title: Myofibrillar Z-discs Are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics.
abstract: 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 myoblastsversusdifferentiated 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.
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Reimann
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Heike
authors.initials: H
authors.lastname: Wiese
authors.affiliation: ¶Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Leber
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Anja N
authors.initials: AN
authors.lastname: Schwäble
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Anna L
authors.initials: AL
authors.lastname: Fricke
authors.affiliation: ¶Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Rohland
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Knapp
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Christian D
authors.initials: CD
authors.lastname: Peikert
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
authors.forename: Friedel
authors.initials: F
authors.lastname: Drepper
authors.affiliation: ¶Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany.
authors.forename: Peter F M
authors.initials: PF
authors.lastname: van der Ven
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.|§BIOSS Centre for Biological Signalling Studies, University of Freiburg.
authors.forename: Gerald
authors.initials: G
authors.lastname: Radziwill
authors.affiliation: ¶Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany.
authors.forename: Dieter O
authors.initials: DO
authors.lastname: Fürst
authors.affiliation: From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany; bettina.warscheid@biologie.uni-freiburg.de.|§BIOSS Centre for Biological Signalling Studies, University of Freiburg.
authors.forename: Bettina
authors.initials: B
authors.lastname: Warscheid
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004558:Electric Stimulation; D064448:Filamins / Q000378:metabolism; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D060066:Protein Interaction Maps; D011493:Protein Kinase C / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012518:Sarcomeres / Q000378:metabolism*
chemical_list: D064448:Filamins; D000255:Adenosine Triphosphate; D011493:Protein Kinase C