Ca2+/calmodulin-dependent protein kinase kinase 1 (CaMKK1) has been implicated in the regulation of skeletal muscle protein synthesis via a mechanistic target of rapamycin complex 1 (mTORC1)-dependent mechanisms. However, the downstream signaling proteins/substrates that connect CaMKK1 to mTORC1 to initiate this beneficial effect on muscle protein homeostasis are currently unknown. The long-term goal of this application is to develop novel drug therapies for muscle atrophy that target constituents of the signaling pathway utilized by CaMKK1 to stimulate muscle protein synthesis. The overall hypothesis is that the phosphorylation status of FKBP3 controls its isomerase activity and subcellular localization; and that these functional and spatial changes facilitate the ability of FKBP3 to regulate mTORC1 signaling and protein synthesis in skeletal muscle. The rationale is based in part on an unbiased, ATP-analog sensitive kinase and quantitative phospho-proteomics approach showing that CaMKK1 phosphorylates FKBP3 on T122, a residue located in its peptidyl-prolyl isomerase domain. To test the overall hypothesis, the following two specific aims were proposed: 1) Determine if FKBP3 regulates mTORC1 signaling and protein synthesis in muscle, and 2) Determine if FKBP3 isomerase activity or localization is regulated by T122 phospho-status. The first aim will use in vivo muscle gene transfer/electroporation to express wild-type and T122 phospho-site mutant proteins in the muscle of male & female mice. The effects of FKBP3 phospho-status on changes in muscle mass/cross-sectional area, mTORC1 signaling proteins, and protein synthesis, will be assessed. The second aim will use purified wild-type CaMKK1 and FKBP3 proteins, cell-free phosphorylation assays, and mass spectrometry to identity all possible CaMKK1- stimulated phospho-sites on FKBP3. Next, FKBP3 T122 phospho-mutant proteins will be generated, and peptidyl-prolyl isomerase activity examined in the presence or absence of rapamycin. We will also use in vivo electroporation to express active CaMKK1 in mouse muscle and then examine FKBP3 isomerase. Last, we will express fluorescently labeled wild-type and T122 phospho-mutant FKBP3 in mouse muscle, and then assess FKBP3 subcellular localization by fluorescence microscopy. The proposed research is innovative because it will determine whether FKBP3 is part of a novel Ca2+/CaMKK1-dependent mechanism that regulates mTORC1 signaling and protein synthesis in skeletal muscle. The proposed research is significant because it will define a part of the cellular mechanism that links intracellular Ca2+ to mTORC1 signaling in muscle. This is a key first step towards the generation of new therapies for muscle atrophy that would target this signaling pathway.