Phosphoprotein CDP138 regulates glucose metabolism

磷蛋白 CDP138 调节葡萄糖代谢

基本信息

批准号：
8750861
负责人：
Zhen Yue Jiang
金额：
$ 26.86万
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/8750861
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 Metabolic 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 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（申请人提供）：已知胰岛素刺激的葡萄糖转运蛋白GLUT4从细胞内储存转移到质膜（PM）需要PI 3激酶-Akt2途径的激活，尽管其分子机制尚未完全清楚。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蛋白质组学方法和基于 siRNA 的功能筛选测定应用于培养的 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质 CDP138 。 CDP138 是一种以前未知的 138 kDa 磷蛋白，由 KIAA0528 编码。有趣的是，CDP138 直接被 Akt2 和 CaMKIId 磷酸化。 CDP138 形成寡聚物并能够结合钙和膜脂。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 易位以及 GLUT4 囊泡和 PM 之间的膜融合至关重要。我们还成功培育了第一个CDP138突变小鼠品系。我们的初步数据显示，CDP138 缺失小鼠（而非其野生型同窝小鼠）在接受高脂肪饮食仅 4 周后会出现高血糖。该项目的重点是研究 CDP138 调节 GLUT4 - PM 融合的分子基础，并使用功能丧失动物模型确定 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，我们将鉴定 C2 结构域中的脂质结合位点，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 与 TBC1D4/TBC1D1 和 RalBP1 相互作用，TBC1D4/TBC1D1 和 RalBP1 分别是 Rab10/Rab8A/Rab13 和 Rac1 的 GTPase 激活蛋白。由于已知这些 GTP 酶参与 GLUT4 易位的调节，我们将检查 CDP138 是否调节它们的活性。此外，我们将通过比较用正常食物或高脂肪饮食喂养的 CDP138 敲除小鼠及其野生型同窝小鼠的胰岛素敏感性、葡萄糖处理、身体成分和代谢率，确定 CDP138 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，我们还将使用敲除小鼠模型检查 CDP138 是否是运动或收缩诱导的葡萄糖转运所必需的。总之，该项目将为 CDP138 作为激酶激活和葡萄糖转运之间的汇聚点的分子机制提供有价值和新颖的见解。