Phosphoprotein CDP138 regulates glucose metabolism

磷蛋白 CDP138 调节葡萄糖代谢

基本信息

批准号：
8852603
负责人：
Zhen Yue Jiang
金额：
$ 35.6万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8852603
关键词：
1-Phosphatidylinositol 3-Kinase Adipocytes Affect Animal Model Animals Binding Binding Sites Biological Assay Body Composition C2 Domain Calcium Binding Calcium ion Cell membrane Cells Cultured Cells Data Diabetes Mellitus Diet Docking Embryo Euglycemic Clamping Exercise Fatty acid glycerol esters Fibroblasts Figs - dietary Fractionation GLUT4 gene GTPase-Activating Proteins Glucose Clamp Glucose Transporter Guanosine Triphosphate Phosphohydrolases Hyperglycemia Insulin Insulin Signaling Pathway Intracellular translocation Knockout Mice Life Link Lipid Binding Lipids Mediating Membrane Membrane Fusion Membrane Lipids Membrane Proteins Molecular Monitor Mus Muscle Mutant Strains Mice Obese Mice Obesity Pathway interactions Phosphoproteins Phosphorylation Phosphorylation Site Phosphotransferases Physiological Process Proteins RNA Interference Regulation Role Signal Pathway Signaling Protein Small Interfering RNA Technology Testing Total Internal Reflection Fluorescent Vesicle Work base blood glucose regulation calmodulin-dependent protein kinase II cell fixing cellular imaging design feeding glucose disposal glucose metabolism glucose transport in vivo insight insulin sensitivity insulin signaling link protein loss of function metabolic rate mouse model mutant novel overexpression protein activation screening therapeutic target trafficking

项目摘要

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.

描述（由申请人提供）：众所周知，胰岛素刺激的葡萄糖转运蛋白转运蛋白GLUT4从细胞内储存到质膜（PM）需要胰岛素刺激的葡萄糖转运蛋白GLUT4易位，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传导蛋白，我们成功地应用了定量磷酸化蛋白质组学方法，并将基于siRNA的功能筛选测定到培养的3T3-L1脂肪细胞上，毫不符合新颖的蛋白质CDP138的要求，以表明新型蛋白质CDP138用于胰岛素刺激Glucose刺激Glucose Compranting and Glut4转化。 CDP138是由KIAA0528编码的138 kDa以前未知的磷蛋白。有趣的是，CDP138直接被AKT2和CAMKIID磷酸化。 CDP138形成低聚物，能够结合钙和膜脂质。我们证明，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囊泡和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。我们的初步数据表明，CDP138无效的小鼠，而不是它们的野生型窝窝，在仅4周的高脂饮食挑战时是高血糖的。该项目的重点是研究CDP138调节Glut4- pm融合的分子基础，并使用功能障碍动物模型来确定CDP138在葡萄糖代谢中的生理意义。首先，我们建议确定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结构域中的脂质结合位点，并测试其在GLUT4易位和GLUT4囊泡中的作用 - 活胶中的PM融合。第三，我们观察到CDP138分别与TBC1D4/TBC1D1和RALBP1，GTPase激活RAB10/RAB8A/RAB13和RAC1的蛋白相互作用。由于已知这些GTPases参与GLUT4易位的调节，因此我们将检查CDP138是否调节其活动。此外，我们将通过比较CDP138基因敲除小鼠中的胰岛素敏感性，葡萄糖处理，身体成分和代谢率来确定CDP138体内CDP138的生理意义及其野生型同变型同变型杂物或高脂饮食。本研究将使用最先进的高胰岛素高血糖夹具技术。最后，我们还将检查CDP138是否对于使用基因敲除小鼠模型进行运动或收缩诱导的葡萄糖转运是必需的。总之，该项目将对CDP138充当激酶激活和葡萄糖转运之间的收敛点的分子机制提供宝贵而新颖的见解。