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Jonathan D. Licht - One of the best experts on this subject based on the ideXlab platform.
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Spry1 and SPRY2 are necessary for eyelid closure.
Developmental biology, 2013Co-Authors: Murali R. Kuracha, Ed Siefker, Jonathan D. Licht, Venkatesh GovindarajanAbstract:Abstract Sproutys (Sprys) are downstream targets and negative feedback regulators of the FGF–Ras–ERK signaling pathway. Our previous studies have shown that Spry1 and SPRY2, through negative modulation of FGF–ERK signaling, allow lens vesicle separation from the overlying ectoderm and regulate corneal epithelial proliferation. Here we show that Spry1 and SPRY2 are necessary for eyelid closure. Murine palpebral conjunctival epithelial cells that differentiate as inner eyelids and adjacent mesenchymal cells express Spry1 and SPRY2 prior to eyelid closure. Conditional deletion of both Spry1 and SPRY2, but not either one alone, in the ocular surface epithelial cells result in the “EOB” (eyes open at birth) phenotype suggesting redundant roles for these proteins during eyelid closure. Spry mutant eyelids show increased proliferation of conjunctival epithelial cells with concomitant induction of FGF targets, Erm, Pea3 and Dusp6 and elevated ERK phosphorylation. Peridermal cells at the leading edge of Spry-mutant eyelids showed reduced c-Jun, but not ERK, phosphorylation, reduced F-actin polymerization and reduced motility in vitro. Spry mutant eyelids also showed disruptions in epithelial mesenchymal interactions reflected in the enhanced mesenchymal Spry1 and Spry4 expression, disaggregation of BMP4-positive mesenchymal cells and loss of Shh in the eyelid epithelium. Spry mutant eyelids also showed increased Wnt signaling and reduced expression of Foxc1 and Foxc2, two transcription factors previously shown to be necessary for eyelid closure. Collectively, our results show that conjunctival epithelial Spry1 and SPRY2 redundantly promote eyelid closure by (a) stimulating ERK-independent, c-Jun-mediated peridermal migration, (b) suppressing conjunctival epithelial proliferation through FGF–ERK signaling, (c) mediating conjunctival epithelial-mesenchymal interactions and (d) maintaining expression of Foxc1 and Foxc2.
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spry1 and SPRY2 are necessary for lens vesicle separation and corneal differentiation
Investigative Ophthalmology & Visual Science, 2011Co-Authors: Murali R. Kuracha, Ed Siefker, Jonathan D. Licht, Daniel Burgess, Jake T Cooper, Michael L Robinson, Venkatesh GovindarajanAbstract:The vertebrate lens is composed of proliferating epithelial cells in the anterior portion and postmitotic, terminally differentiated fiber cells in the posterior part. This polarity is maintained throughout life by precise coordination of proliferation, cell cycle exit, and differentiation. An inductive signal from the retina is thought to induce lens epithelial cells at the equatorial region to withdraw from the cell cycle and initiate the fiber differentiation program.1 In vitro and in vivo studies show that FGF stimulation of lens epithelial cells is sufficient to induce premature fiber differentiation.2–5 Expression of secreted dominant negative FGF receptor 3 (FGFR3) or deletion of all six alleles of FGFR1, -2, and -3 in the lens in transgenic or knockout mice, respectively, is sufficient to inhibit initiation of lens fiber differentiation.6,7 These results suggest that FGFR-meditated signaling is necessary for normal fiber differentiation. Sproutys play an essential role in regulation of Ras-Raf-Erk signaling downstream of FGFR stimulation. Four vertebrate Sprys, Spry1-4, have been identified and have been shown to antagonize Ras-Raf-ERK signaling downstream of FGFR.8–11 During murine embryogenesis, the sites of Spry expression often coincide with centers of FGF signaling.11–15 Spry-knockout mice have revealed interesting roles for these proteins in regulation of organogenesis. Spry1-null mice show kidney and urinary tract defects with ectopic branching of the Wolffian duct and increased ERK activation.16,17 SPRY2-null mice show alterations in inner ear development that are rescued by reduction in FGF8 dosage.18 Spry3 is not expressed in the mouse embryo but is expressed in the adult brain and testes.11 Effects of Spry3 deletion in the murine germline have not been reported. Spry4-null mice show growth retardation and polysyndactyly.19 In addition, SPRY2 and -4 have been shown to be critical for repression of ERK activation and diastema tooth formation.20 In the brain, Spry1 and -2 regulate cortical patterning, proliferation, and differentiation by repression of FGF-ERK signaling.9 These results establish Sprys as critical modulators of FGF-ERK signaling during organogenesis. Expression of Spry1 and -2 at different stages of lens maturation has been described previously.21 Nonetheless, the question of whether Sprys are necessary for lens development has not been addressed. By conditional deletion of Spry1 and -2 in ocular tissues, we show that these genes play critical roles in not only lens but also corneal development.
