Exercise regulates a diverse array of phosphorylation networks which are thought to promote numerous health benefits. Functionally characterising these networks hold great promise to identify new therapeutic targets for a range of diseases including type 2 diabetes, cancer and neurological disorders. We have recently shown that the uncharacterised protein, C18ORF25 is significantly phosphorylated at Ser-67 across human, mouse and rat exercise models in skeletal muscle. In silico analysis reveals that it is a homolog of ARKadia, a regulator of TGF-B/BMP signalling, and is conserved down to bone-jawed fish. Furthermore, machine learning to predict the upstream kinase(s) mediating this phosphorylation event revealed C18ORF25 to be a highly significant hit as an AMPK substrate. Given the well described role of AMPK in metabolic adaptations during exercise, we hypothesise C18ORF25 is a novel regulator of exercise metabolism. Here, we validate C18ORF25 as a novel exercise-regulated AMPK substrate. To characterise the functional role of C18ORF25, we generated a whole-body knockout (KO) mouse model. Our preliminary data reveal KO mice gained similar weight on a chow diet compared to WT littermates but we observed a striking increase in adiposity and subtle decrease in lean mass from 6 weeks of age. These young KO mice showed a slight glucose intolerance and a significant reduction in energy expenditure with no difference in food intake, and they are also not able to run as far following forced treadmill exercise. Furthermore, functional analysis of ex vivo isolated muscles revealed those from KO mice have reduced contractile force and fatigue quicker. To investigate the functional effects of phosphorylation, we overexpressed a wild-type, a Ser-66/67-Ala mutant (to inhibit phosphorylation) or Ser-66/67-Asp mutant (to mimic phosphorylation) in HEK293 cells and performed a proteomic analysis. We observed a surprising remodelling of the proteome induced by these mutations including significant regulation of BMP signalling. Our preliminary data suggest C18ORF25 plays a vital role in AMPK-mediated regulation of BMP-signalling to allow metabolic adaptations to exercise. My project aims to perform further proteomic and phospho-proteomic analysis on C18ORF25 KO tissue to explore upstream and downstream signalling of C18ORF25 and the effect that phosphorylation has on it. We also aim to replicate experiments in metabolically insulted (high fat diet) C18ORF25 KO mice.