Synthesizing Signaling Pathways from Temporal Phosphoproteomic Data
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Epithelial Cell, Cell Culture
DISEASE(S): Disease Free
SUBMITTER: 
Anthony Gitter
LAB HEAD: Anthony Gitter
PROVIDER: PXD006697 | Pride | 2018-09-18
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 86 |
Similar Datasets
Project description:Molecular switches are essential modules in signaling networks by enabling a quick response to environmental changes. Here, we describe a role for small ubiquitin-like-modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compared the endogenous SUMO-proteomes of Hela cells before and after EGF-stimulation. Thereby, we identified a small group of transcriptional repressors, including IRF2BP1, IRF2BP2 and IRF2BPL as novel players in EGFR signaling, which lost SUMO transiently within 15 min of EGF treatment. We found 38 genes whose expression within the first hour of EGF treatment was altered in cells expressing SUMOylation deficient IRF2BP1, including Dual specific phosphatase 1 (DUSP1), an important feedback regulator of EGFR signalling. We show that IRF2BP1 binds to the proximal promoter of DUSP1, whose activation requires IRF2BP1 deSUMOylation. Thus, the SUMO-dependent molecular switch of IRF2BP1 activity is important to finely tune EGFR signaling
2021-04-13 | PXD018049 | Pride
Project description:Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.
2023-03-11 | PXD031988 | Pride
Project description:The Epidermal Growth Factor Receptor (EGFR) has been studied extensively due to its critical role in cellular signaling and association with disease. Previous models have elucidated interactions between EGFR and downstream adaptor proteins, or showed phenotypes affected by EGFR. However, the link between EGFR phosphorylation and phenotypic outcome is still poorly understood. Here, we employed a suite of isogenic cells lines expressing site-specific mutations at each of the EGFR C-terminal phosphorylation sites to interrogate their role in signaling network and cell biological response to stimulation. Our results demonstrate the resilience of the EGFR network, which was largely similar even in the context of multiple Y-to-F mutations in the EGFR C-terminal tail, while also revealing nodes in the network that have not previously been linked to EGFR signaling. Our data-driven model highlights signaling network nodes associated with distinct EGF-driven cell responses, including migration, proliferation, and receptor trafficking. Application of this same approach to less studied RTKs should provide a plethora of novel associations that should lead to a much better understanding of these signaling networks.
2023-05-09 | PXD032403 | Pride
Project description:HELA cells derived from human cervical tumor were subjected to EGF stimulation for 0,20,40,60,120,240 and 480 minutes. We used microarrays to understand the temporal regulation of the cellular EGFR cascade. Experiment Overall Design: HELA cells were grown in DMEM supplemented with 10% FCS and antibiotics. Cells were serum deprived for 24 hours. Following stimulation with 10 ng/ml EGF for 0,20,40,60,120,240 and 480 minutes.
2007-11-02 | E-GEOD-6783 | biostudies-arrayexpress
Project description:Lung cancer is the leading cause of cancer death. Mutations in the kinase domain of EGFR, a predominant driver oncogene, such as L858R missense mutation and a series of deletions spanning the conserved sequence 747LREA750, are associated with sensitivity to tyrosine kinase inhibitors (TKIs). However, patients receiving EGFR-TKIs (gefitinib and erlotinib) develop drug-resistance due to a secondary mutation at the gatekeeper residue (T790M) in about 50-60% of cases, urging for new drug development. Afatinib, a FDA approved second-generation EGFR-TKI that was developed to circumvent T790M-mediated resistance, has not been very effective in clinical trials. In this study, we performed a global phosphoproteomic screen to identify targets that undergo mutant EGFR-dependent tyrosine phosphorylation and their modulation by erlotinib or afatinib. We undertook stable isotope labeling of amino acids in cell culture (SILAC), phosphopeptide enrichment, and quantitative mass spectrometry to identify dynamic changes of phosphorylation downstream of mutant EGFRs in lung adenocarcinoma cells harboring L858R or L858R/T790M mutations and their modulation by erlotinib and afatinib inhibition. We identified and quantified 397, 429, 223, and 594 phosphotyrosine sites in H3255, 11-18,PC9, and H1975 cell lines that were grown in presence of FBS and in presence/absence of TKIs, respectively. These account for a total of 907 unique phospho-tyrosine sites in 496 proteins. Among them, 187 phosphotyrosine sites were found to be in 89 kinases, which may serve as intermediary regulatory kinases in EGFR signalling pathway. Further analysis indicated that in TKI-sensitive H3255 and 11-18 cells, there were 58/111 and 65/101 tyrosine sites that were hypophosphorylated in presence of erlotinib and afatinib, respectively. However, in TKI-resistant H1975 cells, 189 and 264 tyrosine sites were hypophosphorylated in presence of erlotinib and afatinib respectively, indicating that the afatinib-specific additional sites could be validated for identifying potentially new drug targets to counter TKI-resistance. Ingenuity pathway analysis (IPA) of proteins with altered phosphorylation sites demonstrated that several canonical pathways including ephrin receptor signalling and integrin signalling pathways were enriched, which may play important roles in cell growth and proliferation. However, upon EGF stimulation of serum starved H3255 cells in presence or absence of TKIs, 99 tyrosine sites that were hyperphosphorylated upon EGF stimulation were inhibited in presence of erlotinib or afatinib. But in H1975 cells treated with erlotinib, 48 of the above sites were either unchanged or were hyperphosphorylated. These sites include EGFR (Y1197/869/998), JAK1 (Y1034), FRK (Y497), GAB1 (Y657/689), MAPK1 (Y187), MAPK3 (Y204), MET (Y1252/1253). Furthermore, a total of 112 sites that were observed to be hypophosphorylated upon EGF stimulation in H1975 cells, were found to be hyperphosphorylated upon erlotinib inhibition. This could possibly be due to the activation of downstream phosphatases with EGF stimulation. We are now performing in-depth bioinformatic analysis and validation experiments using functional genomics to understand the role of targets of mutant EGFR signalling in lung cancer.
