Regulation of kinase-substrate networks in muscle cells under mechanical stress

精确调节复杂的细胞内信号网络对于满足收缩骨骼肌的高机械要求是必不可少的。蛋白质磷酸化在机械应变引起的直接分子反应中起着核心作用。这种可逆的磷酸化事件受到许多蛋白激酶和磷酸酶的严格控制，并决定细胞反应。然而，仍然有一个很大的知识差距的细胞目标和机制的机械应力反应。因此，在第一个资助期，我们研究了机械应力下骨骼肌管中磷酸化蛋白质组的变化。我们的数据显示MAP激酶JNK 1、p38-α、ERK 1/2以及PKC-α和AKT的强烈激活。通过综合分析，我们确定了film，某些小的热休克蛋白以及BAG 3和分子伴侣辅助的选择性自噬（CASA）机制的成员作为骨骼肌机械应力反应的中心目标。在这个细丝蛋白相关的蛋白质网络中，我们精确地定位了许多磷酸化和去磷酸化的位点。我们确定细丝蛋白C（FLNc）作为机械应力过程中肌管信号传导的节点。我们证明，AKT和PKC-α介导的FLN c在其机械感应结构域20中的双位点磷酸化减少了与细丝蛋白A相互作用蛋白1（FILIP 1）的结合。FLNc通过双位点磷酸化稳定，因为FILIP 1参与其自噬清除。然而，在机械应力期间，这两个位点都变得去磷酸化，这最有可能是由蛋白磷酸酶1介导的。相反，机械应力诱导的FLN c在其他区域的磷酸化对伴侣蛋白结合和细丝蛋白展开动力学有影响。因此，计划工作的中心目标是鉴定和验证负责FLNc不同区域以及BAG 3中可逆磷酸化的激酶和磷酸酶，以及机械应力期间自噬途径的其他参与者。为此，我们将结合定量磷酸蛋白质组学对机械应力激活激酶和磷酸酶进行抑制剂研究。我们将阐明FLNc的去磷酸化是否是急性机械应力下FILIP 1介导的降解的开关。此外，我们将表征FLNC-伴侣相互作用和力诱导的展开动力学中激酶控制的变化。将根据机械应力下肌节稳定性的后果对FLNc磷酸化进行位点特异性评估。这些复杂的激酶和磷酸酶的FLNc及其蛋白质抑制网络的成员的关系获得的知识，最终将使我们能够精确地目标和调制的关键因素和机制的机械应力保护在未来的应用。

The precise regulation of a complex intracellular signaling network is essential to meet the high mechanical demands of contracting skeletal muscles. Protein phosphorylation plays a central role in the immediate molecular reactions elicited by mechanical strain. Such reversible phosphorylation events are tightly controlled by numerous protein kinases and phosphatases and determine cellular responses. Yet, there is still a large knowledge gap about the cellular targets and mechanisms underlying the mechanical stress response. In the first funding period, we therefore studied changes in the phosphoproteome in skeletal myotubes under mechanical stress. Our data revealed a strong activation of the MAP kinases JNK1, p38-alpha, ERK1/2 as well as PKC-alpha and AKT. Through an integrative analysis we determined filamins, certain small heat shock proteins as well as BAG3 and the members of the chaperone-assisted selective autophagy (CASA) machinery as central targets of the mechanical stress response in skeletal muscle. Within this filamin-associated protein network, we precisely localized the numerous sites of phosphorylation and dephosphorylation. We identified filamin C (FLNc) as nodal point of signalling in myotubes during mechanical stress. We demonstrated that AKT- and PKC-alpha-mediated dual-site phosphorylation of FLNc in its mechanosensing domain 20 reduces the binding to the filamin A-interacting protein 1 (FILIP1). FLNc is stabilized by dual-site phosphorylation, since FILIP1 is involved in its autophagic removal. However, during mechanical stress, both sites become dephosphorylated, which is most likely mediated by the protein phosphatase 1. In contrast, mechanical stress-induced phosphorylation of FLNc in other regions had an impact on chaperone binding and filamin unfolding dynamics. Thus, a central objective of the planned work is to identify and verify the kinases and phosphatases that are responsible for the reversible phosphorylations in the different regions of FLNc as well as in BAG3 and further players of the autophagic pathways during mechanical stress. To this end, we will perform inhibitor studies of mechanical stress-activated kinases and phosphatases in combination with quantitative phosphoproteomics. We will elucidate whether dephosphorylation of FLNc is a switch for FILIP1-mediated degradation under acute mechanical stress. In addition, we will characterize kinase-controlled changes in FLNc-chaperone interactions and force-induced unfolding dynamics. FLNc phosphorylations will be site-specifically assessed in terms of consequences on sarcomere stability under mechanical stress. The knowledge obtained on these complex kinase- and phosphatase-relationships of FLNc and the members of its proteostasis network will eventually allow us to precisely target and modulate key factors and mechanisms of mechanical stress protection in future applications.