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David J. Waxman - One of the best experts on this subject based on the ideXlab platform.
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stat5 regulation of sex dependent hepatic cpg methylation at distal regulatory elements mapping to sex biased genes
Molecular and Cellular Biology, 2020Co-Authors: Pengying Hao, David J. WaxmanAbstract:Growth hormone-activated STAT5B is an essential regulator of sex-differential gene expression in mouse liver; however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5B (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were downregulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were derepressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG methylation were seen in STAT5-deficient liver, where sex differences were abolished at 88% of ∼1,500 sex-differentially methylated regions, largely due to increased DNA methylation upon STAT5 loss. STAT5-dependent CpG hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (histone H3 lysine 4 monomethylation [H3K4me1] and histone H3 lysine 27 acetylation [H3K27ac]), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. Thus, the sex-dependent binding of STAT5 to liver chromatin is closely linked to the sex-dependent demethylation of distal regulatory elements linked to STAT5-dependent genes important for liver sex bias.
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stat5 regulation of sex dependent hepatic cpg methylation at distal regulatory elements mapping to sex biased genes
bioRxiv, 2020Co-Authors: Pengying Hao, David J. WaxmanAbstract:Growth hormone-activated STAT5B is an essential regulator of sex-differential gene expression in mouse liver, however, its impact on hepatic gene expression and epigenetic responses is poorly understood. Here, we found a substantial, albeit incomplete loss of liver sex bias in hepatocyte-specific STAT5a/STAT5B (collectively, STAT5)-deficient mouse liver. In male liver, many male-biased genes were down regulated in direct association with the loss of STAT5 binding; many female-biased genes, which show low STAT5 binding, were de-repressed, indicating an indirect mechanism for repression by STAT5. Extensive changes in CpG-methylation were seen in STAT5-deficient liver, where sex differences in DNA methylation were abolished at 88% of ~1,500 differentially-methylated regions, largely due to an increase in methylation at the hypomethylated sites. STAT5-dependent CpG-hypomethylation was rarely found at proximal promoters of STAT5-dependent genes. Rather, STAT5 primarily regulated the methylation of distal enhancers, where STAT5 deficiency induced widespread hypermethylation at genomic regions enriched for accessible chromatin, enhancer histone marks (H3K4me1, H3K27ac), STAT5 binding, and DNA motifs for STAT5 and other transcription factors implicated in liver sex differences. In conclusion, the sex-dependent binding of STAT5 to liver chromatin is closely linked to sex-dependent demethylation of distal regulatory elements mapping to STAT5-dependent genes important for liver sex bias.
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loss of sexually dimorphic liver gene expression upon hepatocyte specific deletion of stat5a STAT5B locus
Endocrinology, 2007Co-Authors: Minita G Holloway, Atsushi Hosui, David J. WaxmanAbstract:Hepatocyte-specific, albumin-Cre recombinase-mediated deletion of the entire mouse Stat5a-STAT5B locus was carried out to evaluate the role of signal transducer and activator of transcription 5a and 5b (STAT5ab) in the sex-dependent transcriptional actions of GH in the liver. The resultant hepatocyte STAT5ab-deficient mice were fertile, and unlike global STAT5B-deficient male mice, postnatal body weight gain was normal, despite a 50% decrease in serum IGF-I. Whole-liver STAT5ab RNA decreased by approximately 65–85%, and residual STAT5 immunostaining was observed in a minority of the hepatocytes, indicating incomplete excision by Cre-recombinase. Quantitative PCR analysis of 20 sexually dimorphic, liver-expressed genes revealed significant down-regulation of 10 of 11 male-specific genes in livers of male hepatocyte STAT5ab-deficient mice. Class I female-specific liver genes were markedly up-regulated (de-repressed), whereas the expression of class II female genes, belonging to the Cyp3a subfamily, was unaf...
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serine phosphorylation of gh activated signal transducer and activator of transcription 5a stat5a and STAT5B impact on stat5 transcriptional activity
Molecular Endocrinology, 2001Co-Authors: Soohee Park, Hiroko Yamashita, David J. WaxmanAbstract:Signal transducer and activator of transcription 5b (STAT5B), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5B DNA-binding and transcriptional activities, the effect of STAT5B serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5B serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5B without inhibiting STAT5B nuclear translocation. Evaluation of the functional consequences of STAT5B serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5B Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity...
