Protein phosphatases as essential regulators of the mechanical stress response

• Applicant: Professorin Dr. Maja Köhn
• Subject Area: Cell Biology
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 388932620

Regulation of protein function by phosphorylation has been shown to play a key role in the cellular mechanical stress response. Many proteins are dephosphorylated under mechanical stress conditions, suggesting that phosphatases are important in this process. However, the phosphatases responsible for the dephosphorylation of important proteins in the mechanical stress response, such as the cochaperone BAG3 and the actin regulator FLNc, are unknown. We applied several approaches in order to identify phosphatases that dephosphorylate functionally important sites on these proteins. BAG3 and FLNc interactomes were measured using pulldown experiments and mass spectrometry read-out. In addition, we have investigated the in vitro ability of phosphatases identified in the interactomes to dephosphorylate certain BAG3 and FLNc sites using phosphopeptides. Furthermore, we have set up a targeted phosphoproteomics method to detect the dephosphorylation of phosphosites on BAG3 using recombinant phosphatases. This was highly important for the project because so far, it was not possible to create selective antibodies for specific phosphosites in BAG3. Together, we narrowed down potential phosphatases of certain BAG3 and FLNc sites. A major aim of the here planned work is to corroborate the identified interactions in vitro and in cells. For the in vitro work, we will apply dephosphorylation assays with phosphopeptides and targeted phosphoproteomics as well as Western blot analysis. To study the cellular dephosphorylation and functional consequences under mechanical stress, we will apply chemical inhibition or activation of a phosphatase. Another aim is to investigate the phosphatase(s) responsible for the newly identified mechanical stress-dependent dephosphorylation of three close phosphorylation sites in the cytoskeleton adaptor SYNPO2, for which early data points to a role in filamin binding. The phosphoproteomic and functional studies will be carried out in close collaboration with experts in the research unit, and the interactions within the unit will allow to relate our results to other cell types and tissues.

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