Functional analysis of the human Sprouty2 gene promoter (original) (raw)
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Evidence that SPROUTY2 functions as an inhibitor of mouse embryonic lung growth and morphogenesis
Mechanisms of Development, 2001
Experimental evidence is rapidly emerging that the coupling of positive regulatory signals with the induction of negative feedback modulators is a mechanism of ®ne regulation in development. Studies in Drosophila and chick have shown that members of the SPROUTY family are inducible negative regulators of growth factors that act through tyrosine kinase receptors. We and others have shown that Fibroblast Growth Factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis. Herein, we provide direct evidence that mSprouty2 is dynamically expressed in the peripheral endoderm in embryonic lung and is downregulated in the clefts between new branches at E12.5. We found that mSprouty2 was expressed in a domain restricted in time and space, adjacent to that of Fgf10 in the peripheral mesenchyme. By E14.5, Fgf10 expression was restricted to a narrow domain of mesenchyme along the extreme edges of the individual lung lobes, whereas mSprouty2 was most highly expressed in the subjacent epithelial terminal buds. FGF10 beads upregulated the expression of mSprouty2 in adjacent epithelium in embryonic lung explant culture. Lung cultures treated with exogenous FGF10 showed greater branching and higher levels of mSpry2 mRNA. Conversely, Fgf10 antisense oligonucleotides reduced branching and decreased mSpry2 mRNA levels. However, treatment with exogenous FGF10 or antisense Fgf10 did not change Shh and FgfR2 mRNA levels in the lungs. We investigated Sprouty2 function during lung development by two different but complementary approaches. The targeted overexpression of mSprouty2 in the peripheral lung epithelium in vivo, using the Surfactant Protein C promoter, resulted in a low level of branching, lung lobe edges abnormal in appearance and the inhibition of epithelial proliferation. Transient high-level overexpression of mSpry2 throughout the pulmonary epithelium by intra-tracheal adenovirus microinjection also resulted in a low level of branching. These results indicate for the ®rst time that mSPROUTY2 functions as a negative regulator of embryonic lung morphogenesis and growth. q
Cell, 1997
with the TATA-box protein accessory factor TAF II 110 (Gill et al., 1994). When bound to distant sites in cis, it Frank Grosveld, and Sjaak Philipsen can interact with itself, thus looping out the intervening Erasmus University Rotterdam DNA (Li et al., 1991; Su et al., 1991). This suggests that Department of Cell Biology Sp1 may establish interactions between promoters and 3000 DR Rotterdam distant regulatory elements in vivo through such a loop-The Netherlands ing mechanism (Ptashne, 1986). Sp1 binding sites appear in numerous promoters and other regulatory sequences of tissue-specific and ubiquitous genes (over Summary 1500 citations). A number of observations imply a wider role for Sp1-Transcription factor Sp1 has been implicated in the like proteins in nuclear processes. Sp1 binding sites are expression of many genes. Moreover, it has been critical for the maintenance of the methylation-free CpG suggested that Sp1 is linked to the maintenance of island of the APRT gene (Brandeis et al., 1994; Macleod methylation-free CpG islands, the cell cycle, and the et al., 1994). Since Sp1 binding sites occur very freformation of active chromatin structures. We have inquently in CpG islands, this suggests that Sp1 prevents activated the mouse Sp1 gene. Sp1 Ϫ/Ϫ embryos are methylation of many CpG islands in the genome. Mainteretarded in development, show a broad range of abnance of the appropriate methylation patterns is essennormalities, and die around day 11 of gestation. In tial for normal development (Li et al., 1992). A second Sp1 Ϫ/Ϫ embryos, the expression of many putative tarlink between Sp1 and nuclear architecture is that Sp1 get genes, including cell cycle-regulated genes, is not may function in remodeling chromatin structures, as was affected, CpG islands remain methylation free, and first observed for the SV40 virus early promoter (Jongactive chromatin is formed at the globin loci. However, stra et al., 1984). Another example is the -globin locus the expression of the methyl-CpG-binding protein control region (LCR) (Grosveld et al., 1987) studied ex-MeCP2 is greatly reduced in Sp1 Ϫ/Ϫ embryos. MeCP2 tensively in our laboratory. The most important part of is thought to be required for the maintenance of differthe LCR, which can activate chromatin in single-copy entiated cells. We suggest that Sp1 is an important transgenic mice (Ellis et al., 1996), is completely depenregulator of this process. dent on the presence of Sp1 binding sites (Philipsen et al., 1993). Third, Sp1 has been implicated in the expres-Introduction sion of cell cycle-regulated genes like thymidine kinase (TK) (Karlseder et al., 1996), dihydrofolate reductase Sp1 was one of the first eukaryotic transcription factors (DHFR) (Lin et al., 1996) and b-myb (Zwicker et al., 1996). to be identified and cloned as a factor that binds to the It has been suggested that Sp1 activity is modulated by SV40 early promoter (Dynan and Tjian, 1983). It contains components of the cell cycle machinery like the cyclins three zinc finger motifs, Cys-2-His-2, which bind to a (Shao and Robbins, 1995) and Rb-like proteins (Shao and Robbins, 1995), and Sp1 has been shown to interact consensus GGG GCG GGG (Letovsky and Dynan, 1989). directly with the growth-and cell cycle-regulated tran-Sp1 is an abundant nuclear protein in most cells, but scription factor E2F in vitro (Karlseder et al., 1996; Lin the level of expression changes during development and et al., 1996). Thus, Sp1 may be intricately linked to the varies in different cell types (Saffer et al., 1991). Sp1 is regulation of the cell cycle and the establishment of the founding member of a growing family of proteins transcriptional competence through the maintenance of with highly homologous zinc-finger domains that bind methylation-free islands and the organization of chro-GC or GT boxes (Hagen et al., 1992; Imataka et al., 1992; matin structure, in addition to its direct role in transcrip-Kingsley and Winoto, 1992; Miller and Bieker, 1993; tional activation. Crossley et al., 1996). This has precluded a straightfor-In order to address these issues directly, we have ward interpretation of expression studies of putative inactivated the mouse Sp1 gene in ES cells and charac-Sp1-responsive genes, and hence, little is known about terized the phenotype of Sp1-deficient mice. Sp1 Ϫ/Ϫ emits biological role. bryos survive until day 9.5 (E9.5) of gestation. They are The molecular properties of Sp1 have been studied severely retarded in growth and show a broad range of in vitro in great detail. The protein is phosphorylated phenotypic abnormalities. Sp1 Ϫ/Ϫ ES cells contribute (Jackson et al., 1990) and highly glycosylated (Jackson extensively to every tissue of chimeras until E9.5 but fail and Tjian, 1988). The N-terminus contains glutamineto contribute after early embryonic development, thus and serine/threonine-rich domains that are essential for demonstrating that the defect in Sp1 Ϫ/Ϫ cells is cell autranscriptional activation (Courey et al., 1989). The tonomous. Furthermore, we show that in Sp1 Ϫ/Ϫ em-C-terminal domain of Sp1 is involved in synergistic actibryos the expression of many putative Sp1 target genes, vation and interaction with other transcription factors including cell cycle-regulated genes, is unaffected, that (Li et al., 1991). Sp1 has been shown to interact directly methylation-free islands are faithfully maintained, and that the globin loci are activated, implying that active chromatin structures can still be formed. However,
Current Biology, 1999
In Drosophila embryos, the loss of sprouty gene function enhances branching of the respiratory system. Three human sprouty homologues (h-Spry1-3) have been cloned recently, but their function is as yet unknown [1]. Here, we show that a murine sprouty gene (mSpry-2), the product of which shares 97% homology with the respective human protein, is expressed in the embryonic murine lung. We used an antisense oligonucleotide strategy to reduce expression of mSpry-2 by 96%, as measured by competitive reverse transcriptase PCR, in E11.