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Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival.
Cell death and differentiation, 2007Co-Authors: Isabelle Gross, Jonathan D. Licht, Olivier Armant, Samir Benosman, J L G De Aguilar, J-n Freund, Michèle Kedinger, Christian Gaiddon, J P LoefflerAbstract:Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of SPRY2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates SPRY2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the SPRY2 promoter. In immature neurons, we show that overexpression of wild-type SPRY2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of SPRY2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, SPRY2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of SPRY2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that SPRY2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.
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SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant.
Cancer research, 2004Co-Authors: Dimitra Tsavachidou, Jonathan D. Licht, Mathew L. Coleman, Galene Athanasiadis, Michael F. Olson, Barbara L. WeberAbstract:BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.
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SPRY2 is an inhibitor of the ras extracellular signal regulated kinase pathway in melanocytes and melanoma cells with wild type braf but not with the v599e mutant
Cancer Research, 2004Co-Authors: Dimitra Tsavachidou, Jonathan D. Licht, Mathew L. Coleman, Galene Athanasiadis, Michael F. Olson, Barbara L. WeberAbstract:BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.
Barbara L. Weber - One of the best experts on this subject based on the ideXlab platform.
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SPRY2 is an inhibitor of the ras/extracellular signal-regulated kinase pathway in melanocytes and melanoma cells with wild-type BRAF but not with the V599E mutant.
Cancer research, 2004Co-Authors: Dimitra Tsavachidou, Jonathan D. Licht, Mathew L. Coleman, Galene Athanasiadis, Michael F. Olson, Barbara L. WeberAbstract:BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.
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SPRY2 is an inhibitor of the ras extracellular signal regulated kinase pathway in melanocytes and melanoma cells with wild type braf but not with the v599e mutant
Cancer Research, 2004Co-Authors: Dimitra Tsavachidou, Jonathan D. Licht, Mathew L. Coleman, Galene Athanasiadis, Michael F. Olson, Barbara L. WeberAbstract:BRAF mutations result in constitutively active BRAF kinase activity and increased extracellular signal-regulated kinase (ERK) signaling and cell proliferation. Initial studies have shown that BRAF mutations occur at a high frequency in melanocytic nevi and metastatic lesions, but recent data have revealed much lower incidence of these mutations in early-stage melanoma, implying that other factors may contribute to melanoma pathogenesis in a wild-type (WT) BRAF context. To identify such contributing factors, we used microarray gene expression profiling to screen for differences in gene expression between a panel of melanocytic and melanoma cell lines with WT BRAF and a group of melanoma cell lines with the V599E BRAF mutation. We found that SPRY2, an inhibitor homologous to SPRY4, which was previously shown to suppress Ras/ERK signaling via direct binding to Raf-1, had reduced expression in WT BRAF cells. Using small interfering RNA-mediated SPRY2 knockdown, we showed that SPRY2 acts as an inhibitor of ERK signaling in melanocytes and WT BRAF melanoma cells, but not in cell lines with the V599E mutation. We also show that SPRY2 and SPRY4 directly bind WT BRAF but not the V599E and other exon 15 BRAF mutants. These data suggest that SPRY2, an inhibitor of ERK signaling, may be bypassed in melanoma cells either by down-regulation of its expression in WT BRAF cells, or by the presence of the BRAF mutation.
