Potts Gregory K, McNally Rachel M, Blanco Rocky, You Jae-Sung, Hebert Alexander S, Westphall Michael S, Coon Joshua J, Hornberger Troy A
Department of Chemistry, University of Wisconsin - Madison, Madison, WI, USA.
Genome Center of Wisconsin, University of Wisconsin - Madison, Madison, WI, USA.
J Physiol. 2017 Aug 1;595(15):5209-5226. doi: 10.1113/JP273904. Epub 2017 Jul 4.
Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill-defined. Here we report a mass spectrometry-based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal-intensity contractions (MICs). Our dataset provides the first comprehensive map of the MIC-regulated phosphoproteome. Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well-known MIC-regulated signalling pathways, as well as to a plethora of novel MIC-regulated events. We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field.
The maintenance of skeletal muscle mass is essential for health and quality of life. It is well recognized that maximal-intensity contractions, such as those which occur during resistance exercise, promote an increase in muscle mass. Yet, the molecules that sense the mechanical information and convert it into the signalling events (e.g. phosphorylation) that drive the increase in muscle mass remain undefined. Here we describe a phosphoproteomics workflow to examine the effects of electrically evoked maximal-intensity contractions (MICs) on protein phosphorylation in mouse skeletal muscle. While a preliminary phosphoproteomics experiment successfully identified a number of MIC-regulated phosphorylation events, a large proportion of these identifications were present on highly abundant myofibrillar proteins. We subsequently incorporated a centrifugation-based fractionation step to deplete the highly abundant myofibrillar proteins and performed a second phosphoproteomics experiment. In total, we identified 5983 unique phosphorylation sites of which 663 were found to be regulated by MIC. GO term enrichment, phosphorylation motif analyses, and kinase-substrate predictions indicated that the MIC-regulated phosphorylation sites were chiefly modified by mTOR, as well as multiple isoforms of the MAPKs and CAMKs. Moreover, a high proportion of the regulated phosphorylation sites were found on proteins that are associated with the Z-disc, with over 74% of the Z-disc proteins experiencing robust changes in phosphorylation. Finally, our analyses revealed that the phosphorylation state of two Z-disc kinases (striated muscle-specific serine/threonine protein kinase and obscurin) was dramatically altered by MIC, and we propose ways these kinases could play a fundamental role in skeletal muscle mechanotransduction.
机械信号在肌肉质量调节中起关键作用，但感知机械信号并将这种刺激转化为调节肌肉质量的生化事件的分子仍不清楚。在此，我们报告了一种基于质谱的工作流程，用于研究小鼠骨骼肌在一轮电诱发最大强度收缩（MICs）后1小时内发生的蛋白质磷酸化变化。我们的数据集提供了首个全面的MIC调节磷酸化蛋白质组图谱。使用无偏见的生物信息学方法，我们证明我们的数据集可识别许多众所周知的MIC调节信号通路，以及大量新的MIC调节事件。我们预计我们的数据集将成为肌肉生物学家的重要基础资源，并有助于为该领域全新的假设奠定基础。
骨骼肌质量的维持对健康和生活质量至关重要。众所周知，最大强度收缩，如阻力运动期间发生的收缩，会促进肌肉质量增加。然而，感知机械信息并将其转化为驱动肌肉质量增加的信号事件（如磷酸化）的分子仍不明确。在此，我们描述了一种磷酸化蛋白质组学工作流程，以研究电诱发最大强度收缩（MICs）对小鼠骨骼肌蛋白质磷酸化的影响。虽然初步的磷酸化蛋白质组学实验成功鉴定了一些MIC调节的磷酸化事件，但这些鉴定中的很大一部分存在于高度丰富的肌原纤维蛋白上。我们随后纳入了基于离心的分级分离步骤以去除高度丰富的肌原纤维蛋白，并进行了第二次磷酸化蛋白质组学实验。我们总共鉴定了5983个独特的磷酸化位点，其中663个被发现受MIC调节。基因本体（GO）术语富集、磷酸化基序分析和激酶-底物预测表明，MIC调节的磷酸化位点主要由mTOR以及丝裂原活化蛋白激酶（MAPKs）和钙/钙调蛋白依赖性蛋白激酶（CAMKs）的多种亚型修饰。此外，在与Z盘相关的蛋白质上发现了高比例的受调节磷酸化位点，超过74%的Z盘蛋白经历了磷酸化的显著变化。最后，我们的分析表明，两种Z盘激酶（横纹肌特异性丝氨酸/苏氨酸蛋白激酶和 obscurin）的磷酸化状态因MIC而发生显著改变，并且我们提出了这些激酶在骨骼肌机械转导中可能发挥重要作用的方式。