Regulation of Sprouty2 stability by mammalian Seven-in-Absentia homolog 2
Journal of Cellular Biochemistry, 2007
Mammalian Sprouty (Spry) gene expression is rapidly induced upon activation of the FGF receptor signaling pathway in multiple cell types including cells of mesenchymal and epithelial origin. Spry2 inhibits FGFdependent ERK activation and thus Spry acts as a feedback inhibitor of FGF-mediated proliferation. In addition, Spry2 interacts with the ring-finger-containing E3 ubiquitin ligase, c-Cbl, in a manner that is dependent upon phosphorylation of Tyr55 of Spry2. This interaction results in the poly-ubiquitination and subsequent degradation of Spry2 by the proteasome. Here, we describe the identification of another E3 ubiquitin ligase, human Seven-in-Absentia homolog-2 (SIAH2), as a Spry2 interacting protein. We show by yeast two-hybrid analysis that the N-terminal domain of Spry2 and the ring finger domain of SIAH2 mediated this interaction. Co-expression of SIAH2 resulted in proteasomal degradation of Spry1, 2, and to a lesser extent Spry4. The related E3 ubiquitin-ligase, SIAH1, had little effect on Spry2 protein stability when coexpressed. Unlike c-Cbl-mediated degradation of Spry2, SIAH2-mediated degradation was independent of phosphorylation of Spry2 on Tyr55. Spry2 was also phosphorylated on Tyr227, and phosphorylation of this residue was also dispensable for SIAH2-mediated degradation of Spry2. Finally, co-expression of SIAH2 with Spry2 resulted in a rescue of FGF2-mediated ERK phosphorylation. These data suggest a novel mechanism whereby Spry2 stability is regulated in a manner that is independent of tyrosine phosphorylation, and provides an addition level of control of Spry2 protein levels.
Sp2 Is a Maternally Inherited Transcription Factor Required for Embryonic Development
Journal of Biological Chemistry, 2010
The Sp family of transcription factors is required for the expression of cell cycle-and developmentally regulated genes, and the deregulated expression of a handful of family members is associated with human tumorigenesis. Sp2 is a relatively poorly characterized member of the Sp family that, although widely expressed, exhibits little or no DNA binding or transcriptional activity in human and mouse cell lines. To begin to address the role(s) played by Sp2 in early metazoan development we have cloned and characterized Sp2 from zebrafish (Danio rerio). We report that 1) the intron/exon organization and amino acid sequence of zebrafish Sp2 is closely conserved with its mammalian orthologues, 2) zebrafish Sp2 weakly stimulates an Sp-dependent promoter in vitro and associates with the nuclear matrix in a DNA-independent fashion, 3) zebrafish Sp2 is inherited as a maternal transcript, is transcribed in zebrafish embryos and adult tissues, and is required for completion of gastrulation, and 4) zebrafish lines carrying transgenes regulated by the Sp2 promoter recapitulate patterns of endogenous Sp2 expression.
The EMBO Journal, 2002
Drosophila Sprouty (dSpry) was genetically identi®ed as a novel antagonist of ®broblast growth factor receptor (FGFR), epidermal growth factor receptor (EGFR) and Sevenless signalling, ostensibly by eliciting its response on the Ras/MAPK pathway. Four mammalian sprouty genes have been cloned, which appear to play an inhibitory role mainly in FGFmediated lung and limb morphogenesis. Evidence is presented herein that describes the functional implications of the direct association between human Sprouty2 (hSpry2) and c-Cbl, and its impact on the cellular localization and signalling capacity of EGFR. Contrary to the consensus view that Spry2 is a general inhibitor of receptor tyrosine kinase signalling, hSpry2 was shown to abrogate EGFR ubiquitylation and endocytosis, and sustain EGF-induced ERK signalling that culminates in differentiation of PC12 cells. Correlative evidence showed the failure of hSpry2DN11 and mSpry4, both de®cient in c-Cbl binding, to instigate these effects. hSpry2 interacts speci®cally with the c-Cbl RING ®nger domain and displaces UbcH7 from its binding site on the E3 ligase. We conclude that hSpry2 potentiates EGFR signalling by speci®cally intercepting c-Cbl-mediated effects on receptor down-regulation.
