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Large-scale in vitro kinase assay with FLNc d23-24 and PKC alpha

ABSTRACT: Large-scale in vitro kinase assay with human and murine FLNc and its site mutants and PKC alpha.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD003185 | Pride | 2017-01-02

REPOSITORIES: Pride

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			Action	DRS
	ELITE-RSLC004476.raw	Raw
	ELITE-RSLC004477.raw	Raw
	ELITE-RSLC004478.raw	Raw
	ELITE-RSLC004479.raw	Raw
	ELITE-RSLC004480.raw	Raw

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Publications

Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics.

Reimann Lena L Wiese Heike H Leber Yvonne Y Schwäble Anja N AN Fricke Anna L AL Rohland Anne A Knapp Bettina B Peikert Christian D CD Drepper Friedel F van der Ven Peter F M PF Radziwill Gerald G Fürst Dieter O DO Warscheid Bettina B

Molecular & cellular proteomics : MCP 20161227 3

The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts <i>versus</i> ...[more]

PMID: 28028127

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Filamin C domain 18-21 in vitro kinase assay

Project description:In vitro kinase assay with human filamin C domain 18-21 recombinantly expressed in E.coli, purified by Ni2+-NTA beads and incubated with PKC alpha, Akt or a combination of both to identify kinase specific phosphorylation sites.

2020-05-25 | PXD008893 | Pride

In vitro calpainolysis assays - Myofibrillar Z-discs are a protein phosphorylation hot spot with PKC α modulating protein dynamics

Project description:In vitro calpainolysis in combination with top down mass spectrometry reveals the calpain-1 cleavage sites in human and murine carboxy terminal regions of FLNc

2017-01-02 | PXD003216 | Pride

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

FLNc Calpain GluC SRM Analysis

Project description:Human filamin C (FLNc) is a target of the protease calpain at Y2625. To confirm the dependency of calpain cleavage from FLNc phosphorylation at position S2623/S2624 by PKCa, FLNc was overexpressed in HEK293 cells and cells were treated with PMA to stimulated kinase activity, with Gö6976 to inhibit kinase activity or mock-treated with DMSO. Subsequently, recombinant calpain-1 and GluC were used and the resulting peptide TVTSSSSRGSSY was monitored by SRM analyses.

2017-01-02 | PXD005097 | Pride

Phosphoproteomics Identifies Dual-Site Phosphorylation in an Extended Basophilic Motif Regulating FILIP1-mediated Degradation of filamin-C FLNc d18-21 BioID experiment

Project description:FLNc, the muscle-specific isoform of the filamin family, is a multi-adaptor protein, comprising 1 amino-terminal actin-binding (ABD) domain followed by 24 immunoglobulin-like (Ig) domains. While FLNc can form homodimers via the last Ig-like domain and thus function as an actin-crosslinker like the other filamins, it features a unique insertion of 82 amino acids (aa) in domain 20. This insert was not only shown to mediate the interaction to several FLNc binding partners, but also to contain an Akt-mediated phosphorylation site at S2234 of mouse FLNc (mFLNc). To reveal novel proteins in the nano-environment of FLNc within myotubes under mild electrical pulse stimulation conditions, we applied a quantitative BioID appraoch.

2021-07-12 | PXD009159 | Pride

FLNc S2233 S2236 PRM analysis

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. Among the significantly regulated phosphopeptides we identified the novel extended basophilic motif RxRxxp[S/T]xxp[S] to be enriched in the set of down-regulated phosphopeptides following inhibition of PI3K/Akt signaling. Using literature-based text mining we identified the kinases Akt, serum and glucocorticoid-regulated kinase 1 (SGK1) and p70S6 kinase to be potentially involved in the phosphorylation of the first serine in the RxRxxp[S/T]xxp[S] motif, whereas no kinase targeting the serine in the +3 position was revealed. In the signaling adapter protein filamin c (FLNc) we found this novel motif in immunoglobulin (Ig)-like domain 20 which is involved in various protein interactions. Through in vitro and in cellulo kinase assays we identified Akt and protein kinase C alpha as the responsible kinases phosphorylating FLNc in this motif at the first and the second serine, respectively.

2020-06-30 | PXD009228 | Pride

Chromatinized PKC-q directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells

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Transcriptome analysis of hiPSC-CMs carrying FLNC mutations

Project description:In this study we performed transcriptome analysis of cardiomyocytes (CMs) derived from two hiPSC lines of patients with V2264M and R1267Q mutations in FLNC gene. hiPSC cell line were obtained from patient’s PBMCs with FLNC mutation using CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific). Detailed information about iPSC line characterization can be found in hiPSCreg database: (FAMRCi011-A and FAMRCi009). Information about FAMRCi009 is earlier published DOI: 10.1016/j.scr.2021.102640.

2024-03-05 | GSE260653 | GEO

FilaminC pull-down experiment from myotube lysates

Project description:This project was designed to identify a difference in the binding affinity of FILIP1 to different FLNc variants. To this end FLNc domain 1-3 and FLNc d18-21 were used as bait for a pull-down analysis.

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A genetically-encoded crosslinker screen identifies SERBP1 as a PKC substrate influencing translation and cell division

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2021-12-09 | PXD029091 | Pride

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