Phosphoprotein CDP138 regulates glucose metabolism

描述（由申请人提供）：已知PI 3-激酶-Akt 2途径的激活是胰岛素刺激的葡萄糖转运蛋白GLUT 4从细胞内储存转运至质膜（PM）所必需的，尽管分子机制尚未完全了解。为了鉴定葡萄糖转运所需的新型胰岛素信号蛋白，我们成功地将定量磷酸蛋白质组学方法和基于siRNA的功能筛选测定应用于培养的3 T3-L1脂肪细胞，明确地证明了胰岛素刺激葡萄糖转运和GLUT 4易位需要新型蛋白质CDP 138。CDP 138是一种138 kDa的未知磷蛋白，由KIAA 0528编码。有趣的是，CDP 138被Akt 2和CaMKIId直接磷酸化。CDP 138形成寡聚体，并能够结合钙和膜脂质。我们证明了CDP 138中的C2结构域和Akt磷酸化位点Ser 197对于胰岛素诱导的GLUT 4易位和GLUT 4囊泡与PM之间的膜融合至关重要。我们还成功开发了第一个CDP 138突变小鼠系。我们的初步数据显示，当用高脂肪饮食攻击仅4周时，CDP 138无效小鼠，而不是它们的野生型同窝小鼠，是高血糖的。本项目的重点是研究CDP 138调节GLUT 4- PM融合的分子基础，并利用功能丧失动物模型确定CDP 138在葡萄糖代谢中的生理意义。首先，我们建议确定磷酸化和寡聚化的CDP 138是否影响其细胞内分布，与钙离子和脂质膜的相互作用，以及GLUT 4易位。其次，我们将鉴定C2结构域中的脂质结合位点，并测试它们在肝细胞中GLUT 4易位和GLUT 4囊泡- PM融合中的作用。第三，我们观察到CDP 138与TBC 1D 4/TBC 1D 1和RalBP 1相互作用，GT3分别激活Rab 10/Rab 8A/Rab 13和Rac 1。由于已知这些GTP酶参与GLUT 4易位的调节，我们将检查CDP 138是否调节它们的活性。此外，我们将通过比较CDP 138基因敲除小鼠和用正常食物或高脂饮食喂养的野生型同窝小鼠中的胰岛素敏感性、葡萄糖处置、身体组成和代谢率来确定CDP 138在体内的生理意义。本研究将使用最先进的高胰岛素-正常血糖钳夹技术。最后，我们还将使用基因敲除小鼠模型检查CDP 138是否是运动或收缩诱导的葡萄糖转运所必需的。总之，该项目将提供有价值的和新的见解的分子机制，其中CDP 138作为一个点之间的收敛激酶激活和葡萄糖转运。 公共卫生相关性：我们确定了一种新的磷蛋白CDP 138作为胰岛素刺激的葡萄糖转运的重要因素，其蛋白水平在肥胖动物中显着降低。该项目旨在了解CDP 138如何调节培养细胞和功能丧失小鼠模型中的葡萄糖转运。此外，该项目将为糖尿病的潜在治疗靶点提供新的见解。

DESCRIPTION (provided by applicant): It is known that activation of the PI 3-kinase - Akt2 pathway is required for insulin-stimulated glucose transporter GLUT4 translocation from intracellular storage to the plasma membrane (PM) although the molecular mechanism is not fully understood. To identify novel insulin signaling proteins required for glucose transport, we have successfully applied quantitative phosphoproteomic approaches and siRNA-based functional screening assays to cultured 3T3-L1 adipocytes, demonstrating unequivocally the requirement of a novel protein CDP138 for insulin stimulation of glucose transport and GLUT4 translocation. CDP138 is a 138 kDa previously unknown phosphoprotein encoded by KIAA0528. Interestingly, CDP138 is phosphorylated by both Akt2 and CaMKIId directly. CDP138 forms oligomers and is capable of binding calcium and membrane lipids. We demonstrated that both the C2 domain and Akt phosphorylation site Ser197 in CDP138 are critical for insulin-induced GLUT4 translocation and membrane fusion between the GLUT4 vesicles and the PM. We have also successfully developed the first CDP138 mutant mouse line. Our preliminary data shows that CDP138 null mice, but not their wild-type littermates, are hyperglycemic when challenged with a high- fat diet for only 4 weeks. The focus of this project is to study the molecular basis by which CDP138 regulates GLUT4 - PM fusion and to determine physiological significance of CDP138 in glucose metabolism using the loss-of-function animal model. First, we propose to determine if phosphorylation and oligomerization of CDP138 affect its intracellular distribution, interactions with calcium ion and lipid membranes, and GLUT4 translocation. Second, we wil identify lipid-binding sites in the C2 domain and test their role in GLUT4 translocation and GLUT4 vesicle - PM fusion in live cels. Third, we observed that CDP138 interacts with TBC1D4/TBC1D1 and RalBP1, GTPase activating proteins for Rab10/Rab8A/Rab13 and Rac1, respectively. Since those GTPases are known to be involved in the regulation of GLUT4 translocation, we will examine if CDP138 regulates their activities. Furthermore, we will determine the physiological significance of CDP138 in vivo, by comparing insulin sensitivity, glucose disposal, body composition and metabolic rate in CDP138 knockout mice and their wild-type littermates fed with a normal chow or a high-fat diet. State-of-the art hyperinsulinemic-euglycemic clamp technology will be used in this study. Finally, we will also examine if CDP138 is necessary for exercise- or contraction-induced glucose transport using the knockout mouse model. Together, this project will provide valuable and novel insight into the molecular mechanisms by which CDP138 acts as a point of convergence between kinase activation and glucose transport. PUBLIC HEALTH RELEVANCE: We identified a novel phosphoprotein CDP138 as an essential factor for insulin-stimulated glucose transport and its protein level is significantly reduced in obese animals. This project is designed to understand how CDP138 regulates glucose transport in both cultured cells and a loss-of-function mouse model. In addition, this project will provide new insight into potential therapeutic targets for diabetes.