Graeme R. Guy - One of the best experts on this subject based on the ideXlab platform.
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Intersectin 1 Enhances Cbl Ubiquitylation of Epidermal Growth Factor Receptor through Regulation of Sprouty2-Cbl Interaction
Molecular and cellular biology, 2011Co-Authors: Mustafa N. Okur, Natalia Martínez, José M. Rojas, Graeme R. Guy, Chee Wai Fong, Jolene Ooi, Carlota A. García-domínguez, John P. O'bryanAbstract:Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463–1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (SPRY2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and SPRY2 and that interaction of ITSN1 with SPRY2 disrupts SPRY2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to SPRY2 through point mutation of the Pro-rich ITSN1 binding site in SPRY2 results in enhanced Cbl-SPRY2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with SPRY2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and SPRY2.
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Sprouty2 Interacts with Protein Kinase Cδ and Disrupts Phosphorylation of Protein Kinase D1
The Journal of biological chemistry, 2009Co-Authors: Soah Yee Chow, Graeme R. GuyAbstract:The Sprouty (Spry) proteins act as inhibitors of the Ras/ERK pathway downstream of receptor tyrosine kinases. In this study, we report a novel interaction between protein kinase C δ (PKCδ) and SPRY2. Endogenous PKCδ and SPRY2 interact in cells upon basic fibroblast growth factor stimulation, indicating a physiological relevance for the interaction. This interaction appeared to require the full-length SPRY2 protein and was conformation-dependent. Conformational constraints were released upon FGFR1 activation, allowing the interaction to occur. Although this interaction did not affect the phosphorylation of PKCδ by another kinase, it reduced the phosphorylation of a PKCδ substrate, protein kinase D1 (PKD1). SPRY2 was found to interact more strongly with PKCδ with increasing amounts of PKD1, which indicated that instead of competing with PKD1 for binding with PKCδ, it was more likely to form a trimeric complex with both PKCδ and PKD1. Formation of the complex was found to be dependent on an existing PKCδ-PKD1 interaction. By disrupting the interaction between PKCδ and PKD1, SPRY2 was unable to associate with PKCδ to form the trimeric complex. As a consequence of this trimeric complex, the existing interaction between PKCδ and PKD1 was increased, and the transfer of phosphate groups from PKCδ to PKD1 was at least partly blocked by SPRY2. The action of SPRY2 on PKCδ resulted in the inhibition of both ERK phosphorylation and invasion of PC-3 cells via PKCδ signaling. By disrupting the capacity of PKCδ to phosphorylate its cognate substrates, SPRY2 may serve to modulate PKCδ signaling downstream of receptor tyrosine kinases and to regulate the physiological outcome.
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Tesk1 Interacts with SPRY2 to Abrogate Its Inhibition of ERK Phosphorylation Downstream of Receptor Tyrosine Kinase Signaling
The Journal of biological chemistry, 2007Co-Authors: Sumana Chandramouli, Permeen Yusoff, Dieu-hung Lao, Hwei Fen Leong, Kensaku Mizuno, Graeme R. GuyAbstract:The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of SPRY2 function. Endogenous Tesk1 and SPRY2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes SPRY2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of SPRY2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by SPRY2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of SPRY2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to SPRY2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of SPRY2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of SPRY2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.
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Direct binding of PP2A to Sprouty2 and phosphorylation changes are a prerequisite for ERK inhibition downstream of fibroblast growth factor receptor stimulation.
