IFN exposure. The phenotype was associated with preferential type 1 IFN activation of STAT4 compared with STAT1. Stimulation through the TCR induced elevated STAT4 expression, and STAT4 was required for peak expansion of antigen-specific CD8 T cells, low STAT1 levels, and resistance to type 1 IFN-mediated inhibition of proliferation. Thus, a mechanism is discovered for regulating the consequences of type 1 IFN exposure in CD8 T cells, with STAT4 acting as a key molecule in driving optimal antigen-specific responses and overcoming STAT1-dependent inhibition of proliferation.
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high basal STAT4 balanced by stat1 induction to control type 1 interferon effects in natural killer cells
Journal of Experimental Medicine, 2007Co-Authors: Takuya Miyagi, Pilar M Gil, Xin Wang, Jennifer Louten, Wenming Chu, Christine A BironAbstract:The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-γ induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN–mediated STAT4 activation for IFN-γ production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-γ, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-γ production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.
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critical role for STAT4 activation by type 1 interferons in the interferon γ response to viral infection
Science, 2002Co-Authors: Khuong B Nguyen, John J Oshea, Wendy T. Watford, Rachelle Salomon, Sigrun R Hofmann, Gary C Pien, Akio Morinobu, Massimo Gadina, Christine A BironAbstract:Interferons (IFNs) are essential for host defense. Although the antiviral effects of the type 1 IFNs IFN-α and IFN-β (IFN-α/β) have been established, their immunoregulatory functions, especially their ability to regulate IFN-γ production, are poorly understood. Here we show that IFN-α/β activate STAT4 directly (STAT, signal transducers and activators of transcription) and that this is required for IFN-γ production during viral infections of mice, in concert with T cell receptor–derived signals. In contrast, STAT1 appears to negatively regulate IFN-α/β induction of IFN-γ. Thus, type 1 IFNs, in addition to interleukin-12, provide pathways for innate regulation of adaptive immunity, and their immunoregulatory functions are controlled by modulating the activity of individual STATs.
Theresa L. Murphy - One of the best experts on this subject based on the ideXlab platform.
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Role of the STAT4 N domain in receptor proximal tyrosine phosphorylation.
Molecular and cellular biology, 2000Co-Authors: Theresa L. Murphy, E. D. Geissal, J. D. Farrar, Kenneth M. MurphyAbstract:The STAT (signal transducers and activators of transcription) family of transcription factors reside as latent cytoplasmic monomers which are phosphorylated on a conserved tyrosine residue in response to ligand-induced receptor activation (5, 15, 38). Following tyrosine phosphorylation, STAT proteins undergo homo- and heterodimerization via reciprocal interactions involving their conserved SH2 domains, followed by STAT dimer nuclear translocation and participation in transcriptional regulation of various cytokine responsive genes. STAT dimers bind the palindromic gamma interferon (IFN-γ)-activated sequence (GAS) TTCNmGAA, where m equals 3 for all STAT proteins except Stat6 (6, 7, 14, 19). STAT-specific binding site preferences have been identified involving both the central nucleotide core and sequences flanking the core palindrome (10, 25, 33, 34). Furthermore, higher-order interactions between STAT dimers, in which a tetrameric complex of two STAT dimers cooperatively binds two adjacent GAS elements, have been described for Stat1, STAT4, and Stat5 (42, 43, 47). It has been suggested that such tetramer formation is facilitated through interactions between the highly conserved N-terminal domain (N domain), facilitating STAT dimer binding to low-affinity, nonconsensus STAT binding sites (47). Other functions for the N domain have also been suggested, including roles in nuclear localization and binding to CREB-binding protein, or p300 (39, 48). The specificity of STAT activation by cytokines is in part mediated by the selective interaction of their SH2 domains with distinct tyrosine-containing motifs located within the cytoplasmic domains of specific cytokine receptors. In addition, each STAT protein has private physiologic functions exerted presumably by selective activation of distinct target genes. One important STAT-dependent biologic function involves T helper differentiation. In particular, STAT4 activation plays a significant role in