PROJECT SUMMARY/ABSTRACT The US population continues to age and tens of millions of people will likely be affected by aging-related muscle atrophy and weakness (i.e., muscle dysfunction) at some point in life. This represents an enormous unmet medical need because aging-related muscle dysfunction lacks effective therapy, compromises independence, prolongs hospitalization and increases all-cause mortality. Impaired proteostasis is a major contributor to aging in mammals. Skeletal muscle fibers are long-living cells thereby being particularly susceptible to proteotoxic stress and relying on efficient protein turnover (i.e., degradation of dysfunctional proteins and organelles coupled to proper protein synthesis) to remain functional. Indeed, protein turnover is defective in aging-related muscle dysfunction, but the mechanisms involved remain insufficiently understood hindering the identification of potential therapeutic targets for this condition. The research proposed here would help to address this issue by investigating the protein ULK2, which we have recently demonstrated to be required for maintenance of effective protein turnover in skeletal muscle. Our follow-up studies uncovered that ULK2 deficient muscles had several features reminiscent of aging muscle, such as impaired protein degradation evidenced by accumulation of insoluble p62-associated ubiquitinated protein aggregates, increased eIF2α (S51) phosphorylation, which limits protein synthesis, as well as atrophy and weakness. Conversely, enhanced ULK2 expression appears to increase strength in aged muscle. These observations strongly support a model where insufficient ULK2 activity contributes to aging-related muscle dysfunction and that ULK2-dependent signaling may be targeted for therapy. To this matter, we have identified 3 unique phosphorylation sites at skeletal muscle ULK2 pontentially modulating its function and have mapped a conserved ULK phoshorylation motif to FIP200 potentially modulating its interaction with p62. This interaction has been shown to be required for autophagosome formation at ubiquitinated protein aggregates and their subsequent degradation. In addition, we determined that ULK2 interacts with GCN1 pontentially inhibiting its stimulation of the eIF2α kinase GCN2. Our proposed studies will build upon these important initial findings and use mouse models to address 3 specific aims in adult and aged muscle. In Aim 1, we will establish how gain and loss of function of ULK2 modulate protein turnover and muscle function in comparison with gain and loss of function of its paralog ULK1. In Aim 2, we will study mutated forms of ULK2 and FIP200 that either block or mimic their phosphorylation to establish their role in regulating protein degradation. In Aim 3, we will use gain and loss of function of GCN1 to establish its role in ULK2-mediated regulation of protein synthesis. Through these studies, we hope to advance our understanding on the mechanisms regulating skeletal...