The Journal of biological chemistry, 2007Co-Authors: Dieu-hung Lao, Sumana Chandramouli, Permeen Yusoff, Robin J. Philp, Chee Wai Fong, Rebecca A. Jackson, Tzuen Yih Saw, Graeme R. GuyAbstract:Abstract In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (SPRY2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of SPRY2 is revealed upon stimulation. The activation of SPRY2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to SPRY2 around residues 50-60. We show that human SPRY2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to SPRY2, but not to SPRY2Δ50-60 (Δ50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on SPRY2. We show that there are at least two distinct pools of SPRY2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets SPRY2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for SPRY2 to act as a Ras/ERK pathway inhibitor in FGF signaling.
Owen J. Sansom - One of the best experts on this subject based on the ideXlab platform.
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SPRY2 loss enhances erbb trafficking and pi3k akt signalling to drive human and mouse prostate carcinogenesis
Embo Molecular Medicine, 2012Co-Authors: Meiling Gao, Rachana Patel, Imran Ahmad, Janis Fleming, Joanne Edwards, Stuart Mccracken, Kanagasabai Sahadevan, Morag Seywright, Jim C. Norman, Owen J. SansomAbstract:Loss of SPRY2 and activation of receptor tyrosine kinases are common events in prostate cancer (PC). However, the molecular basis of their interaction and clinical impact remains to be fully examined. SPRY2 loss may functionally synergize with aberrant cellular signalling to drive PC and to promote treatment-resistant disease. Here, we report evidence for a positive feedback regulation of the ErbB-PI3K/AKT cascade by SPRY2 loss in in vitro as well as pre-clinical in vivo models and clinical PC. Reduction in SPRY2 expression resulted in hyper-activation of PI3K/AKT signalling to drive proliferation and invasion by enhanced internalization of EGFR/HER2 and their sustained signalling at the early endosome in a PTEN-dependent manner. This involved p38 MAPK activation by PI3K to facilitate clathrin-mediated ErbB receptor endocytosis. Finally, in vitro and in vivo inhibition of PI3K suppressed proliferation and invasion, supporting PI3K/AKT as a target for therapy particularly in patients with PTEN-haploinsufficient-, low SPRY2- and ErbB-expressing tumours. In conclusion, SPRY2 is an important tumour suppressor in PC since its loss drives the PI3K/AKT pathway via functional interaction with the ErbB system.
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SPRY2 loss enhances ErbB trafficking and PI3K/AKT signalling to drive human and mouse prostate carcinogenesis
EMBO molecular medicine, 2012Co-Authors: Meiling Gao, Rachana Patel, Imran Ahmad, Janis Fleming, Joanne Edwards, Stuart Mccracken, Kanagasabai Sahadevan, Morag Seywright, Jim C. Norman, Owen J. SansomAbstract:Loss of SPRY2 and activation of receptor tyrosine kinases are common events in prostate cancer (PC). However, the molecular basis of their interaction and clinical impact remains to be fully examined. SPRY2 loss may functionally synergize with aberrant cellular signalling to drive PC and to promote treatment-resistant disease. Here, we report evidence for a positive feedback regulation of the ErbB-PI3K/AKT cascade by SPRY2 loss in in vitro as well as pre-clinical in vivo models and clinical PC. Reduction in SPRY2 expression resulted in hyper-activation of PI3K/AKT signalling to drive proliferation and invasion by enhanced internalization of EGFR/HER2 and their sustained signalling at the early endosome in a PTEN-dependent manner. This involved p38 MAPK activation by PI3K to facilitate clathrin-mediated ErbB receptor endocytosis. Finally, in vitro and in vivo inhibition of PI3K suppressed proliferation and invasion, supporting PI3K/AKT as a target for therapy particularly in patients with PTEN-haploinsufficient-, low SPRY2- and ErbB-expressing tumours. In conclusion, SPRY2 is an important tumour suppressor in PC since its loss drives the PI3K/AKT pathway via functional interaction with the ErbB system.
Tarun B. Patel - One of the best experts on this subject based on the ideXlab platform.