directing development of T helper type 1 (Th1) T cells from naive CD4+ precursors (16, 18, 41). Control of Th1 differentiation is exerted both by the tissue-restricted expression of STAT4 and by the limited activation of STAT4 by only certain cytokines. STAT4 is activated by interleukin 12 (IL-12) (1, 16), a cytokine which potently induces Th1 development (13, 22) through recruitment to a tyrosine-based motif in the IL-12 receptor β2 (IL-12Rβ2) subunit via the STAT4 SH2 domain (26). IL-12 activates STAT4 in all species examined, and the requirement for STAT4 in Th1 development has been confirmed by targeted deletion of STAT4 in mice (18, 41). IFN-α also activates STAT4 and induces Th1 development in human T cells (1, 32), but not mouse T cells (32, 46). STAT4 activation by IFN-α, however, does not involve direct binding to the cytoplasmic domain of the IFN-α receptor (IFN-αR), but instead occurs through an intermediate step (8). First, IFN-α signaling leads to phosphorylation of a conserved tyrosine in the receptor cytoplasmic domain that acts to recruit Stat2, which is subsequently phosphorylated on a conserved tyrosine 690. Stat2 serves as an adapter that binds the SH2 domains of both Stat1 and STAT4. Stat1 binds to tyrosine 690 of Stat2; however, STAT4 binds to a distinct region of Stat2, specifically to the most carboxy-terminal regions of Stat2. In summary, IL-12 and IFN-α each induce Th1 development and activate STAT4, but the STAT4 SH2 domain interaction differs between these receptor pathways. How STAT4 promotes Th1 development is unclear. STAT4 could directly regulate activity of the IFN-γ gene. Recombinant STAT4 produces a footprint on specific sites within the IFN-γ gene promoter and first intron, sites which are low affinity and nonconsensus. The cooperative interaction of adjacent sites with STAT4 dimers binding as a tetramer via adjacent amino termini was suggested as a mechanism for augmenting IFN-γ gene expression (47), although the requirement for these sites in regulation of the native IFN-γ gene has not been established. Alternately, STAT4 may regulate expression of other signaling molecules or transcription factors that act in Th1 development. For example, STAT4 is required for the Th1-specific expression of the Ets transcription family member ERM (28), although a role for ERM in IFN-γ gene expression has not been demonstrated. In addressing potential STAT4 tetramer interactions for IFN-γ regulation, we became interested in the role of the STAT4 N domain in mediating STAT dimer-dimer interactions. The structure of the STAT N domain was determined from the isolated N domain from STAT4, which was found to naturally pack as a dimer in the crystal (43). In the STAT4 N domain, composed of eight alpha helices which form a hook-like structure, a conserved tryptophan residue, W37, was shown to be engaged in critical internal polar interactions between interacting helices of reciprocal N domain subunits (43). For functional analysis, the role of this tryptophan was evaluated in Stat1 rather than STAT4; however, mutation of this tryptophan prevented tetramer formation of recombinant Stat1 protein and caused the loss of an IFN-γ augmentation of a synthetic promoter composed of multimerized GAS elements. Furthermore, mutation of this conserved tryptophan in Stat5 was recently shown to prevent the ability of Stat5 to undergo tetramer formation on the adjacent STAT sites present in the IL-2Rα chain promoter (17). So far, the role of this tryptophan-mediated N domain dimerization had not been evaluated for STAT4 nor evaluated in a system where native physiologic responses to STAT4 activation could be observed. To this end, we have carried out a mutational analysis of the STAT4 N domain for IFN-α- and IL-12-induced STAT4 activation using cell lines that lack STAT4 expression and primary T cells derived from STAT4-deficient mice. Surprisingly, our results point to additional roles of the STAT4 N domain beyond mediating tetramer formation on DNA. Our results indicate that the STAT4 N domain also can influence the ability of STAT proteins to undergo successful interactions with cytokine receptor complexes. Importantly, the W37A mutation within the N domain of STAT4 interferes with IFN-α-induced tyrosine phosphorylation of the STAT4 monomer, interrupting STAT4 activation before formation of the STAT4 dimer. This result precludes any conclusions regarding functional tetramer activity based on this mutation for STAT4. Finally, the data suggest that N-domains may be involved in targeting certain STATs to receptors, influencing their suitability as substrates for receptor-dependent kinases.
