Myogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.

Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.

We have developed the underrepresented post-translational modification (PTM) database (urPTMdb), a PTM gene set database to accelerate the discovery of enriched protein modifications in experimental data. urPTMdb provides curated lists of proteins reported to be substrates of underrepresented modifications. Their enrichment in proteomics datasets can reveal unexpected PTM regulations. urPTMdb can be implemented in existing workflows, or used in TeaProt, an online Shiny tool that integrates upstream transcription factor enrichment analysis with downstream pathway analysis through an easy-to-use interactive interface. TeaProt annotates user-uploaded data with drug-gene interactions, subcellular localizations, phenotypic functions, gene-disease associations, and enzyme-gene interactions. TeaProt enables gene set enrichment analysis (GSEA) to discover enrichments in gene sets from various resources, including MSigDB, CHEA, and urPTMdb. We demonstrate the utility of urPTMdb and TeaProt through the analysis of a previously published Western diet-induced remodeling of the tongue proteome, which revealed altered cellular processes associated with energy metabolism, interferon alpha/gamma response, adipogenesis, HMGylation substrate enrichment, and transcription regulation through PPARG and CEBPA. Additionally, we analyzed the interactome of ADP-ribose glycohydrolase TARG1, a key enzyme that removes mono-ADP-ribosylation. This analysis identified an enrichment of ADP-ribosylation, ribosomal proteins, and proteins localized in the nucleoli and endoplasmic reticulum. TeaProt and urPTMdb are accessible at https://tea.coffeeprot.com/.