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growth hormone but not prolactin maintains low level activation of stat5a and STAT5B in female rat liver
Endocrinology, 1999Co-Authors: Hee K Choi, David J. WaxmanAbstract:STAT5B, a member of the signal transducers and activators of transcription family, is activated in rat liver in response to the intermittent (pulsatile) plasma pattern of GH that is characteristic of adult males. Previous studies have shown that the near-continuous plasma GH pattern of adult female rats is associated with a dramatic down-regulation of the STAT5 activation pathway. The present study demonstrates the presence of a low-level STAT5 DNA-binding activity in adult female rat liver and investigates the hormonal factors required for its maintenance. PRL is not responsible for this low-level STAT5 activity, as demonstrated in experiments involving estrus cycle monitoring (to investigate a possible role of the proestrus PRL surge), implantation of bromocriptine pellets (to eliminate PRL release by the pituitary), and direct injection of purified PRL. Rather, the low-level STAT5 activity is shown to result from chronic plasma GH stimulation, as demonstrated by GH infusion studies carried out in hypophysectomized rats. Furthermore, gel mobility supershift experiments demonstrate that the same STAT5-containing DNA-binding complexes are formed by both male and female adult rat liver extracts, albeit at approximately 10- to 20-fold lower levels by the female extracts. This DNA-binding activity is primarily comprised of STAT5B but also contains STAT5a, which is shown to be preferentially activated by the male plasma GH pattern in a manner similar to STAT5B. Thus, the dominance of activated STAT5B, compared with STAT5a, in the strong DNA-binding complexes formed in adult male rat liver nuclear extracts, is a reflection of the relative abundance in liver of the two STAT5 forms and is not attributable to an intrinsic, preferential activation of STAT5B by plasma GH pulses. The physiological significance of the low-level activated STAT5a and STAT5B seen in female rat liver and its effects on liver gene expression are uncertain but may involve the activation of femaleexpressed cytochromes P450 and other liver genes. (Endocrinology 140: 5126 ‐5135, 1999)
David S Finbloom - One of the best experts on this subject based on the ideXlab platform.
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stat5a deficient mice demonstrate a defect in granulocyte macrophage colony stimulating factor induced proliferation and gene expression
Blood, 1997Co-Authors: Gerald M Feldman, Mark P Hayes, Anthony Wynshawboris, Louis A. Rosenthal, David S FinbloomAbstract:Responses of cells to cytokines typically involve the activation of a family of latent DNA binding proteins, referred to as signal transducers and activators of transcription (STAT) proteins, which are critical for the expression of early response genes. Of the seven known STAT proteins, STAT5 (originally called mammary gland factor) has been shown to be activated by several cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5, which are known to play important roles in growth and differentiation of hematopoietic precursors. In this report we have used mice that are deficient in STAT5A (one of two homologues of STAT5) to study the role of STAT5A in GM-CSF stimulation of cells. When bone marrow–derived macrophages were generated by differentiation with macrophage-CSF (M-CSF), exposure of cells from wild-type mice to GM-CSF resulted in a typical pattern of assembly of DNA binding proteins specific for the gamma activation sequence (GAS) element within the β-casein promoter. However, in cells from the STAT5A null mouse one of the shifted bands was absent. Immunoblotting analysis in the null mice showed that lack of STAT5A protein resulted in no alteration in activation of STAT5B by tyrosine phosphorylation. Proliferation experiments revealed that, when exposed to increasing concentrations of GM-CSF, cells derived from the null mice grew considerably more slowly than cells derived from the wild-type mice. Moreover, expression of GM-CSF–dependent genes, CIS and A1, was markedly inhibited in cells derived from null mice as compared with those of wild-type mice. The decreased expression observed with A1, a bcl-2 like gene, may account in part for the suppression of growth in cells from the null mice. These data suggest that the presence of STAT5A during the GM-CSF–induced assembly of STAT5 dimers is critical for the formation of competent transcription factors that are required for both gene expression and cell proliferation.