Molecular Biology of the Cell, 2004
Sprouty proteins are recently identified receptor tyrosine kinase (RTK) inhibitors potentially involved in many developmental processes. Here, we report that Sprouty proteins become tyrosine phosphorylated after growth factor treatment. We identified Tyr55 as a key residue for Sprouty2 phosphorylation and showed that phosphorylation was required for Sprouty2 to inhibit RTK signaling, because a mutant Sprouty2 lacking Tyr55 augmented signaling. We found that tyrosine phosphorylation of Sprouty2 affected neither its subcellular localization nor its interaction with Grb2, FRS2/SNT, or other Sprouty proteins. In contrast, Sprouty2 tyrosine phosphorylation was necessary for its binding to the Src homology 2-like domain of c-Cbl after fibroblast growth factor (FGF) stimulation. To determine whether c-Cbl was required for Sprouty2-dependent cellular events, Sprouty2 was introduced into c-Cbl-wild-type and -null fibroblasts. Sprouty2 efficiently inhibited FGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 in c-Cbl-null fibroblasts, thus indicating that the FGF-dependent binding of c-Cbl to Sprouty2 was dispensable for its inhibitory activity. However, c-Cbl mediates polyubiquitylation/proteasomal degradation of Sprouty2 in response to FGF. Last, using Src-family pharmacological inhibitors and dominant-negative Src, we showed that a Src-like kinase was required for tyrosine phosphorylation of Sprouty2 by growth factors. Thus, these data highlight a novel negative and positive regulatory loop that allows for the controlled, homeostatic inhibition of RTK signaling. ‡ Corresponding authors.
Spatial signal repression as an additional role of Sprouty2 protein variants
Cellular Signalling, 2019
Sprouty2 (Spry2) is a prominent member of a protein family with crucial functions in the modulation of signal transduction. One of its main actions is the repression of mitogenactivated protein kinase (MAPK) pathway in response to growth factor-induced signalling. A common single nucleotide polymorphism within the Spry2 gene creates two protein variants where a proline adjacent to the serine rich domain is converted to an additional serine. Both protein variants perform similar functions although their efficiency in fulfilling these tasks varies. In this report, we used biochemical fractionation methods as well as confocal microscopy to analyse quantitative and qualitative differences in the distribution of Spry2 variants. We found that Spry2 proteins localize not solely to the plasma membrane, but also to other membrane engulfed compartments like for example the Golgi apparatus. In these less dense organelles, predominantly slower migrating forms reside indicating that posttranslational modification contributes to the distribution profile of Spry2. However there is no significant difference in the distribution of the two variants. Additionally, we found that Spry2 could be found exclusively in membrane fractions irrespective of the mitogen availability and the phosphorylation status. Considering the interference of extracellular signal-regulated kinase (ERK) activation in the cytoplasm, both Spry2 variants inhibited the levels of phosphorylated ERK (pERK) significantly to a similar extent. In contrast, the induction profiles of pERK levels were completely different in the nuclei. Again, both Spry2 variants diminished the levels of pERK. While the proline variant lowered the activation throughout the observation period, the serine variant failed to interfere with immediate accumulation of nuclear pERK levels, but the signal duration was shortened. Since the extent of the pERK inhibition in the nuclei was drastically more pronounced than in the cytoplasm, we conclude that Spry2in addition to its known functions as a repressor of general ERK phosphorylationfunctions as a spatial repressor of nucleic ERK activation. Accordingly, a dominant negative version of Spry2 was only able to enhance the pERK levels of serumdeprived cells in the cytosol, while in the nucleus the intensity of the pERK signal in response to serum addition was significantly increased.
Intermolecular Interactions of Sprouty Proteins and Their Implications in Development and Disease
Molecular Pharmacology, 2009
Receptor tyrosine kinase (RTK) signaling is spatially and temporally regulated by a number of positive and negative regulatory mechanisms. These regulatory mechanisms control the amplitude and duration of the signals initiated at the cell surface to have a normal or aberrant biological outcome in development and disease, respectively. In the past decade, the Sprouty (Spry) family of proteins has been identified as modulators of RTK signaling in normal development and disease. This review