Exercise regulates a diverse array of phosphorylation networks thought to promote numerous health benefits. These networks hold great promise as novel therapeutic targets. Here, we show that the uncharacterised protein, C18ORF25 is phosphorylated at Ser-67 across various human, mouse and rat exercise types in skeletal muscle. Machine-learning revealed Ser-67 as a probable substrate of the AMP-activated protein kinase (AMPK). Using biochemical, pharmacological and genetic approaches, we show that C18ORF25 is a direct substrate of AMPK.
To functionally characterise C18ORF25, we generated a whole-body knockout (KO) mouse model. KO mice gained similar weight on a chow diet compared to wild type (WT) littermates but display an increase in adiposity and decrease in lean mass. Interestingly, KO mice on a chow or high-fat diet displayed no differences in whole body glucose tolerance or skeletal muscle insulin sensitivity but fatigue quicker than WT mice during treadmill exercise. KO mice also have significant reductions in skeletal muscle force production and fatiguability when electrically stimulated ex vivo.
To investigate a potential role of C18ORF25 phosphorylation, muscles were transduced with a Ser-66/67-Asp (phospho-mimetic) mutant or a Ser-66/67-Ala (phospho-dead) mutant using adeno-associated virus. Muscles expressing the phospho-mimetic displayed significantly increased force production following ex vivo electrical stimulation compared to those expressing phospho-dead mutants. Taken together, our data suggests that phosphorylation of C18ORF25 plays a vital role in skeletal muscle function during exercise and contraction.