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granulocyte macrophage colony stimulating factor preferentially activates the 94 kd stat5a and an 80 kd stat5a isoform in human peripheral blood monocytes
Blood, 1996Co-Authors: R L Rosen, K D Winestock, David S FinbloomAbstract:Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces immediate effects in monocytes by activation of the Janus kinase (JAK2) and STAT transcription factor (STAT5) pathway. Recent studies have identified homologues of STAT5, STAT5A, and STAT5B, as well as lower molecular weight variants of STAT5. To define the activation of the STAT5 homologues and lower molecular weight variant in human monocytes and monocytes differentiated into macrophages by culture in macrophage- CSF (M-CSF), we measured the GM-CSF induced tyrosine phosphorylation of STAT5A, STAT5B, and any lower molecular weight STAT5 isoforms. Freshly isolated monocytes expressed 94-kD STAT5A, 92-kD STAT5B, and an 80-kD STAT5A molecule. Whereas 94-kD STAT5A was clearly tyrosine phosphorylated and bound to the enhancer element, the gamma response region (GRR), of the Fc gamma RI gene, substantially less tyrosine phosphorylated STAT5B bound to the immobilized GRR element. Macrophages lost their ability to express the 80-kD STAT5A protein, but retained their ability to activate STAT5A. STAT5A-STAT5A homodimers and STAT5A- STAT5B heterodimers formed in response to GM-CSF. Therefore, activation of STAT5A predominates compared to STAT5B when assayed by direct immunoprecipitation and by evaluation of bound STATs to immobilized GRR. Selective activation of STAT5 homologues in addition to generation of lower molecular isoforms may provide specificity and control to genes expressed in response to cytokines such as GM-CSF.
Gertraud W Robinson - One of the best experts on this subject based on the ideXlab platform.
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signal transducer and activator of transcription 5 stat5 paralog dose governs t cell effector and regulatory functions
eLife, 2016Co-Authors: Alejandro V Villarino, Gertraud W Robinson, Arian Laurence, Michael Bonelli, Barbara Dema, Behdad Afzali, Hanyu Shih, Hongwei Sun, Stephen R Brooks, Lothar HennighausenAbstract:The immune system in mammals is one of the most complex networks in the animal kingdom. One way that its many components communicate is via proteins called cytokines, which are released by cells and detected by receptors on the surface of other cells. This leads to the activation of signals inside the responding cells that alter the activity of genes and, ultimately, direct how they behave. STAT5 is a signal protein that is activated when certain cytokines bind to receptors on the cell surface. Consequently, it is an attractive target for drug therapies that seek to alter immune responses and there is keen interest in understanding how it works. It is an unusual protein in that there are two versions – termed STAT5A and STAT5B – that are produced by two separate genes. Together, STAT5A and STAT5B are fundamental to the immune system but there is considerable debate about whether they perform the same job or have distinct roles. Villarino et al. used a combination of genetic and genomic approaches to investigate how both versions of STAT5 work in mice. The experiments show that STAT5B plays a much bigger role in immune cells than STAT5A. Unexpectedly, the experiments indicate that the disparity is not due to differences in protein activity, but is caused by differences in the amount of these proteins in cells. Villarino et al.’s findings resolve longstanding questions about the relationship between STAT5A and STAT5B within the immune system. A logical next step is to find the molecular mechanisms responsible for causing different amounts of STAT5A and STAT5B to be produced in immune cells. Future work will also compare the roles of STAT5A and STAT5B in non-immune cells and explore whether it might be possible to develop therapies that specifically target one version and not the other.
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loss of ezh2 results in precocious mammary gland development and activation of stat5 dependent genes
Nucleic Acids Research, 2015Co-Authors: Kyung Hyun Yoo, Keunsoo Kang, Gertraud W Robinson, Tim Hensel, Lothar HennighausenAbstract:Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activator EZH2 controls the differentiation clock of mammary epithelium. Ablation of Ezh2 from mammary stem cells resulted in precocious differentiation of alveolar epithelium during pregnancy and the activation of mammary-specific STAT5 target genes. This coincided with enhanced occupancy of these loci by STAT5, EZH1 and RNA Pol II. Limited activation of differentiation-specific genes was observed in mammary epithelium lacking both EZH2 and STAT5, suggesting a modulating but not mandatory role for STAT5. Loss of EZH2 did not result in overt changes in genome-wide and gene-specific H3K27me3 profiles, suggesting compensation through enhanced EZH1 recruitment. Differentiated mammary epithelia did not form in the combined absence of EZH1 and EZH2. Transplantation experiments failed to demonstrate a role for EZH2 in the activity of mammary stem and progenitor cells. In summary, while EZH1 and EZH2 serve redundant functions in the establishment of H3K27me3 marks and the formation of mammary alveoli, the presence of EZH2 is required to control progressive differentiation of milk secreting epithelium during pregnancy.