2017-03-29 | PXD004373 | Pride
Project description:Cylindromatosis tumor suppressor protein (CYLD) is deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, the deficiency of CYLD impairs the interaction of EGFR with Cbl-b, which leads to a reduced ubiquitination of the receptor followed by its decreased degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors pressor for the negative regulation of a tumorigenic signaling pathway.
2017-06-05 | PXD006390 | Pride
Project description:Expression profile of human Flp-In-293-WT-FBXO25 or Flp-In-293-∆F-FBXO25 cells comparing non treated cells vs. tetraciclyne treated for 24 and 48 hours. Flp-In-293-∆F-FBXO25 cells when induced express the non functional protein (without F-box domain). The objective of the study was to identify genes up or down-regulated when Fbxo25 wild-type or Fbxo25 lacking F-box domain is superexpressed.
2012-05-15 | GSE37380 | GEO
Project description:Aberrant activation of Hedgehog (HH) signaling has been identified as a key etiologic factor of many human malignancies. Signal strength, target gene specificity, and oncogenic activity of HH signaling profoundly depend on interactions with other pathways such as epidermal growth factor receptor-mediated signaling which has been shown to cooperate with HH/GLI in basal cell carcinoma and pancreatic cancer. We demonstrate that the human medulloblastoma cell line Daoy possesses a fully inducible endogenous HH pathway. Treatment of Daoy cells with Sonic Hedgehog or Smoothened agonist induced expression of GLI1 protein and prevented processing of GLI3 to its repressor form. To study interactions between HH- and EGF-induced signaling in greater detail, time-resolved measurements were carried out and analyzed on the transcriptomic as well as proteomic level. Daoy cells responded to the co-treatment by downregulating GLI1, PTCH, and HHIP on the transcript level which was also seen when Amphiregulin (AREG) was used instead of EGF. The finding that EGFR signaling silences proteins acting as negative regulators of HH signaling is firstly described here as a novel crosstalk mechanism. Furthermore, combined EGFR/HH signaling maintains high GLI1 protein levels contrasting its downregulation on the transcript level. On the other hand, high level synergism was observed with respect to a strong and significant upregulation of numerous canonical EGF-targets with putative tumor-promoting properties such as MMP7, VEGFA, and IL-8. In conclusion, synergistic effects between EGFR and HH signaling can selectively induce a switch from a canonical HH/GLI profile to a modulated specific target gene profile pointing to more wide-spread, yet context-dependent, interactions between HH/GLI and growth factor receptor signaling in human malignancies. To study interactions between HH- and EGF-induced signaling, time-resolved measurements were carried out over a period of 24 h at 14 different time points after stimulation by EGF with and without additional stimulation by SHH. Furthermore, as a control, cells without any stimulation by EGF and SHH (control) and cells in the presents of SHH were analyzed. Overall, three biological replicates of 60 different treatment/timepoints were analyzed yielding 180 different samples.
2014-07-31 | E-GEOD-46045 | biostudies-arrayexpress
Project description:Growth factor receptor-bound protein 2 or Grb2 is an adaptor protein involved in signal transduction. It is widely expressed and integral to several cellular functions, including cell proliferation. We designed this study to capture interacting partners of Grb2 in absence and presence of epidermal growth factor (EGF). HEK 293 cells, expressing tagged Grb2, were cultured with SILAC labels in presence and absence of EGF. Grb2 interactome was enriched by immunoprecipitation and subjected to mass spectrometry. This data will help in understanding the regulatory effect of EGF stimulation on Grb2 protein and its interactors.
2019-03-05 | PXD012957 | Pride
Project description:MCF10A cells derived from spontaneously immortalized normal human mammary epithelia were subjected to EGF/SERUM stimulation for 0,20,40,60,120,240 and 480 minutes. We used microarrays to understand the temporal regulation of the cellular EGFR cascade. Experiment Overall Design: MCF10A cells were grown in DME:F12 medium supplemented with antibiotics, [10 mg/ml insulin, 0.1 mg/ml cholera toxin, 0.5 mg/ml hydrocortisone, heat-inactivated horse serum (5%); defined as 'serum' in the text] and 10ng/ml EGF. Cells were serum deprived for 24 hours. Following stimulation with EGF/SERUM for 0,20,40,60,120,240 and 480 minutes.
2007-11-01 | E-GEOD-6784 | biostudies-arrayexpress