EGF-induced FoxO3a nuclear-cytoplasmic pulsing dynamics in mammary epithelial cell lines following treatment with increasing doses of a MEK inhibitor. Dataset 1 of 3: single-cell reporter measurements.
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ABSTRACT:
SUBMITTER: Somponnat Sampattavanich, Bernhard Steiert
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.
Project description:Phosphoproteomic analysis of EGF stimulated MCF7 cells. This dataset is the basis for the development of modeling of signaling networks. A fundamental challenge in biology is to delineate the signaling pathways that govern cellular responses to genetic and environmental cues. Phosphoproteomics is an emerging technology that provides key data on activity levels of proteins under conditions of interest. However, the interpretation of these data is hampered by the lack of methods that can translate site-specific information into global maps of active proteins and signaling networks. To meet this challenge, we propose PHOTON, a method for integrating phosphorylation data with protein-protein interaction networks to identify active proteins and pathways and pinpoint functional phosphosites. We demonstrate the utility of PHOTON by applying it to interpret existing and novel phosphoproteomic datasets related to EGF and insulin responses. PHOTON substantially outperforms the widely-used cutoff approach, providing highly reproducible predictions that are more in line with current biological knowledge
Project description:In the present work we propose a new therapy for NRAS mutant melanoma. Simultaneous inhibition of MEK and ROCK caused induction of BimEL , PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.
Project description:To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response. Keywords: time-course analysis Synchronized human mammary epithelial cells were stimulated into mitosis using epidermal growth factor and samples were harvested at 1, 4, 8, 13, 18 and 24hrs for parallel analysis by microarray, global proteomics and western blot analysis.
Project description:Activation of the epidermal growth factor receptor (EGFR) during tumor development can trigger the MEK signaling pathway. In the present study, we investigated the MEK signaling pathway in non‑small cell lung cancer (NSCLC) cells with respect to the effect of epidermal growth factor (EGF) on expression of Ret finger protein like 3 (RFPL3) and human telomerase reverse transcriptase (hTERT), and the effect of RFPL3 overexpression on other MEK signaling proteins. In vitro, A549 and H1299 cells were treated with different concentrations of EGF for 24 h or 48 h. Expression of RFPL3 and hTERT at the mRNA and protein levels was determined by real‑time quantitative PCR (RT‑qPCR) and western blot analysis; cell viability was detected by MTT assay, and apoptosis was assayed via flow cytometry. We also pretreated A549 and H1299 cells with EGFR tyrosine kinase inhibitors, AG1478 and erlotinib, and MEK‑specific inhibitor (PD98059) in the presence of EGF. We used western blot analysis to assess the expression levels of RFPL3, hTERT and related MEK‑pathway proteins in A549 and H1299 cells transfected with RFPL3‑overexpression plasmids. EGF significantly upregulated RFPL3 and hTERT protein levels in the NSCLC cells. RFPL3 and hTERT proteins upregulation by EGF were attenuated by pretreatment with AG1478 and erlotinib. EGF promoted proliferation and inhibited apoptosis; PD98059 decreased RFPL3 and hTERT protein expression; and RFPL3 overexpression increased the expression of hTERT and related MEK‑pathway proteins. EGF upregulated RFPL3 and hTERT protein expression in NSCLC cells via the MEK pathway, promoted cell proliferation and inhibited apoptosis. RFPL3 overexpression increased expression of hTERT and related MEK signaling proteins (Ras, Raf, ERK and p‑ERK), which implies that RFPL3 is a potential therapeutic target for NSCLC.