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

• Applicant: Professorin Dr. Bettina Warscheid
• Subject Area: Cell Biology ; Biochemistry
• Term: since 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 388932620

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.

• DFG Programme: Research Units
• Subproject of: FOR 2743: Mechanical Stress Protection