The Z-disc is a protein-rich structure critically important for the development and integrity of myofibrils, which are the contractile organelles of cross-striated muscle cells. We here used mouse C2C12 myoblast, which were differentiated into myotubes, followed by electrical pulse stimulation (EPS) to generate contracting myotubes comprising mature Z-discs. Using a quantitative proteomics approach, we found significant changes in the relative abundance of 387 proteins in myoblasts versus differ ...[more]
Project description:This project was designed to identify a difference in the binding affinity of FILIP1 to different FLNc variants. To this end FLNc domain 1-3 and FLNc d18-21 were used as bait for a pull-down analysis.
Project description:In vitro kinase assay with human filamin C domain 18-21 recombinantly expressed in E.coli, purified by Ni2+-NTA beads and incubated with PKC alpha, Akt or a combination of both to identify kinase specific phosphorylation sites.
Project description:The Z-disc is a protein-rich structure critically important for myofibril development and integrity. Since a role of the Z-disc for signal integration and transduction was recently suggested, its precise phosphorylation landscape warranted in-depth analysis. We therefore established a site-resolved protein phosphorylation map of the Z-disc in skeletal myocytes and found that it is a phosphorylation hotspot in living cells.
Project description:Skeletal muscle is known to adapt dynamically to changes in workload by regulatory processes of the phosphatidylinositide 3-kinase (PI3K)/Akt pathway. We performed a global quantitative phosphoproteomics analysis of contracting mouse C2 myotubes treated with insulin growth factor 1 (IGF-1) or LY294002 to activate or inhibit PI3K/Akt signaling, respectively. Among the significantly regulated phosphopeptides we identified the novel extended basophilic motif RxRxxp[S/T]xxp[S] to be enriched in the set of down-regulated phosphopeptides following inhibition of PI3K/Akt signaling. Using literature-based text mining we identified the kinases Akt, serum and glucocorticoid-regulated kinase 1 (SGK1) and p70S6 kinase to be potentially involved in the phosphorylation of the first serine in the RxRxxp[S/T]xxp[S] motif, whereas no kinase targeting the serine in the +3 position was revealed. In the signaling adapter protein filamin c (FLNc) we found this novel motif in immunoglobulin (Ig)-like domain 20 which is involved in various protein interactions. Through in vitro and in cellulo kinase assays we identified Akt and protein kinase C alpha as the responsible kinases phosphorylating FLNc in this motif at the first and the second serine, respectively.
Project description:FLNc, the muscle-specific isoform of the filamin family, is a multi-adaptor protein, comprising 1 amino-terminal actin-binding (ABD) domain followed by 24 immunoglobulin-like (Ig) domains. While FLNc can form homodimers via the last Ig-like domain and thus function as an actin-crosslinker like the other filamins, it features a unique insertion of 82 amino acids (aa) in domain 20. This insert was not only shown to mediate the interaction to several FLNc binding partners, but also to contain an Akt-mediated phosphorylation site at S2234 of mouse FLNc (mFLNc). To reveal novel proteins in the nano-environment of FLNc within myotubes under mild electrical pulse stimulation conditions, we applied a quantitative BioID appraoch.
Project description:Filamin C is mainly expressed in striated muscle cells where it localizes to Z-discs, myotendinous junctions and intercalated discs. Recent studies have revealed numerous mutations in the FLNC gene causing familiar and sporadic myopathies and cardiomyopathies with marked clinical variability. The most frequent myopathic mutation, p.W2710X, which is associated with myofibrillar myopathy, deletes the carboxy-terminal 16 amino acids from filamin C and abolishes the dimerization property of Ig-like domain 24. We previously characterized "knock-in" mice heterozygous for this mutation (p.W2711X), and have now investigated homozygous mice using protein and mRNA expression analyses, mass spectrometry, and extensive immunolocalization and ultrastructural studies. Although the latter mice display a relatively mild myopathy under normal conditions, our analyses identified major mechanisms causing the pathophysiology of this disease: (i) the expression level of filamin C protein is drastically reduced; (ii) mutant filamin C is relocalized from Z-discs to particularly mechanically strained parts of muscle cells, i.e. myotendinous junctions and myofibrillar lesions; (iii) the number of lesions is greatly increased and these lesions lack BAG3; (iv) the expression of HSPB7 is almost completely abolished. These findings indicate grave disturbances of BAG3-dependent and -independent autophagy pathways that are required for efficient lesion repair. In addition, our studies reveal general mechanisms of lesion formation and demonstrate that defective filamin C dimerization via its carboxy-terminal domain does not disturb assembly and basic function of myofibrils. An alternative dimerization site might compensate for that loss. Since filamins function as stress sensors, our data further substantiate that filamin C is important for mechanosensing in the context of Z‑disc stabilization and maintenance.
Project description:We performed cross-linking mass spectrometry experiments on intact mitochondria isolated from mouse heart in two conditions, native-state and high-salt treatment to disrupt electrostatic interactions. Both conditions were provided in biological replicates.
Project description:In this project we cloned 141 mammalian RhoGAPs/GEFs (112 human, 26 mouse, 2 rat, 1 chimpanzee) and performed a systematic study of their interactome, localisation and specificity. Here we submit the mass spectrometry data from the interactome screen.