Myofibrillar Z-discs are a protein phosphorylation hot spot with PKC α 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 analyzed changes in the relative abundance of 2,588 proteins, of which 387 were significantly regulated in myoblasts versus myotubes. Changes in protein expression generally reflected the drastic phenotypic conversion of the 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. Since 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 exemplary fashion, we analyzed the actin-binding multi-adaptor 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 dataset provides an invaluable 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.