Dataset Information

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

ABSTRACT: 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

INSTRUMENT(S): LTQ Orbitrap Elite

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

SUBMITTER: Friedel Drepper

LAB HEAD: Bettina Warscheid

PROVIDER: PXD003216 | Pride | 2017-01-02

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	ELITE-RSLC008108.raw	Raw
	ELITE-RSLC008108_XT_00001_M_fullscan.raw	Raw
	ELITE-RSLC008109.raw	Raw
	ELITE-RSLC008109_XT_00001_M_fullscan.raw	Raw
	ELITE-RSLC008110.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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Project description: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 versus differentiated myotubes, reflecting the drastic phenotypic conversion of these cells during myogenesis. Interestingly, EPS of differentiated myotubes to induce Z-disc assembly and maturation resulted in increased levels of proteins involved in ATP synthesis, presumably to fulfill the higher energy demand of contracting myotubes. Because an important role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape further warranted in-depth analysis. We therefore established, by global phosphoproteomics of EPS-treated contracting myotubes, a comprehensive site-resolved protein phosphorylation map of the Z-disc and found that it is a phosphorylation hotspot in skeletal myocytes, underscoring its functions in signaling and disease-related processes. In an illustrative fashion, we analyzed the actin-binding multiadaptor protein filamin C (FLNc), which is essential for Z-disc assembly and maintenance, and found that PKCα phosphorylation at distinct serine residues in its hinge 2 region prevents its cleavage at an adjacent tyrosine residue by calpain 1. Fluorescence recovery after photobleaching experiments indicated that this phosphorylation modulates FLNc dynamics. Moreover, FLNc lacking the cleaved Ig-like domain 24 exhibited remarkably fast kinetics and exceedingly high mobility. Our data set provides research community resource for further identification of kinase-mediated changes in myofibrillar protein interactions, kinetics, and mobility that will greatly advance our understanding of Z-disc dynamics and signaling.