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Hypoxia inducible factors regulate the transcription of the sprouty2 gene and expression of the sprouty2 protein.
PloS one, 2017Co-Authors: Xianlong Gao, Kristin C. Hicks, Paul Neumann, Tarun B. PatelAbstract:Receptor Tyrosine Kinase (RTK) signaling plays a major role in tumorigenesis and normal development. Sprouty2 (SPRY2) attenuates RTK signaling and inhibits processes such as angiogenesis, cell proliferation, migration and survival, which are all upregulated in tumors. Indeed in cancers of the liver, lung, prostate and breast, SPRY2 protein levels are markedly decreased correlating with poor patient prognosis and shorter survival. Thus, it is important to understand how expression of SPRY2 is regulated. While prior studies have focused on the post-translation regulation of SPRY2, very few studies have focused on the transcriptional regulation of SPRY2 gene. Here, we demonstrate that in the human hepatoma cell line, Hep3B, the transcription of SPRY2 is inhibited by the transcription regulating hypoxia inducible factors (HIFs). HIFs are composed of an oxygen regulated alpha subunit (HIF1α or HIF2α) and a beta subunit (HIF1β). Intriguingly, silencing of HIF1α and HIF2α elevates SPRY2 mRNA and protein levels suggesting HIFs reduce the transcription of the SPRY2 promoter. In silico analysis identified ten hypoxia response elements (HREs) in the proximal promoter and first intron of SPRY2. Using chromatin immunoprecipitation (ChIP), we show that HIF1α/2α bind near the putative HREs in the proximal promoter and intron of SPRY2. Our studies demonstrated that not only is the SPRY2 promoter methylated, but silencing HIF1α/2α reduced the methylation. ChIP assays also showed DNA methyltransferase1 (DNMT1) binding to the proximal promoter and first intron of SPRY2 and silencing HIF1α/2α decreased this association. Additionally, silencing of DNMT1 mimicked the HIF1α/2α silencing-mediated increase in SPRY2 mRNA and protein. While simultaneous silencing of HIF1α/2α and DNMT1 increased SPRY2 mRNA a little more, the increase was not additive suggesting a common mechanism by which DNMT1 and HIF1α/2α regulate SPRY2 transcription. Together these data suggest that the transcription of SPRY2 is inhibited by HIFs, in part, via DNMT1- mediated methylation.
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Sprouty2 Protein Regulates Hypoxia-inducible Factor-α (HIFα) Protein Levels and Transcription of HIFα-responsive Genes
The Journal of biological chemistry, 2016Co-Authors: Kristin C. Hicks, Tarun B. PatelAbstract:The α-subunits of hypoxia-inducible factors (HIF1α and HIF2α) promote transcription of genes that regulate glycolysis and cell survival and growth. Sprouty2 (SPRY2) is a modulator of receptor tyrosine kinase signaling and inhibits cell proliferation by a number of different mechanisms. Because of the seemingly opposite actions of HIFα subunits and SPRY2 on cellular processes, we investigated whether SPRY2 regulates the levels of HIF1α and HIF2α proteins. In cell lines from different types of tumors in which the decreased protein levels of SPRY2 have been associated with poor prognosis, silencing of SPRY2 elevated HIF1α protein levels. Increases in HIF1α and HIF2α protein levels due to silencing of SPRY2 also up-regulated HIFα target genes. Using HIF1α as a prototype, we show that SPRY2 decreases HIF1α stability and enhances the ubiquitylation of HIF1α by a von Hippel-Lindau protein (pVHL)-dependent mechanism. SPRY2 also exists in a complex with HIF1α. Because SPRY2 can also associate with pVHL, using a mutant form of SPRY2 (3P/3A-SPRY2) that binds HIF1α, but not pVHL, we show that WT-SPRY2, but not the 3P/3A-SPRY2 decreases HIF1α protein levels. In accordance, expression of WT-SPRY2, but not 3P/3A-SPRY2 results in a decrease in HIF1α-sensitive glucose uptake. Together our data suggest that SPRY2 acts as a scaffold to bring more pVHL/associated E3 ligase in proximity of HIF1α and increase its ubiquitylation and degradation. This represents a novel action for SPRY2 in modulating biological processes regulated by HIFα subunits.