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sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on stat5a b concentration
Nucleic Acids Research, 2013Co-Authors: Daisuke Yamaji, Keunsoo Kang, Gertraud W RobinsonAbstract:The transcription factors Signal Transducer and Activator of Transcription (STAT) 5A/B mediate prolactin-induced mammary development during pregnancy. However, it is not clear how the different processes, expansion and maturation of alveolar precursor cells and the differential induction of milk protein genes are regulated on a molecular level. We have used mouse genetics and genome-wide analyses to determine how altering concentrations of STAT5A and STAT5B impacts mammary epithelial development during pregnancy and the regulation of target genes. The presence of only a single Stat5a or STAT5B allele was sufficient for the establishment of histologically undifferentiated alveolar units and two alleles permitted the execution of a differentiation program similar to that found with all four alleles. While one copy of Stat5 induced limited expression of target genes, two copies activated a lactation-like gene signature. Using ChIP-seq analyses on intact tissue under physiological conditions, we found that highly expressed and regulated genes were bound by STAT5 in their promoter proximal regions, whereas upstream binding had minor biological consequences. Remarkably, 80% of the genes bound by STAT5 in vivo were not under STAT5 control. RNA polymerase II intensity was directly proportional to STAT5 concentration only on STAT5 regulated genes providing mechanistic insight by which STAT5 activates mammary specific genes.
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interpretation of cytokine signaling through the transcription factors stat5a and STAT5B
Genes & Development, 2008Co-Authors: Gertraud W RobinsonAbstract:Transcription factors from the family of Signal Transducers and Activators of Transcription (STAT) are activated by numerous cytokines. Two members of this family, STAT5A and STAT5B (collectively called STAT5), have gained prominence in that they are activated by a wide variety of cytokines such as interleukins, erythropoietin, growth hormone, and prolactin. Furthermore, constitutive STAT5 activation is observed in the majority of leukemias and many solid tumors. Inactivation studies in mice as well as human mutations have provided insight into many of STAT5’s functions. Disruption of cytokine signaling through STAT5 results in a variety of cell-specific effects, ranging from a defective immune system and impaired erythropoiesis, the complete absence of mammary development during pregnancy, to aberrant liver function. On a molecular level, STAT5 has been linked to cell specification, proliferation, differentiation, and survival. Evidence is growing that the diverse outcomes of STAT5 signaling are not only determined by the expression of specific receptors but also by the interaction of STAT5 with cofactors and the cell-specific activity of members of the SOCS family, which negatively regulate STAT function. In this review, we focus on emerging concepts and challenges in the field of Janus kinase (JAK)–STAT5 signaling. First, we discuss unique functions of STAT5 in three distinct systems: mammary epithelial cells, hepatocytes, and regulatory T cells. Second, we present an example of how STAT5 can achieve cell specificity in hepatocytes through a physical and functional interaction with the glucocorticoid receptor. Third, we focus on the relevance of STAT5 in the development and progression of leukemia. Next, we discuss lessons derived from human mutations and disease. Finally, we address an emerging issue that the interpretation of experiments from STAT5-deficient mice and cells might be compromised as these cells might reroute and reprogram cytokine signals to the “wrong” STATs and thus acquire inappropriate cues. We propose that mice with mutations in various components of the JAK–STAT signaling pathway are living laboratories, which will provide insight into the versatility of signaling hardware and the adaptability of the software.
