Characterizing the Role of CLASP2 in Insulin-Stimulated Glucose Transport

表征 CLASP2 在胰岛素刺激的葡萄糖转运中的作用

• 批准号: 9135353
• 负责人: Paul R Langlais
• 金额: $ 12.11万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-25 至 2016-10-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9135353
• 关键词: 1-Phosphatidylinositol 3-Kinase; Actins; Adipocytes; Affect; Antibodies; Area; Binding; Biological Assay; Cardiovascular Diseases; Cell membrane; Control Animal; Data; Detection; Disease; Distal; Effectiveness; Event; Fatty acid glycerol esters; Feedback; Glucose; Glucose Transporter; Glycogen Synthase Kinase 3; Health; Human; Image; Immunoprecipitation; In Vitro; Insulin; Insulin Resistance; Investigation; Mass Spectrum Analysis; Mediating; Membrane; Microtubules; Mitosis; Modeling; Molecular; Mus; Muscle; Muscle Fibers; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphotransferases; Positioning Attribute; Protein Isoforms; Proteins; Proteomics; Public Health; Publishing; Regulation; Research; Role; Series; Site; Small Interfering RNA; System; Techniques; Tertiary Protein Structure; Testing; Tissues; Travel; Vesicle; Western Blotting; base; cell cortex; cell motility; design; feeding; glucose transport; glucose uptake; inhibitor/antagonist; knock-down; mutant; overexpression; protein expression; protein protein interaction; research study; trafficking

DESCRIPTION (provided by the applicant): Insulin stimulates the mobilization of glucose transporter 4 (GLUT4) storage vesicles from intracellular pools to the plasma membrane, resulting in an influx of glucose into target tissues such as muscle and fat. This system is disturbed in insulin resistance and type 2 diabetes. Insulin-stimulated insertion of GLUT4 into the plasma membrane is the end product of a series of protein-protein interactions and dynamic cytoskeletal events that are still under investigation. In the first submission, we showed that CLIP-associating protein 2 (CLASP2), a protein previously unassociated with insulin action, is responsive to insulin. Using a mass spectrometry-based proteomics approach combined with phosphoserine antibody immunoprecipitation from L6 myotubes, we detected a 4.8-fold increase of CLASP2 in the insulin stimulated anti-phosphoserine immunoprecipitates as compared to basal. Western blotting of CLASP2 immunoprecipitates with the phospho- antibody confirmed that CLASP2 undergoes insulin-stimulated phosphorylation. Confocal imaging of L6 myotubes revealed that CLASP2 is positioned at the plasma membrane within areas of insulin-mediated cortical actin remodeling. Since insulin-induced cortical actin reorganization is a target for GLUT4 translocation, we tested for detection of both CLASP2 and GLUT4 within the plasma membrane ruffle generated by insulin- stimulated dynamic actin remodeling. Confocal imaging revealed that CLASP2 colocalizes with GLUT4 at insulin-stimulated plasma membrane ridges. CLASP2 is known to direct the distal end of microtubules to the cell cortex, and it has been shown that GLUT4 travels along microtubule tracks. In support of the prospect that CLASP2 directs microtubule-based delivery of GLUT4 to cell cortex landing zones important for insulin action, siRNA mediated knockdown of CLASP2 in L6 myotubes inhibited insulin-stimulated GLUT4 translocation to the plasma membrane. Furthermore, siRNA mediated knockdown of CLASP2 in 3T3-L1 adipocytes inhibits insulin- stimulated glucose transport. The revised proposal presents new preliminary data from the previously proposed endogenous CLASP2 interactome studies from 3T3-L1 adipocytes, in which several known CLASP2 interacting proteins were identified, validating the approach. Mouse tissue was analyzed and an isoform- specific CLASP2 protein expression pattern in insulin-sensitive tissues such as muscle and fat was discovered. New preliminary data has identified insulin-regulated endogenous CLASP2 phosphorylation within L6 myotubes, including phosphorylation that is sensitive to inhibition of either glycogen synthase kinase 3 (GSK3) or PI 3-kinase (PI 3-K). Within systems such as cell migration, CLASP2 is known to be negatively regulated by GSK3. We therefore propose to test the overall hypothesis that CLASP2 is negatively regulated by GSK3- mediated phosphorylation in the basal state. Inactivation of GSK3 by insulin through PI 3-K relieves CLASP2, allowing for the distal end of microtubules to situate at the specific CLASP2-targeted landing zones on the cell cortex, whereby situating GLUT4 containing vesicles proximal to the plasma membrane.

描述(申请人提供)：胰岛素刺激葡萄糖转运蛋白4(GLUT4)存储囊泡从细胞内池到质膜的动员，导致葡萄糖流入靶组织，如肌肉和脂肪。这一系统在胰岛素抵抗和2型糖尿病中受到干扰。胰岛素刺激的GLUT4插入质膜是一系列蛋白质-蛋白质相互作用和动态细胞骨架事件的最终产物，这些事件仍在研究中。在第一篇论文中，我们展示了片段相关蛋白2(CLASP2)，一种以前与胰岛素作用无关的蛋白质，对胰岛素有反应。使用基于质谱学的蛋白质组学方法，结合L6肌管中的磷丝氨酸抗体免疫沉淀，我们检测到胰岛素刺激的抗磷丝氨酸免疫沉淀物中CLASP2的表达是基础水平的4.8倍。用磷酸化抗体对CLASP2免疫沉淀物进行免疫印迹证实，CLASP2经历了胰岛素刺激的磷酸化。L6肌管的共聚焦成像显示，CLASP2位于胰岛素介导的皮质肌动蛋白重塑区域的质膜上。由于胰岛素诱导的皮质肌动蛋白重组是一个目标 对于GLUT4易位，我们测试了由胰岛素刺激的动态肌动蛋白重塑产生的质膜褶皱中CLASP2和GLUT4的检测。共聚焦成像显示CLASP2与GLUT4共定位于胰岛素刺激的质膜脊部。已知CLASP2将微管的远端定向到细胞皮质，并且已经证明GLUT4沿着微管的轨迹移动。为了支持CLASP2引导基于微管的GLUT4转运到对胰岛素作用重要的细胞皮质着陆区的前景，siRNA介导的L6肌管中CLASP2的敲除抑制了胰岛素刺激的GLUT4到质膜的移位。此外，siRNA介导的CLASP2在3T3-L1脂肪细胞中的下调抑制了胰岛素刺激的葡萄糖转运。修订后的提案提供了来自先前提出的来自3T3-L1脂肪细胞的内源性CLASP2相互作用组研究的新的初步数据，其中鉴定了几个已知的CLASP2相互作用蛋白，验证了该方法。对小鼠组织进行了分析，发现在肌肉和脂肪等胰岛素敏感组织中存在异构体特异的CLASP2蛋白表达模式。新的初步数据证实，胰岛素调节的内源性CLASP2在L6肌管内的磷酸化，包括对糖原合成酶激酶3(GSK3)或PI 3-K(PI 3-K)抑制敏感的磷酸化。在细胞迁移等系统中，已知CLASP2受到GSK3的负调控。因此，我们建议检验CLASP2在基础状态下受GSK3介导的磷酸化负调控的总体假设。胰岛素通过PI 3-K使GSK3失活，解除了CLASP2，使微管的远端位于细胞皮质上特定的CLASP2靶向着陆区，从而使含有GLUT4的小泡位于质膜附近。