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

Ca 2 +/钙调蛋白依赖性蛋白激酶激酶1（CaMKK 1）参与了细胞凋亡的调节。 通过雷帕霉素复合物1（mTORC 1）依赖性机制靶点的骨骼肌蛋白质合成 机制等然而，连接CaMKK 1和mTORC 1的下游信号蛋白/底物， 引发这种对肌肉蛋白质体内平衡有益作用是目前未知的。长期目标是 应用是开发新的药物治疗肌肉萎缩的目标成分的信号通路 利用CaMKK 1刺激肌肉蛋白质合成。总的假设是， FKBP 3状态控制其异构酶活性和亚细胞定位;这些功能和空间定位 这些变化促进FKBP 3调节骨骼肌中mTORC 1信号传导和蛋白质合成的能力。 基本原理部分基于无偏的、ATP类似物敏感的激酶和定量磷酸化蛋白质组学 一种方法显示CaMKK 1磷酸化T122上的FKBP 3，T122是位于其肽基脯氨酰中的残基， 异构酶结构域。为了检验总体假设，提出了以下两个具体目标：1）确定 FKBP 3是否调节肌肉中的mTORC 1信号传导和蛋白质合成，以及2）确定FKBP 3异构酶 活性或定位受T122磷酸化状态调节。第一个目标将使用体内肌肉基因 转移/电穿孔以在雄性和雌性的肌肉中表达野生型和T122磷酸化位点突变蛋白， 雌性老鼠FKBP 3磷酸化状态对肌肉质量/横截面积，mTORC 1变化的影响 将评估信号蛋白和蛋白质合成。第二个目标将使用纯化的野生型CaMKK 1 和FKBP 3蛋白，无细胞磷酸化测定，和质谱鉴定所有可能的CaMKK 1- FKBP 3上的刺激磷酸化位点。接下来，将产生FKBP 3 T122磷酸突变蛋白， 在存在或不存在雷帕霉素的情况下检测肽基-脯氨酰异构酶活性。我们还将在体内使用 电穿孔以在小鼠肌肉中表达活性CaMKK 1，然后检测FKBP 3异构酶。最后，我们将 在小鼠肌肉中表达荧光标记的野生型和T122磷酸突变型FKBP 3，然后评估 FKBP 3亚细胞定位荧光显微镜。这项研究是创新的，因为它将 确定FKBP 3是否是调节mTORC 1的新型Ca 2 +/CaMKK 1依赖性机制的一部分 信号传导和蛋白质合成。这项研究很重要，因为它将定义一个 连接细胞内Ca 2+与肌肉中mTORC 1信号传导的细胞机制的一部分。这是关键的第一步 针对这一信号通路的肌肉萎缩的新疗法。

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.