Phosphoprotein CDP138 regulates glucose metabolism

磷蛋白 CDP138 调节葡萄糖代谢

• 批准号: 8222368
• 负责人: Zhen Yue Jiang
• 金额: $ 42.41万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 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/8222368
• 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; Screening procedure; 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; therapeutic target; trafficking

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.

描述(申请人提供)：已知，胰岛素刺激的葡萄糖转运体GLUT4从细胞内储存到质膜(PM)需要激活PI 3-K-Akt2途径，尽管分子机制尚不完全清楚。为了确定葡萄糖转运所需的新的胰岛素信号蛋白，我们成功地将定量磷酸蛋白质组学方法和基于siRNA的功能筛选分析应用于培养的3T3-L1脂肪细胞，明确地证明了新的蛋白质CDP138在胰岛素刺激葡萄糖转运和GLUT4易位方面是必需的。CDP138是由KIAA0528编码的一个138 kDa的未知磷蛋白。有趣的是，CDP138被Akt2和CaMKIId直接磷酸化。CDP138形成低聚体，并能结合钙和膜脂。我们证明CDP138的C2结构域和Akt磷酸化位点Ser197在胰岛素诱导的GLUT4转位和GLUT4囊泡与PM之间的膜融合中都是关键的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缺失的小鼠，但不是它们的野生型小鼠，当被高脂饮食挑战仅4周时，就会出现高血糖。本课题的重点是研究CDP138调节GLUT4-PM融合的分子基础，并利用功能丧失动物模型确定CDP138在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在细胞内的分布、与钙离子和脂膜的相互作用以及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TbC1D4/TbC1D1和RalBP1相互作用，分别与Rab10/Rab8A/Rab13和rac1的GTP酶激活蛋白结合。由于已知这些GTP酶参与了GLUT4易位的调节，我们将研究CDP138是否调节它们的活性。此外，我们将通过比较CDP138基因敲除小鼠及其野生型小鼠在正常饮食或高脂饮食下的胰岛素敏感性、葡萄糖处置、身体成分和代谢率，来确定CDP138在体内的生理意义。在这项研究中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的小鼠模型来检验CDP138是否对运动或收缩诱导的葡萄糖运输是必需的。总之，该项目将为CDP138作为激酶激活和葡萄糖运输之间的聚合点的分子机制提供有价值的新见解。 公共卫生相关性：我们发现了一种新的磷蛋白CDP138，它是胰岛素刺激的葡萄糖运输的关键因子，其蛋白水平在肥胖动物中显著降低。该项目旨在了解CDP138如何在培养细胞和功能丧失的小鼠模型中调节葡萄糖运输。此外，该项目将为糖尿病的潜在治疗目标提供新的见解。