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loss of signal transducer and activator of transcription 5 leads to hepatosteatosis and impaired liver regeneration
Hepatology, 2007Co-Authors: Yongzhi Cui, Gertraud W Robinson, Atsushi Hosui, Oksana Gavrilova, Rui Sun, Kezhen Shen, Weiping Chen, Margaret C Cam, Bin Gao, Lothar HennighausenAbstract:Growth hormone controls many facets of a cell's biology through the transcription factors Stat5a and STAT5B (Stat5). However, whole body deletion of these genes from the mouse does not provide portentous information on cell-specific cytokine signaling. To explore liver-specific functions of Stat5, the entire Stat5 locus was deleted in hepatocytes using Cre-mediated recombination. Notably, Stat5-mutant mice developed fatty livers and displayed impaired proliferation of hepatocytes upon partial hepatectomy (PHx). Loss of Stat5 led to molecular consequences beyond the reduced expression of Stat5 target genes, such as those encoding suppressor of cytokine signaling 2 (SOCS2), Cish, and insulin-like growth factor 1 (IGF-1). In particular, circulating growth hormone levels were increased and correlated with insulin resistance and increased insulin levels. Aberrant growth hormone (GH)-induced activation of the transcription factors Stat1 and Stat3 was observed in mutant livers. To test whether some of the defects observed in liver-specific Stat5 deficient mice were due to aberrant Stat1 expression and activation, we generated Stat1−/− mice with a hepatocyte-specific deletion of Stat5. Concomitant loss of both Stat5 and Stat1 restored cell proliferation upon PHx but did not reverse fatty liver development. Thus the molecular underpinnings of some defects observed in the absence of Stat5 are the consequence of a deregulated activation of other signal transducers and activators of transcription (STAT) family members. Conclusion: Aberrant cytokine-Stat5 signaling in hepatocytes alters their physiology through increased activity of Stat1 and Stat3. Such cross-talk between different pathways could add to the complexity of syndromes observed in disease. (HEPATOLOGY 2007.)
Robert A. Kirken - One of the best experts on this subject based on the ideXlab platform.
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specific inhibition of stat5a b promotes apoptosis of il 2 responsive primary and tumor derived lymphoid cells
Journal of Immunology, 2003Co-Authors: Fariba Behbod, Zsuzsanna S Nagy, James G Karras, Charlene R Johnson, Stanislaw M. Stepkowski, David W Jarvis, Robert A. KirkenAbstract:Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2′- O -methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in ≥55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas STAT5B required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for STAT5B. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and STAT5B, both proteins preferentially mediate an antiapoptotic survival influence.
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functional uncoupling of the janus kinase 3 stat5 pathway in malignant growth of human t cell leukemia virus type 1 transformed human t cells
Journal of Immunology, 2000Co-Authors: Rebecca A Erwin, Robert A. Kirken, Lihua Wang, Yuling Wang, William L FarrarAbstract:Human T cell leukemia virus type 1 (HTLV-1) transforms cytokine-dependent T lymphocytes and causes adult T cell leukemia. Janus tyrosine kinase (Jak)3 and transcription factors Stat5a and STAT5B are essential for the proliferation of normal T cells and are constitutively hyperactivated in both HTLV-1-transformed human T cell lines and lymphocytes isolated from HTLV-1-infected patients; therefore, a critical role for the Jak3-Stat5 pathway in the progression of this disease has been postulated. We recently reported that tyrphostin AG-490 selectively blocked IL-2 activation of Jak3/Stat5 and growth of murine T cell lines. Here we demonstrate that disruption of Jak3/Stat5a/b signaling with AG-490 (50 μM) blocked the proliferation of primary human T lymphocytes, but paradoxically failed to inhibit the proliferation of HTLV-1-transformed human T cell lines, HuT-102 and MT-2. Structural homologues of AG-490 also inhibited the proliferation of primary human T cells, but not HTLV-1-infected cells. Disruption of constitutive Jak3/Stat5 activation by AG-490 was demonstrated by inhibition of 1) tyrosine phosphorylation of Jak3, Stat5a (Tyr 694 ), and STAT5B (Tyr 699 ); 2) serine phosphorylation of Stat5a (Ser 726 ) as determined by a novel phosphospecific Ab; and 3) Stat5a/b DNA binding to the Stat5-responsive β-casein promoter. In contrast, AG-490 had no effect on DNA binding by p50/p65 components of NF-κB, a transcription factor activated by the HTLV-1-encoded phosphoprotein, Tax. Collectively, these data suggest that the Jak3-Stat5 pathway in HTLV-1-transformed T cells has become functionally redundant for proliferation. Reversal of this functional uncoupling may be required before Jak3/Stat5 inhibitors will be useful in the treatment of this malignancy.
