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Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes

  • Anna L. Fricke
    Anna L. Fricke
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
    Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, 97074 Würzburg, Germany
  • Wignand W. D. Mühlhäuser
    Wignand W. D. Mühlhäuser
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
  • Lena Reimann
    Lena Reimann
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
    More by Lena Reimann
  • Johannes P. Zimmermann
    Johannes P. Zimmermann
    Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, 97074 Würzburg, Germany
  • Christa Reichenbach
    Christa Reichenbach
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
  • Bettina Knapp
    Bettina Knapp
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
  • Christian D. Peikert
    Christian D. Peikert
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
  • Alexander M. Heberle
    Alexander M. Heberle
    Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, 6020 Innsbruck, Austria
  • Erik Faessler
    Erik Faessler
    Jena University Language & Information Engineering (JULIE) Lab, Friedrich Schiller University Jena, 07743 Jena, Germany
  • Sascha Schäuble
    Sascha Schäuble
    Jena University Language & Information Engineering (JULIE) Lab, Friedrich Schiller University Jena, 07743 Jena, Germany
    Systems Biology and Bioinformatics Unit, Leibniz Institute for Natural Product Research and Infection Biology─Leibniz-HKI, 07745 Jena, Germany
  • Udo Hahn
    Udo Hahn
    Jena University Language & Information Engineering (JULIE) Lab, Friedrich Schiller University Jena, 07743 Jena, Germany
    More by Udo Hahn
  • Kathrin Thedieck
    Kathrin Thedieck
    Institute of Biochemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, 6020 Innsbruck, Austria
    Department of Pediatrics, Section Systems Medicine of Metabolism and Signaling, University of Groningen, University Medical Center Groningen, Groningen 9700 RB, The Netherlands
    Department for Neuroscience, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg 26129, Germany
  • Gerald Radziwill
    Gerald Radziwill
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
    Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104 Freiburg, Germany
  • , and 
  • Bettina Warscheid*
    Bettina Warscheid
    Biochemistry and Functional Proteomics, Institute of Biology II, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany
    Biochemistry II, Theodor Boveri-Institute, Biocenter, University of Würzburg, 97074 Würzburg, Germany
    Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104 Freiburg, Germany
    *Email: [email protected]
Cite this: J. Proteome Res. 2023, 22, 3, 768–789
Publication Date (Web):February 10, 2023
https://doi.org/10.1021/acs.jproteome.2c00505
Copyright © 2023 The Authors. Published by American Chemical Society

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    Abstract

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    Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.

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    Supporting Information

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.2c00505.

    • Supplemental experimental section; antibodies used in this study (Supplemental Experimental Table S1); uncropped Western blot images of Figure 1 (Experimental Figure 1a–e); uncropped Western blot and Coomassie images of Figure S4 (Supplemental Experimental Figure 2a–d); mapping of PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways (Figure S1); evaluation of the global quantitative phosphoproteome after pathway inhibition using MK-2206, LY294002, U0126, and Torin1 (Figure S2); enriched motifs within downregulated phosphopeptides upon PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathway inhibition (Figure S3); activation and inhibition time course for AKT, S6K, and RSK signaling by pharmacological intervention (Figure S4); evaluation of the global label-free phosphoproteome after combined pathway activation and inhibition (Figure S5) (PDF)

    • Proof of concept based on known kinase–substrate relationships (Supplemental Table 1); data of the global MK/U0/LY/To SILAC phosphoproteomics experiment (Supplemental Table 2); evaluation of regulated motifs and kinases in the global MK/U0/LY/To SILAC phosphoproteomics experiment (Supplemental Table 3); motif clusterings of the global MK/U0/LY/To SILAC phosphoproteomics experiment (Supplemental Table 4); text mining results for the RxRxxp[ST] clustering of the global MK/U0/LY/To SILAC phosphoproteomics experiment (Supplemental Table 5); data of the global label-free MK/BI/PF phosphoproteomics experiment (Supplemental Table 6); KSEA and motif clustering of the label-free MK/PF/BI phosphoproteomics experiment (Supplemental Table 7); quantitative MS data of the kinase target validation assay using targeted MS (Supplemental Table 8); summary of the RxRxxp[ST]-containing phosphopeptides (Supplemental Table 9) (ZIP)

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