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selective loss of type i interferon induced STAT4 activation caused by a minisatellite insertion in mouse stat2
Nature Immunology, 2000Co-Authors: David J Farrar, Theresa L. Murphy, Janice D. Smith, Stewart Leung, George R Stark, Kenneth M. MurphyAbstract:The use of murine systems to model pathogen-induced human diseases presumes that general immune mechanisms between these species are conserved. One important immunoregulatory mechanism involves linkage of innate and adaptive immunity to direct the development of T helper subsets, for example toward subset 1 (TH1) development through STAT4 activation. In analyzing type I interferon signaling, we uncovered a difference between murine and human cells which may affect how these two species control linkage between innate and adaptive immunity. We show that in humans, type I interferons induce TH1 development and can activate STAT4 by recruitment to the IFN-alpha receptor complex specifically via the carboxy-terminus of STAT2. However, the mouse Stat2 gene harbors a minisatellite insertion that has altered the carboxy-terminus and selectively disrupted its capacity to activate STAT4, but not other STATs. This defect in murine Stat2 suggests that the signals leading to STAT4 activation and TH1 development in CD4+ T cells are different between mice and humans.
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Recruitment of STAT4 to the Human Interferon-α/β Receptor Requires Activated Stat2
The Journal of biological chemistry, 2000Co-Authors: J. David Farrar, Theresa L. Murphy, Janice D. Smith, Kenneth M. MurphyAbstract:Abstract STAT4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although STAT4 is activated by interleukin (IL)-12 in both human and murine T cells, STAT4 is activated by interferon (IFN)-α only in human, but not murine, CD4+ T cells. This species-specific difference in cytokine activation of STAT4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of STAT4 recruitment and activation by the human IFN-α receptor. Analysis of phosphopeptide binding analysis suggests that STAT4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-α receptor complex. Expression of murine STAT4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-α-induced STAT4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of STAT4 by the IL-12 receptor, STAT4 activation by the human IFN-α receptor occurs through indirect recruitment by intermediates involving Stat2.
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recruitment of STAT4 to the human interferon α β receptor requires activated stat2
Journal of Biological Chemistry, 2000Co-Authors: David J Farrar, Theresa L. Murphy, Janice D. Smith, Kenneth M. MurphyAbstract:Abstract STAT4 activation is involved in differentiation of type 1 helper (Th1) T cells. Although STAT4 is activated by interleukin (IL)-12 in both human and murine T cells, STAT4 is activated by interferon (IFN)-α only in human, but not murine, CD4+ T cells. This species-specific difference in cytokine activation of STAT4 underlies critical differences in Th1 development in response to cytokines and is important to the interpretation of murine models of immunopathogenesis. Here, we sought to determine the mechanism of STAT4 recruitment and activation by the human IFN-α receptor. Analysis of phosphopeptide binding analysis suggests that STAT4 does not interact directly with tyrosine-phosphorylated amino acid residues within the cytoplasmic domains of either of the subunits of the IFN-α receptor complex. Expression of murine STAT4 in the Stat1-deficient U3A and the Stat2-deficient U6A cell lines shows that IFN-α-induced STAT4 phosphorylation requires the presence of activated Stat2 but not Stat1. Thus, in contrast to the direct recruitment of STAT4 by the IL-12 receptor, STAT4 activation by the human IFN-α receptor occurs through indirect recruitment by intermediates involving Stat2.
