Role of FK506-Binding Protein 3 (FKBP3) as a Novel Regulator of Skeletal Muscle Protein Synthesis

钙/钙调蛋白依赖的蛋白激酶1(CAMKK1)参与细胞外信号转导的调控。 依赖雷帕霉素复合体1(MTORC1)的机制靶点合成骨骼肌蛋白质 机械装置。然而，连接CAMKK1和mTORC1的下游信号蛋白/底物可以 启动这种对肌肉蛋白质动态平衡的有益影响目前尚不清楚。这样做的长期目标是 应用是开发针对信号通路成分的肌肉萎缩的新药物疗法 被CAMKK1用来刺激肌肉蛋白质的合成。总体假设是，磷酸化 FKBP3的状态控制着它的异构酶活性和亚细胞定位，以及这些功能和空间 这些变化促进了FKBP3调节骨骼肌mTORC1信号和蛋白质合成的能力。 其理论基础部分是基于无偏见的、ATP类似物敏感的激酶和定量的磷酸蛋白质组学。 CAMKK1使T122上的FKBP3磷酸化的方法，T122是位于其肽基-脯氨基上的残基 异构酶结构域。为了检验整个假设，提出了以下两个具体目标：1)确定 如果FKBP3调节肌肉中的mTORC1信号和蛋白质合成，以及2)确定FKBP3异构酶 活性或定位受T122磷酸化状态的调节。第一个目标是使用体内肌肉基因 转移/电穿孔技术在公猪肌肉中表达野生型和T122磷酸化突变蛋白 雌性老鼠。FKBP3磷酸化状态对肌肉质量/横截面积mTORC1变化的影响 将对信号蛋白和蛋白质合成进行评估。第二个目标是使用纯化的野生型CAMKK1 和FKBP3蛋白，无细胞磷酸化分析和质谱学鉴定所有可能的CAMKK1- FKBP3上受刺激的磷酸化位点。接下来，将产生FKBP3 T122磷酸化突变蛋白，并 在存在或不存在雷帕霉素的情况下，检查肽基-脯氨酰异构酶活性。我们还将在体内使用 电穿孔在小鼠肌肉中表达活性CAMKK1，然后检测FKBP3异构酶。最后，我们会 在小鼠肌肉中表达荧光标记的野生型和T122磷酸化突变体FKBP3，并对 荧光显微镜下FKBP3亚细胞定位。这项拟议的研究具有创新性，因为它将 确定FKBP3是否是新的钙/CAMKK1依赖机制的一部分，该机制调节mTORC1 骨骼肌中的信号和蛋白质合成。拟议的研究具有重要意义，因为它将定义一个 细胞机制的一部分，将细胞内的钙离子与肌肉中的mTORC1信号联系起来。这是关键的第一步 以此信号通路为靶点的肌肉萎缩新疗法的产生。

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.