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differential control of the phosphorylation state of proline juxtaposed serine residues ser725 of stat5a and ser730 of STAT5B in prolactin sensitive cells
Journal of Biological Chemistry, 1998Co-Authors: Hiroko Yamashita, Rebecca A Erwin, William L Farrar, Jun Xu, Robert A. KirkenAbstract:Abstract Transcription factors of the Stat family are controlled by protein kinases. Phosphorylation of a positionally conserved tyrosine residue is obligatory for Stat dimerization, nuclear translocation, and specific DNA binding. Studies of Stat1 and Stat3 have suggested that serine phosphorylation may also regulate function. We now identify serine residues located in a conserved PSP motif of Stat5a (Ser725) and STAT5B (Ser730) as major phosphorylation sites, using mutagenesis, phosphoamino acid analysis, and site-specific anti-Stat5-phosphoserine antibodies. Unexpectedly, phosphorylation control of this PSP motif differed between the highly homologous Stat5a and STAT5B proteins. Whereas Ser725 of Stat5a was constitutively phosphorylated both in COS-7 cells and Nb2 lymphocytes, phosphorylation of Ser730of STAT5B was markedly stimulated by prolactin. The data also suggested the existence of a second major serine phosphorylation site in Stat5a. Interestingly, constitutive phosphorylation of the PSP motif was suppressed by PD98059 but not by staurosporine under conditions in which both agents inhibited mitogen-activated protein kinases. Furthermore, pretreatment of cells with staurosporine, PD98059, H7, or wortmannin did not prevent either Stat5a or STAT5B from becoming maximally serine-phosphorylated after prolactin exposure. We propose that two pathways regulate Stat5 serine phosphorylation, one that is prolactin-activated and PD98059-resistant and one that is constitutively active and PD98059-sensitive and preferentially targets Stat5a. Finally, phosphorylation of the PSP motif of Stat5a or STAT5B was not essential for DNA binding or transcriptional activation of a β-casein reporter gene in COS-7 cells, suggesting that serine kinase control of Stat5 activity differs from that of Stat1 and Stat3.
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two discrete regions of interleukin 2 il2 receptor beta independently mediate il2 activation of a pd98059 rapamycin wortmannin insensitive stat5a b serine kinase
Journal of Biological Chemistry, 1997Co-Authors: Robert A. Kirken, Rebecca A Erwin, Maria Grazia Malabarba, Luis Dasilva, Jun Xu, William L FarrarAbstract:Abstract Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and STAT5B proteins lack this putative mitogen-activated protein kinase phosphorylation site. The present study set out to establish whether Stat5a and STAT5B are under the control of an interleukin-2 (IL2)-activated Stat5 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and STAT5B in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1–5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of STAT5B with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced STAT5B response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor β (IL2Rβ) that could independently restore the ability of a truncated IL2Rβ mutant to mediate Stat5a/b phosphorylation and DNA binding to the γ-activated site of the β-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rβ region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation.
Soohee Park - One of the best experts on this subject based on the ideXlab platform.
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serine phosphorylation of gh activated signal transducer and activator of transcription 5a stat5a and STAT5B impact on stat5 transcriptional activity
Molecular Endocrinology, 2001Co-Authors: Soohee Park, Hiroko Yamashita, David J. WaxmanAbstract:Signal transducer and activator of transcription 5b (STAT5B), the major liver-expressed STAT5 form, is phosphorylated on both tyrosine and serine in GH-stimulated cells. Although tyrosine phosphorylation is known to be critical for the dimerization, nuclear translocation, and activation of STAT5B DNA-binding and transcriptional activities, the effect of STAT5B serine phosphorylation is uncertain. Presently, we identify Ser730 as the site of STAT5B serine phosphorylation in GH-stimulated liver cells. We additionally show that the serine kinase inhibitor H7 partially blocks the GH-stimulated formation of (Ser,Tyr)-diphosphorylated STAT5B without inhibiting STAT5B nuclear translocation. Evaluation of the functional consequences of STAT5B serine phosphorylation by mutational analysis revealed an approximately 50% decrease in GH-stimulated luciferase reporter gene activity regulated by an isolated STAT5-binding site when STAT5B Ser730 was mutated to alanine and under conditions where STAT5 DNA-binding activity...
