Phosphoprotein CDP138 regulates glucose metabolism

描述（由申请人提供）：众所周知，胰岛素刺激的葡萄糖转运体GLUT4从细胞内储存转运到质膜（PM）需要激活PI 3-激酶- Akt2途径，尽管其分子机制尚不完全清楚。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化蛋白质组学方法和基于sirna的功能筛选试验应用于培养的3T3-L1脂肪细胞，明确证明了胰岛素刺激葡萄糖转运和GLUT4易位需要一种新型蛋白CDP138。CDP138是由KIAA0528编码的一种138 kDa的未知磷酸化蛋白。有趣的是，CDP138被Akt2和CaMKIId直接磷酸化。CDP138形成低聚物，能够结合钙和膜脂。我们证明了CDP138中的C2结构域和Akt磷酸化位点Ser197对于胰岛素诱导的GLUT4易位和GLUT4囊泡与PM之间的膜融合至关重要。我们还成功地开发了第一个CDP138突变小鼠系。我们的初步数据显示，CDP138缺失的小鼠，而不是它们的野生型幼崽，在高脂肪饮食仅仅4周的情况下就会出现高血糖。本项目重点研究CDP138调控GLUT4 - PM融合的分子基础，并利用功能缺失动物模型确定CDP138在糖代谢中的生理意义。首先，我们打算确定CDP138的磷酸化和寡聚化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GLUT4易位。其次，我们将确定C2结构域的脂质结合位点，并测试它们在活细胞中GLUT4易位和GLUT4囊泡- PM融合中的作用。第三，我们观察到CDP138分别与Rab10/Rab8A/Rab13和Rac1的GTPase激活蛋白TBC1D4/TBC1D1和RalBP1相互作用。由于已知这些gtpase参与GLUT4易位的调控，我们将研究CDP138是否调控它们的活性。此外，我们将通过比较CDP138基因敲除小鼠的胰岛素敏感性、葡萄糖处理、身体组成和代谢率，确定CDP138在体内的生理意义，这些小鼠分别饲喂正常食物和高脂肪食物。本研究将采用最先进的高胰岛素-血糖钳技术。最后，我们还将使用敲除小鼠模型检查CDP138是否对运动或收缩诱导的葡萄糖运输是必要的。总之，该项目将为CDP138作为激酶激活和葡萄糖转运之间的交汇点的分子机制提供有价值的和新颖的见解。

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.