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

机械应力下肌肉细胞激酶底物网络的调节

• 批准号: 401376272
• 负责人: Professorin Dr. Bettina Warscheid
• 金额: --
• 依托单位: Biozentrum der Universität Würzburg
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/401376272?language=en
• 关键词: Regulation; kinase; substrate; networks; muscle

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.

复杂的细胞内信号网络的精确调节对于满足收缩骨骼肌的高机械需求至关重要。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。这种可逆磷酸化事件受到多种蛋白激酶和磷酸酶的严格控制，并决定细胞反应。然而，关于机械应力反应背后的细胞靶点和机制仍然存在很大的知识差距。因此，在第一个资助期间，我们研究了机械应力下骨骼肌管中磷酸蛋白质组的变化。我们的数据显示 MAP 激酶 JNK1、p38-α、ERK1/2 以及 PKC-α 和 AKT 被强烈激活。通过综合分析，我们确定细丝蛋白、某些小热休克蛋白以及 BAG3 和分子伴侣辅助选择性自噬 (CASA) 机制的成员是骨骼肌机械应力反应的中心目标。在这个细丝相关蛋白网络中，我们精确定位了许多磷酸化和去磷酸化位点。我们将细丝蛋白 C (FLNc) 确定为机械应力期间肌管中信号传导的节点。我们证明，AKT 和 PKC-α 介导的 FLNc 在其机械传感结构域 20 中的双位点磷酸化减少了与丝蛋白 A 相互作用蛋白 1 (FILIP1) 的结合。 FLNc 通过双位点磷酸化而稳定，因为 FILIP1 参与其自噬清除。然而，在机械应激过程中，两个位点都会去磷酸化，这很可能是由蛋白磷酸酶 1 介导的。相反，机械应激诱导的 FLNc 在其他区域的磷酸化对分子伴侣结合和纤丝蛋白解折叠动力学有影响。因此，计划工作的中心目标是识别和验证负责 FLNc 不同区域以及 BAG3 和机械应力期间自噬途径的其他参与者的可逆磷酸化的激酶和磷酸酶。为此，我们将结合定量磷酸蛋白质组学对机械应力激活的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 FILIP1 介导的降解的开关。此外，我们将描述 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。 FLNc 磷酸化将根据机械应力下对肌节稳定性的影响进行位点特异性评估。关于 FLNc 及其蛋白质稳态网络成员的这些复杂激酶和磷酸酶关系所获得的知识最终将使我们能够在未来的应用中精确定位和调节机械应力保护的关键因素和机制。