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IGF-1 stimulated C2 skeletal myotube phosphoproteome

ABSTRACT: Skeletal muscle is known to adapt dynamically to changes in workload by regulatory processes of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. We performed a global quantitative phosphoproteomics analysis of contracting mouse C2 myotubes treated with insulin growth factor 1 (IGF-1) as control and additionally MK-2206 or LY294002 to inhibit PI3K/Akt signaling, respectively.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture, Myotube

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD016721 | Pride | 2020-05-25

REPOSITORIES: Pride

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			Action	DRS
	MaxQuant_result_files.zip	Other
	QEplus024398.raw	Raw
	QEplus024400.raw	Raw
	QEplus024402.raw	Raw
	QEplus024404.raw	Raw

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Publications

Phosphoproteomics identifies dual-site phosphorylation in an extended basophilic motif regulating FILIP1-mediated degradation of filamin-C.

Reimann Lena L Schwäble Anja N AN Fricke Anna L AL Mühlhäuser Wignand W D WWD Leber Yvonne Y Lohanadan Keerthika K Puchinger Martin G MG Schäuble Sascha S Faessler Erik E Wiese Heike H Reichenbach Christa C Knapp Bettina B Peikert Christian D CD Drepper Friedel F Hahn Udo U Kreutz Clemens C van der Ven Peter F M PFM Radziwill Gerald G Djinović-Carugo Kristina K Fürst Dieter O DO Warscheid Bettina B

Communications biology 20200522 1

The PI3K/Akt pathway promotes skeletal muscle growth and myogenic differentiation. Although its importance in skeletal muscle biology is well documented, many of its substrates remain to be identified. We here studied PI3K/Akt signaling in contracting skeletal muscle cells by quantitative phosphoproteomics. We identified the extended basophilic phosphosite motif RxRxxp[S/T]xxp[S/T] in various proteins including filamin-C (FLNc). Importantly, this extended motif, located in a unique insert in Ig- ...[more]

PMID: 32444788

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C2 skeletal myotube phosphoproteome

Project description:Skeletal muscle is known to adapt dynamically to changes in workload by regulatory processes of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. We performed a global quantitative phosphoproteomics analysis of contracting mouse C2 myotubes treated with insulin growth factor 1 (IGF-1) or LY294002 to activate or inhibit PI3K/Akt signaling, respectively.

2020-05-25 | PXD009117 | Pride

Identification of RNA binding proteins that interact with AU-rich elements of Lif 3`UTR

Project description:Leukemia inhibitory factor (LIF) is an important endogenous factor for photoreceptor protection. Lif is known to be upregulated both in induced and inherited disease models of photoreceptor degeneration only in a subset of Muller cells. Since Lif is induced in Muller cells in response to photoreceptor injury, we studied its regulation in Muller cells. We have recently identified several AU-rich elements (AREs) within the 3′ untranslated region (UTR) of Lif . We also showed that as a result of these elements Lif encodes a highly unstable mRNA. mRNAs containing AREs in the 3`UTR undergo rapid ARE-mediated mRNA decay in the cytoplasm which is mediated by the RNA–binding proteins. In order to identify the RNA-binding proteins that are involved in the regulation of Lif transcripts, we will use the biotin-labelled RNA. By using this methodology, we will isolate the related RNA binding proteins. We plan to analyze the isolated proteins by mass spectrometry, and compare the mass spectrometry results of different regions with or without AREs . Once we identify the specific proteins that bind only to AREs, we will do the functional characterization of these proteins by cotransfecting the constructs with AREs and the siRNA that inhibits the expression of related RNA binding protein.

2016-07-20 | PXD001463 | Pride

CRISPR/Cas9-mediated knockout and endogenous complex analysis of Cluap1/ IFT38

Project description:CRISPR/Cas9-mediated gene-editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interaction, and in contrast to an exogenous expression of a protein, these can be studied maintaining physiological stochiometries, topology and context. We have used CRISPR/Cas9-mediated recombination to Cluap1/ IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis which was then correlated to the corresponding interactome determined by ectopic expression.

2018-04-04 | PXD008343 | Pride

Quantitative phosphoproteome of contracting C2 myotubes with pharmacological intervention of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK cascades

Project description:Various protein kinases are regulating the intracellular signaling network of skeletal muscle cells. Despite that many of the involved kinases are known, their downstream targets have remained largely unexplored. To deepen our understanding of the PI3K-AKT-mTOR-S6K and the RAF-MEK-ERK-RSK signaling network in myotubes, we globally analyzed changes in protein phosphorylation levels upon kinase inhibition within these pathways. The phosphoproteomics data were used to define potential targets of the kinases AKT, RSK and S6K, which share the substrate recognition motif RxRxxp[ST].

