C18ORF25 is a homolog of Arkadia (RNF111), an E3 ubiquitin ligase with SUMO-interaction motifs (SIMs) (PMID: 31417085). However, C18ORF25 lacks the entire C-terminal RING domain of RNF111 which is required for ubiquitin binding suggesting it lacks ubiquitination activity and may therefore act as an adaptor or signalling scaffold (PMID: 26283374). We have previously shown that mice lacking C18Orf25 throughout the entire body have increased adiposity, decreased lean mass, lower exercise capacity and significantly reduced ex vivo skeletal muscle force production (PMID: 35882232). Skeletal muscle isolated from C18Orf25 knockout (KO) mice have reduced cAMP-dependent protein kinase A (PKA) levels, and reduced phosphorylation of several contractile proteins and proteins involved in calcium handling. Furthermore, analysis of single muscle fibres from C18Orf25 KO mice revealed impaired SR calcium cycling in fast-twitch fibres only (PMID: 35882232). We also previously showed that the exercise-regulated phosphorylation of S67 on C18ORF25 is directly catalysed by AMPK. This phosphorylation site plays an important role in the function of C18ORF25 as re-expression of a phospho-mimetic S66/67D but not phospho-dead S66/67A in C18Orf25 KO mice is able to rescue skeletal muscle contractile defects PMID: 35882232). In the present study, we further investigated the potential molecular functions of C18ORF25. We performed affinity purification coupled to tandem mass spectrometry (AP-MS) to probe the protein:protein interaction (PPI) landscape of C18ORF25. Included in this analysis was also an investigation of potential S67 phosphorylation-dependent PPIs.