1. SILAC based secretome analysis of WT and tKO cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive Plus
ORGANISM(S): Mus Musculus (mouse)
TISSUE(S): Skin, Fibroblast
SUBMITTER: 
Prerana Wagle
LAB HEAD: Thomas Krieg
PROVIDER: PXD044683 | Pride | 2023-08-21
REPOSITORIES: Pride
Similar Datasets
Project description:human lung fibroblasts WT and ATG7KO (two clones) were used for unbiased SILAC/AHA-based secretome and proteome measurements.
2021-09-10 | PXD025001 | Pride
Project description:In cells, several cargoes are delivered to the cell surface or the extracellular space via unconventional secretion routes. GRASP55 is a Golgi protein that regulates unconventional secretion of distinct proteins and controls the assembly and membrane stacking of Golgi cisternae. Recent work suggested that the role of GRASP55 in unconventional secretion may involve its relocalization to other organelles. However, the stimuli that drive GRASP55-dependent unconventional secretion, the signaling events that regulate GRASP55 function, the subcellular locations where GRASP55 acts, and the cargoes that follow this route for secretion remain unclear. Here, we show that mTORC1 directly phosphorylates GRASP55 at multiple sites to maintain its Golgi localization. Cellular stresses or drugs that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to autophagosomal / MVB structures. Using secretome and surfactome analyses in GRASP55-null cells, we identify for the first time numerous -previously unknown- cargoes that rely on this unconventional secretory pathway.
2021-09-09 | PXD020331 | Pride
Project description:MASTL (Microtubule associated serine-threonine kinase-like) is a relatively recently identified mitotic accelerator. There are also indications of its role in cancer progression, especially in breast cancer, but the molecular mechanisms behind this and the cell cycle independent functions of MASTL are poorly understood. Regulation of cell adhesion and actin dynamics play a central role in governing cell contractility, migration and cell division in normal and cancer cells. Here, we identify MASTL as regulator of cell contractility and activator of the transcriptional co-activator MTRF-A. We show that depletion of MASTL increases cell contact with the extracellular matrix, reduces contractile actin stress fibers in normal and breast cancer cells. Importantly, depletion of MASTL also leads to strongly impairs motility of breast cancer cells. Our transcriptome and proteome profiling revealed that depletion of MASTL impairs transcription and expression of several MRTF-A target genes implicated in cellular movement and actomyosin contraction, including Rho Guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and tropomyosin 4.2 (TPM4). Mechanistically, we find that MASTL is necessary for serum-induced activation of SRF/MTRF-A transcription and that exogenous expression of GEF-H1 in MASTL depleted cells is sufficient to restore cell contractility. Taken together, our results suggest that MASTL is a previously undescribed key regulator of cell morphology and the actin cytoskeleton through transcriptional control of multiple adhesion and actin cytoskeleton regulating genes with key roles in contractility, cell adhesion and migration. MASTL (Microtubule associated serine-threonine kinase-like) is a relatively recently identified mitotic accelerator. There are also indications of its role in cancer progression, especially in breast cancer, but the molecular mechanisms behind this and the cell cycle independent functions of MASTL are poorly understood. Regulation of cell adhesion and actin dynamics play a central role in cell migration, morphology and cancer progression, but the role of MASTL in these processes has not been evaluated. Here, we show that depletion of MASTL increases cell contact with the extracellular matrix in breast cancer cells. Importantly, depletion of MASTL also leads to reduction of contractile actin stress fibers and decreased cell motility. Further, our transcriptome and proteome profiling revealed that mechanistically depletion of MASTL impairs transcription and expression of regulators of cellular movement and actomyosin contraction, including Rho Guanine nucleotide exchange factor 2 (GEF-H1, ARHGEF2) and tropomyosin 4.2 (TPM4). Further, we show that the transcriptional changes are caused by defectiveness of the serum response factor (SRF) signalling. Importantly, this study reveals advanced role for MASTL in interphase cells that activates a transcriptional program strongly impacting on the expression of regulators of cellular motility and actomyosin contraction.
2020-04-14 | PXD013757 | Pride
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
Project description:Biogenesis of complex IV of the mitochondrial respiratory chain requires assembly factors for subunit maturation, co-factor attachment and stabilization of intermediate assemblies. A pathogenic mutation in Coa6, leading to substitution of a conserved tryptophan to a cysteine residue, results in a loss of complex IV activity and cardiomyopathy. Here we demonstrate that the complex IV defect correlates with a severe loss in complex IV assembly in patient heart but not fibroblasts. Complete loss of Coa6 activity using gene-editing in HEK293T cells resulted in a profound growth defect due to complex IV deficiency, caused by impaired biogenesis of the copper-bound mtDNA encoded subunit COX2 and subsequent accumulation of complex IV assembly intermediates. We show that the pathogenic mutation in Coa6 does not affect its import into mitochondria but impairs its maturation and stability. Furthermore we find that Coa6 binds copper with a high affinity and also associates with mitochondrial copper chaperones, revealing that Coa6 is intricately involved in the copper-dependent biogenesis of COX2.
2016-07-28 | PXD001958 | Pride
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
Project description:Intraepithelial T cells (IET) provide surveillance of the intestinal epithelium, but little is known about how epithelial-derived signals regulate IET. Analyzing mice with a deficiency for the herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily (TNFRSF14), we showed HVEM maintains the survival of subsets of small intestine IET through interactions with TNFSF14 (LIGHT). RNA-seq and analysis of organoids showed that epithelial cell expressed HVEM provided signals that were required for optimal synthesis of collagen IV. Collagen IV supported IET survival in vitro via interactions with IET β1 integrins. Furthermore, absence of β1 integrin expression in T lymphocytes decreased TCR αβ+ IET in vivo. Intravital microscopy showed that the patrolling movement of all IET was reduced without epithelial HVEM, and we hypothesize that collagen IV interactions with β1 integrins could have been responsible. As likely consequences of decreased total IET number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM binding partners CD160 or LIGHT, or β1 integrins. Therefore, these data defined a circuit whereby epithelial HVEM regulated IET indirectly, through basement membrane synthesis that interacts with T cell integrins, with consequences for mucosal immunity.
2022-06-17 | GSE206183 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-02-16 | E-GEOD-65410 | biostudies-arrayexpress
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) as control and additionally MK-2206 or LY294002 to inhibit PI3K/Akt signaling, respectively.
2020-05-25 | PXD016721 | Pride
Project description:The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network around mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and analyzed in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.<br><br>Additional contact details:<br><a href="mailto:k.thedieck@umcg.nl">Prof. Dr. Kathrin Thedieck</a><br>Department of Pediatrics, University of Groningen, University Medical Center Groningen (UMCG), 9713 AV Groningen, The Netherlands.<br>Faculty VI - School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, 26111 Oldenburg, Germany.<br><br>Prof. Dr. Bettina Warscheid<br>Faculty of Biology, Institute of Biology II, University of Freiburg, 79104 Freiburg, Germany.<br>BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.<br>
2015-04-28 | PXD001244 | Pride