2023-02-22 | PXD018667 | Pride

Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane treated human breast epithelial cells

Project description:Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study transcriptomic and proteomic changes in the estrogen receptor negative, non tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R=0.679, P<0.05) and KEAP1 knockdown (R=0.853, P<0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and proteomic level. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast. MCF10A cells were treated with SFN or had KEAP1 knocked down by siRNA.

2011-04-22 | E-GEOD-28813 | biostudies-arrayexpress

C-terminal FLNc in vivo kinase assay

Project description:The Z-disc is a protein-rich structure critically important for myofibril development and integrity. Since a role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape warranted in-depth analysis. We therefore established a site-resolved protein phosphorylation map of the Z-disc in skeletal myocytes and found that it is a phosphorylation hotspot in living cells, underscoring its functions in signalling and disease-related processes. In an exemplary fashion, we analysed the actin-binding multi-adaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKC phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. With this quantitative in vivo kinase assay, we show that the phosphorylation site S2625 in mouse FLNc is significantly down-regulated upon treatment of C2C12 myotubes with the PKCα inhibitor Gö6976.

2017-01-02 | PXD004151 | Pride

MicroRNA profiling by array of mouse skeletal muscle cells treated with TNF-alpha or IGF-I

Project description:Murine skeletal muscle cells pmi28 were cultured in growth medium (HamM-^Rs F10, supplemented with 20 % FCS, 2 mM L-glutamine, and 1 % Penicillin / Streptomycin). Myoblasts were cultured on laminin-1 coated dishes for an additional 24 h before switching a fraction of dishes to differentiation medium (DMEM medium containing 2 % horse serum, 2 mM L glutamine, and 1 % Penicillin / Streptomycin) and to differentiation medium containing either 2 x 103 U/ml mouse recombinant TNF-? (Roche Applied Science), or 5 M-5g/ml mouse recombinant IGF-I (Sigma-Aldrich), or carrier only. pmi28 cells (TNF-? and IGF-I treatments as well as controls) were harvested 24 h after the induction of differentiation. The experiments were performed in quadruplicates or triplicates, respectively.

2013-05-15 | E-MTAB-1114 | biostudies-arrayexpress

mTORC2 regulates amino acid metabolism in cancer by phosphorylation of the cystine-glutamate antiporter xCT

Project description:Mutations in cancer reprogram amino acid metabolism to drive tumor growth, but the molecular mechanisms are not well understood. Using an unbiased proteomic screen, we identified mTORC2 as a critical regulator of amino acid metabolism in cancer via phosphorylation of the cystine-glutamate antiporter xCT. mTORC2 phosphorylates serine 26 at the cytosolic N-terminus of xCT, inhibiting its activity. Genetic inhibition of mTORC2, or pharmacologic mTOR kinase inhibition, promotes glutamate secretion, cystine uptake and incorporation into glutathione linking growth factor receptor signaling with amino acid uptake and utilization. These results identify an unanticipated mechanism of amino acid metabolic reprogramming in cancer, enabling tumor cells to adapt to changing environmental conditions.

2017-06-28 | PXD006461 | Pride

Dynamic profiling of the protein life cycle in response to pathogens (protein)

Project description:Protein expression is regulated by production and degradation of mRNAs and proteins, but their specific relationships remain unknown. We combine measurements of protein production and degradation and mRNA dynamics to build a quantitative genomic model of the differential regulation of gene expression in LPS stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for over half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction of novel cellular functions and remodeling of preexisting functions through the protein life cycle. Mouse primary dendritic cells were treated with LPS or mock stimulus and profiled over a 12-hour time course. Cells were grown in M-labeled SILAC media, which was replaced with H-labeled SILAC media at time 0. Aliquots were taken at 0, 0.5, 1, 2, 3, 4, 5, 6, 9, and 12 hours post-stimulation and added to equal volumes of a master mix of unlabeled (L) cells for the purpose of normalization. RNA-Seq was performed at 0, 1, 2, 4, 6, 9, and 12 hours post-stimulation.

2015-03-06 | E-GEOD-59792 | biostudies-arrayexpress

ER/PR RIME Experiments in MCF7 and T47D cell lines

Project description:Immuno-precipitation followed by MS was performed using the RIME protocol in this study. Estrogen Receptor (ER) and Progestorone Receptor (PR) were targetted using antibodies. The experiments were performed in a quantitative manner using SILAC labelling. Cells grown in complete serum (estrogenic) conditions were compared against an cells in similar complete media, however supplemented with Progesterone (PG) or R5020 for 4 hours. Experiments were performed in MCF7 and T47D cell lines

2015-05-26 | PXD002104 | Pride