Mutational analysis reveals separable DNA binding and trans-activation of Drosophila STAT92E (original) (raw)
Related papers
EMBO reports, 2008
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission. In vertebrates, seven signal transducer and activator of transcription (STAT) proteins bind to palindromic sites separated by spacers of two or three nucleotides (STAT1), four nucleotides (STAT6) or three nucleotides (STAT2 to STAT5a/b). This diversity of binding sites provides specificity to counter semiredundancy and was thought to be a recent evolutionary acquisition. Here, we examine the natural DNA-binding sites of the single Drosophila Stat and show that this is not the case. Rather, Drosophila Stat92E is able to bind to and activate target gene expression through both 3n and 4n spaced sites. Our experiments indicate that Stat92E has a higher binding affinity for 3n sites than for 4n sites and suggest that the levels of target gene expression can be modulated by insertion and/or deletion of single bases. Our results indicate that the ancestral STAT protein had the capacity to bind to 3n and 4n sites and that specific STAT binding preferences evolved with the radiation of the vertebrate STAT family.
EMBO Reports, 2008
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without specific permission. In vertebrates, seven signal transducer and activator of transcription (STAT) proteins bind to palindromic sites separated by spacers of two or three nucleotides (STAT1), four nucleotides (STAT6) or three nucleotides (STAT2 to STAT5a/b). This diversity of binding sites provides specificity to counter semiredundancy and was thought to be a recent evolutionary acquisition. Here, we examine the natural DNA-binding sites of the single Drosophila Stat and show that this is not the case. Rather, Drosophila Stat92E is able to bind to and activate target gene expression through both 3n and 4n spaced sites. Our experiments indicate that Stat92E has a higher binding affinity for 3n sites than for 4n sites and suggest that the levels of target gene expression can be modulated by insertion and/or deletion of single bases. Our results indicate that the ancestral STAT protein had the capacity to bind to 3n and 4n sites and that specific STAT binding preferences evolved with the radiation of the vertebrate STAT family.
The Drosophila JAK-STAT pathway
JAK-STAT, 2013
The molecular characterization of the JAK-STAT pathway has recently celebrated its 20th birthday, a milestone that marks the discovery of the tyrosine kinases TYK2 and JAK1 and the STAT1 and STAT2 transcription factors as the key factors underlying the cellular response to type I interferons. The initial description of the first JAK-STAT pathway components sparked a flurry of activity in multiple labs, which rapidly characterized a wide range of ligands, receptors, four JAKs, and seven STATs present in vertebrate cells. Collectively these factors signal through fundamentally similar mechanisms; a JAK-STAT pathway that now serves as a textbook example of how extracellular ligands can act at the cell surface to modulate nuclear gene expression. Hopscotch-the fly JAK. In contrast to the key role played by the Drosophila system in identifying many other key cellular signaling cascades, the initial steps leading to the identification of the Drosophila JAK-STAT pathway components lagged pioneering work being undertaken in vertebrate cell-based systems. However, the ball was set rolling in 1994 when the Perrimon lab cloned a novel gene termed Hopscotch (Hop), which encodes a maternally supplied protein required for the patterning of the embryonic cuticle (for an example of the LOF phenotype see ) and the proliferation of diploid imaginal cells. 5,6 Cloning of Hop identified it as a 1177 amino acid non-receptor tyrosine kinase, expressed throughout development, with a kinase domain, sharing 39% identity with JAK1, JAK2, and Tyk2, and an overall identity of 27% to JAK2. While not necessarily apparent at the time, the identification of JAK and the characteristic segmentation phenotype associated with pathway mutants represented a key insight and the first step on the path toward identifying the rest of the pathway.