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Regulation of cellular levels of Sprouty2 protein by prolyl hydroxylase domain and von Hippel-Lindau proteins.
The Journal of biological chemistry, 2011Co-Authors: Kimberly Anderson, Kyle A. Nordquist, Xianlong Gao, Kristin C. Hicks, Bo Zhai, Steven P. Gygi, Tarun B. PatelAbstract:Sprouty (Spry) proteins modulate the actions of receptor tyrosine kinases during development and tumorigenesis. Decreases in cellular levels of Spry, especially Sprouty2 (SPRY2), have been implicated in the growth and progression of tumors of the breast, prostate, lung, and liver. During development and tumor growth, cells experience hypoxia. Therefore, we investigated how hypoxia modulates the levels of Spry proteins. Hypoxia elevated the levels of all four expressed Spry isoforms in HeLa cells. Amounts of endogenous SPRY2 in LS147T and HEP3B cells were also elevated by hypoxia. Using SPRY2 as a prototype, we demonstrate that silencing and expression of prolyl hydroxylase domain proteins (PHD1-3) increase and decrease, respectively, the cellular content of SPRY2. SPRY2 also preferentially interacted with PHD1-3 and von Hippel-Lindau protein (pVHL) during normoxia but not in hypoxia. Additionally, SPRY2 is hydroxylated on Pro residues 18, 144, and 160, and substitution of these residues with Ala enhanced stability of SPRY2 and abrogated its interactions with pVHL. Silencing of pVHL increased levels of SPRY2 by decreasing its ubiquitylation and degradation and thereby augmented the ability of SPRY2 to inhibit FGF-elicited activation of ERK1/2. Thus, prolyl hydroxylase mediated hydroxylation and subsequent pVHL-elicited ubiquitylation of SPRY2 target it for degradation and, consequently, provide a novel mechanism of regulating growth factor signaling.
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HECT Domain-containing E3 Ubiquitin Ligase Nedd4 Interacts with and Ubiquitinates Sprouty2
The Journal of biological chemistry, 2009Co-Authors: Francis Edwin, Kimberly Anderson, Tarun B. PatelAbstract:Abstract Sprouty (Spry) proteins are important regulators of receptor tyrosine kinase signaling in development and disease. Alterations in cellular Spry content have been associated with certain forms of cancers and also in cardiovascular diseases. Thus, understanding the mechanisms that regulate cellular Spry levels are important. Herein, we demonstrate that Spry1 and SPRY2, but not Spry3 or Spry4, associate with the HECT domain family E3 ubiquitin ligase, Nedd4. The SPRY2/Nedd4 association involves the WW domains of Nedd4 and requires phosphorylation of the Mnk2 kinase sites, Ser112 and Ser121, on SPRY2. The phospho-Ser112/121 region on SPRY2 that binds WW domains of Nedd4 is a novel non-canonical WW domain binding region that does not contain Pro residues after phospho-Ser. Endogenous and overexpressed Nedd4 polyubiquitinate SPRY2 via Lys48 on ubiquitin and decrease its stability. Silencing of endogenous Nedd4 increased the cellular SPRY2 content and attenuated fibroblast growth factor-elicited ERK1/2 activation that was reversed when elevations in SPRY2 levels were prevented by SPRY2-specific small interfering RNA. Mnk2 silencing decreased SPRY2-Nedd4 interactions and also augmented the ability of SPRY2 to inhibit fibroblast growth factor signaling. This is the first report demonstrating the regulation of cellular Spry content and its ability to modulate receptor tyrosine kinase signaling by a HECT domain-containing E3 ubiquitin ligase.