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distinctive roles of stat5a and STAT5B in sexual dimorphism of hepatic p450 gene expression impact of stat5a gene disruption
Journal of Biological Chemistry, 1999Co-Authors: Soohee Park, Helen W Davey, David J. WaxmanAbstract:Abstract STAT5B gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239–7244). In the present study, disruption of the mouse Stat5a gene, whose coding sequence is ∼90% identical to the STAT5B gene, resulted in no loss of expression in male mice of several sex-dependent, GH-regulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5B feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16α-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6β-hydroxylase). Since STAT5a is thus nonessential for these male GH responses, STAT5B homodimers, but not STAT5a-STAT5B heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either Stat5a or STAT5Bled to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. Stat5a or STAT5B gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5a, which is much less abundant in liver than STAT5B, and STAT5B are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and gene-specific effects that GH has on the liver.
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distinctive roles of stat5a and STAT5B in sexual dimorphism of hepatic p450 gene expression
1999Co-Authors: Soohee Park, Helen W Davey, David J. WaxmanAbstract:STAT5B gene disruption leads to an apparent growth hormone (GH) pulse insensitivity associated with loss of male-characteristic body growth rates and male-specific liver gene expression (Udy, G. B., Towers, R. P., Snell, R. G., Wilkins, R. J., Park, S. H., Ram, P. A., Waxman, D. J., and Davey, H. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 7239 ‐7244). In the present study, disruption of the mouse Stat5a gene, whose coding sequence is ;90% identical to the STAT5B gene, resulted in no loss of expression in male mice of several sex-dependent, GHregulated liver cytochrome P450 (CYP) enzymes. By contrast, the loss of STAT5B feminized the livers of males by decreasing expression of male-specific CYPs (CYP2D9 and testosterone 16a-hydroxylase) while increasing to female levels several female-predominant liver CYPs (CYP3A, CYP2B, and testosterone 6b-hydroxylase). Since STAT5a is thus nonessential for these male GH responses, STAT5B homodimers, but not STAT5aSTAT5B heterodimers, probably mediate the sexually dimorphic effects of male GH pulses on liver CYP expression. In female mice, however, disruption of either Stat5a or STAT5B led to striking decreases in several liver CYP-catalyzed testosterone hydroxylase activities. Stat5a or STAT5B gene disruption also led to the loss of a female-specific, GH-regulated hepatic CYP2B enzyme. STAT5a, which is much less abundant in liver than STAT5B, and STAT5B are therefore both required for constitutive expression in female but not male mouse liver of certain GH-regulated CYP steroid hydroxylases, suggesting that STAT5 protein heterodimerization is an important determinant of the sex-dependent and genespecific effects that GH has on the liver.
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regulation of signal transducer and activator of transcription stat 5b activation by the temporal pattern of growth hormone stimulation
Molecular Endocrinology, 1997Co-Authors: Carol A Gebert, Soohee Park, David J. WaxmanAbstract:Plasma GH profiles, intermittent in adult male and continuous in adult female rats, respectively, activate unique patterns of gene transcription in male and female rat liver. Pulsatile, but not continuous, GH exposure activates liver STAT5 (signal transducer and activator of transcription-5) by tyrosine phosphorylation, leading to nuclear translocation, and is proposed to play a key role in GH pulse-regulated male-specific liver gene expression. The mechanisms underlying the GH pattern dependence of STAT5 activation are presently investigated using a rat hepatocyte-derived cell line. Rat GH stimulated tyrosine phosphorylation followed by serine or threonine phosphorylation, leading to activation of the DNA-binding activity of STAT5B, the major STAT5 form present in these cells. Maximal STAT5B activation required a full 20 min at a receptor-saturating GH concentration of 50 ng/ml, suggesting that hormone binding leading to receptor dimerization is a relatively slow process. Repeat cycles of GH pulsation le...
