Reimann Lena, Wiese Heike, Leber Yvonne, Schwäble Anja N, Fricke Anna L, Rohland Anne, Knapp Bettina, Peikert Christian D, Drepper Friedel, van der Ven Peter F M, Radziwill Gerald, Fürst Dieter O, Warscheid Bettina
From the ‡Department of Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.
¶Department of Molecular Cell Biology, Institute for Cell Biology, University of Bonn, 53121 Bonn, Germany.
Mol Cell Proteomics. 2017 Mar;16(3):346-367. doi: 10.1074/mcp.M116.065425. Epub 2016 Dec 27.
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 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.
Z盘是一种富含蛋白质的结构，对肌原纤维的发育和完整性至关重要，肌原纤维是横纹肌细胞的收缩细胞器。我们在此使用小鼠C2C12成肌细胞，将其分化为肌管，然后进行电脉冲刺激（EPS）以产生包含成熟Z盘的收缩性肌管。使用定量蛋白质组学方法，我们发现成肌细胞分化为肌管过程中387种蛋白质的相对丰度发生了显著变化，反映了这些细胞在肌生成过程中的剧烈表型转变。有趣的是，对分化的肌管进行EPS诱导Z盘组装和成熟，导致参与ATP合成的蛋白质水平升高，推测这是为了满足收缩性肌管更高的能量需求。由于最近有人提出Z盘在信号整合和转导中起重要作用，因此其精确的磷酸化图谱值得进一步深入分析。因此，我们通过对EPS处理的收缩性肌管进行全局磷酸蛋白质组学分析，建立了Z盘的全面位点解析蛋白质磷酸化图谱，发现它是骨骼肌细胞中的一个磷酸化热点，突出了其在信号传导和疾病相关过程中的功能。作为一个示例，我们分析了肌动蛋白结合多接头蛋白细丝蛋白C（FLNc），它对Z盘的组装和维持至关重要，发现在其铰链2区域不同丝氨酸残基处的PKCα磷酸化可防止其被钙蛋白酶1在相邻酪氨酸残基处切割。光漂白后荧光恢复实验表明，这种磷酸化调节了FLNc的动力学。此外，缺乏切割的免疫球蛋白样结构域24的FLNc表现出非常快的动力学和极高的流动性。我们的数据集为进一步鉴定激酶介导的肌原纤维蛋白相互作用、动力学和流动性变化提供了研究社区资源，这将极大地推进我们对Z盘动力学和信号传导的理解。