Opposing roles for Drosophila JAK/STAT signalling during cellular proliferation
Oncogene, 2005
The JAK/STAT signalling pathway mediates both antiproliferative responses following interferon stimulation and cellular proliferation in response to cytokines such as interleukins and growth factors. Central to these responses are the seven vertebrate STAT molecules, misregulation of which is implicated in a variety of malignancies. We have investigated the proliferative role of the single Drosophila STAT92E, part of the evolutionarily conserved JAK/STAT cascade. During second instar larval wing disc development pathway activity is both necessary and sufficient to promote proliferation of this epithelial cell type. However by later stages, endogenous STAT92E is stimulated by a noncannonical mechanism to exert pronounced antiproliferative effects. Ectopic canonical activation is sufficient to further decrease proliferation and leads to the premature arrest of cells in the G2 phase of the cell cycle. The single STAT92E present in Drosophila therefore mediates both proproliferative functions analogous to vertebrate interleukin-stimulated STAT3 and antiproliferative functions analogous to interferon-stimulated STAT1. Pro-and antiproliferative roles therefore represent ancestral activities conserved through evolution and subsequently assigned to distinct molecules.
A Drosophila PIAS homologue negatively regulates stat92E
Proceedings of the National Academy of Sciences, 2001
Transcriptional activation by, and therefore the physiologic impact of, activated tyrosine-phosphorylated STATs ( s ignal t ransducers and a ctivators of t ranscription) may be negatively regulated by proteins termed PIAS ( p rotein i nhibitors of a ctivated s tats), as shown by previous experiments with mammalian cells in culture. Here, by using the genetic modifications in Drosophila , we demonstrate the in vivo functional interaction of the Drosophila homologues stat92E and a Drosophila PIAS gene ( dpias ). To this end we use a LOF allele and conditionally overexpressed dpias in JAK-STAT pathway mutant backgrounds. We conclude that the correct dpias / stat92E ratio is crucial for blood cell and eye development.
Cellular and Molecular Life Sciences (CMLS), 1999
The seven mammalian members of the signal paper describes several important discoveries linked to transducer and activator of transcription (STAT) family mechanistic aspects of STAT transcription factor function. These include regulated serine phosphorylation of share a common core structure which reflects their shared mechanism of activation, dimerization, and DNA bind-the transactivating domain, promoter-dependent interactions of STATs with each other, or of STATs with other ing. By contrast, the STAT C termini containing the transcription factors, and with transcriptional co-activa-sequences required for transcriptional activation are tors. The basis, background, and implications of these much less homologous, suggesting different ways by which individual STATs activate their target genes. This molecular events will be summarized and discussed.
Journal of Biological Chemistry, 2000
The Drosophila raf (D-raf) gene promoter contains a recognition consensus sequence for Drosophila STAT (D-STAT). By band mobility shift assay, we detected a factor binding to the D-STAT-recognition sequence in extracts of cultured Drosophila cells treated with vanadate peroxide. UV-cross-linking analyses suggested the size of the binding factor to be almost same as that of D-STAT. Furthermore, the binding activity was increased in cells cotransfected with HOP and D-STAT expression plasmids. These results strongly suggest that D-STAT binds to the D-STAT recognition sequence in the D-raf gene promoter. Transient luciferase expression assay using Schneider 2 cells indicated that the D-raf gene promoter is activated by D-STAT through the D-STAT-binding site. Furthermore, analyses with transgenic flies carrying Draf-lacZ fusion genes with and without mutations in the D-STAT-binding site pointed to an important role in D-raf gene promoter activity throughout development. We also found that the D-STAT-binding site is required for injury-induced activation of the D-raf gene promoter. Here we propose that D-STAT can participate in regulation of the mitogenactivated protein kinase cascade through D-raf gene activation.
Expression and characterization of two STAT isoforms from Sf9 cells
Developmental & Comparative Immunology, 2008
In invertebrates, the JAK-STAT signaling pathway is involved in the anti-bacterial response and is part of an anti-viral response in Drosophila. In this study, we show that two STAT transcripts are generated by alternative splicing and encode two isoforms of Sf-STAT with different C-terminal ends. These two isoforms were produced and purified using the recombinant baculovirus technology. Both purified isoforms showed similar DNA-binding activity and displayed weak but significant transactivation potential toward a Drosophila promoter that contained a STAT-binding motif. No significant activation of the Sf-STAT protein in Sf9 cells was found by infection with baculovirus AcMNPV.
GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo
Gene Expression Patterns, 2007
JAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reXects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs diVer by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reXect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP Xuorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are speciWcally activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reXect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.