Study of Z-disc protein interaction and signaling networks
Final Report Abstract
The myofibrillar Z-disc is a large multi-protein assembly of high dynamics with crucial functions in signal integration and signal transduction in cross-striated muscle cells. The central aim of this projects was to systematically study the protein phosphorylation landscape of the myofibrillar Z-disc in fully differentiated contracting skeletal muscle cells and to elucidate how interactions, dynamics, mobility and/or the stability of Z-disc proteins are reversibly modulated by kinase-mediated phosphorylation events. Through global phosphoproteomic analysis, we thoroughly mapped sarcomeric protein phosphorylation pattern at a site-specific level, which showed that the myofibrillar Z-disc serves a central phosphorylation-dependent signaling platform in skeletal muscle cells. In this complex Z-disc (phospho-)protein network, the actin-binding multi-adaptor protein filamin C (FLNc) and its various binding partners exhibited a multitude of phosphorylation sites, many of which occurring in clusters within distinct protein regions. Detailed functional studies showed that FLNc is phosphorylated at specific serine residues in its flexible hinge 2 region by protein kinase c alpha (PKCα), which protect it from cleavage at an adjacent tyrosine residue by the cysteine protease calpain 1. Furthermore, we found that this phosphorylation precisely controls FLNc dynamics, which is essential for its function as signaling adaptor protein in cross-striated muscle cells. In addition to PKCα signaling at the level of the Z-disc, we investigated phosphoinositide-3-kinase (PI3K)/Akt signaling in fully differentiated contracting skeletal muscle cells using quantitative phosphoproteomics. Based on our quantitative phosphoproteome data, we identified the extended basophilic phosphorylation motif RxRxxp[S/T]xxp[S/T] in FLNc. Notably, the motif is located in the 82-amino acid insert in domain 20 of FLNc, which is lacking in its other family members filamin A and filamin B. Dual-site phosphorylation of FLNc within the extended motif in domain 20 is mediated by Akt and PKCα. Our data show that these phosphorylations reduce the interaction of FLNc with filamin A-interacting protein 1 (FILIP1), which we identified and confirmed as novel FLNc binding partner through quantitative proximity proteomics and interaction studies. Since we demostrated that FILIP1 induces degradation of filamin in skeletal muscle cells, dual-site phosphorylation of FLNc provides a mechanism to specifically protect FLNc, but not FLNa, from FILIP1-mediated degradation. In addition, we found that dual-site phosphorylation is required for fast dynamics and high mobility of FLNc in cross-striated muscle cells.
Publications
- New insights into myosin phosphorylation during cyclic nucleotide-mediated smooth muscle relaxation. J Muscle Res Cell Motil 2012; 33: 471-483
Puetz S, Schroeter MM, Piechura H, Reimann L, Hunger MS, Lubomirov LT, Metzler D, Warscheid B, Pfitzer G
(See online at https://doi.org/10.1007/s10974-012-9306-9) - SILAC for the study of mammalian cell lines and yeast protein complexes. Methods Mol Biol 2012; 893: 201-221
Piechura H, Oeljeklaus S, Warscheid B
(See online at https://doi.org/10.1007/978-1-61779-885-6_14) - Comparison of alternative MS/MS and bioinformatics approaches for confident phosphorylation site localization. J Proteome Res 2014; 13: 1128-1137
Wiese H, Kuhlmann K, Wiese S, Stoepel NS, Pawlas M, Meyer HE, Stephan C, Eisenacher M, Drepper F, Warscheid B
(See online at https://doi.org/10.1021/pr400402s) - Functional proteomics identifies acinus L as a direct insulin- and amino acid-dependent mammalian target of rapamycin complex 1 (mTORC1) substrate, Mol Cell Proteomics 2015; 8:2042-2055
Schwarz J, Wiese H, Tölle RC, Zarei M, Dengjel J, Warscheid B, Thedieck K
(See online at https://doi.org/10.1074/mcp.M114.045807) - Structural insights into Ca2+- calmodulin regulation of plectin 1a-integrin β4 interaction in hemidesmosomes. Structure 2015; 23: 558-570
Song J-G, Kostan J, Drepper F, Knapp B, de Almeida Ribeiro Jr E, Konarev PV, Grishkovskaya I, Wiche G, Gregor M, Svergun DI, Warscheid B, Djinović Carugo K
(See online at https://doi.org/10.1016/j.str.2015.01.011) - A systems study reveals concurrent activation of AMPK and mTOR by amino acids. Nat Commun 2016; 7: 13254
Dalle Pezze P, Ruf S, Sonntag AG, Langelaar-Makkinje M, Hall P, Heberle AM, Navas PR, Van Eunen K, Tölle RC, Schwarz JJ, Wiese H, Warscheid B, Deitersen J, Storck B, Faessler E, Schäuble S, Hahn U, Horvatovich P, Shanley DP, Thedieck K
(See online at https://doi.org/10.1038/ncomms13254) - Optogenetic clustering of CNK1 reveals mechanistic insights in RAF and AKT signalling controlling cell fate decisions. Sci Rep 2016; 6: 38155
Fischer A, Warscheid B, Weber W, Radziwill G
(See online at https://doi.org/10.1038/srep38155) - Xilmass: a new approach toward the identification of cross-linked peptides. Anal Chem 2016; 88: 9949-9957
Yılmaz Su, Drepper F, Hulstaert N, Černič Ma, Gevaert K, Economou A, Warscheid B, Martens L, Vandermarliere E
(See online at https://doi.org/10.1021/acs.analchem.6b01585) - AKT-dependent phosphorylation of the SAM domain induces oligomerization and activation of the scaffold protein CNK1. Biochim Biophys Acta – Mol Cell Res 2017; 1864: 89-100
Fischer A, Weber W, Warscheid B, Radziwill G
(See online at https://doi.org/10.1016/j.bbamcr.2016.10.009) - Myofibrillar Z-discs are a Protein Phosphorylation Hot Spot with Protein Kinase C (PKCα) Modulating Protein Dynamics. Mol Cell Proteomics 2017; 16: 346-367
Reimann L, Wiese H, Leber Y, Schwäble AN, Fricke AL, Rohland A, Knapp B, Peikert CD, Drepper F, van der Ven PF, Radziwill G, Fürst DO, Warscheid B
(See online at https://doi.org/10.1074/mcp.M116.065425)
