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

基本信息

批准号：
9341235
负责人：
Paul R Langlais
金额：
$ 34.54万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-25 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9341235
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 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 Role Series Site Small Interfering RNA System Techniques Tertiary Protein Structure Testing Tissues Travel Vesicle Western Blotting base cell cortex cell motility design experimental study glucose transport glucose uptake inhibitor/antagonist knock-down mutant overexpression protein expression protein protein interaction public health relevance trafficking

项目摘要

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（由申请人提供）：胰岛素刺激葡萄糖转运蛋白4（GLUT4）从细胞内池到质膜的储存囊泡的动员，从而导致葡萄糖流入肌肉和脂肪等靶组织中。该系统因胰岛素抵抗和2型糖尿病而受到干扰。胰岛素刺激的GLUT4插入质膜是一系列蛋白质 - 蛋白质相互作用和仍在研究的动态细胞骨架事件的最终产物。在第一次提交中，我们表明夹子缔解蛋白2（CLASP2）是一种先前与胰岛素作用无关的蛋白质，对胰岛素有反应。使用基于L6肌管的磷serine抗体免疫沉淀的基于质谱的蛋白质组学方法，我们检测到与基础相比，在胰岛素刺激的抗磷酸氨基免疫沉淀中，CLASP2的CLASP2增加了4.8倍。 CLASP2免疫沉淀的蛋白质印迹用磷酸抗体证实了CLASP2经历胰岛素刺激的磷酸化。 L6肌管的共聚焦成像表明，CLASP2位于胰岛素介导的皮质肌动蛋白重塑区域内的质膜。由于胰岛素诱导的皮质肌动蛋白重组是一个靶标对于GLUT4易位，我们测试了通过胰岛素刺激的动态肌动蛋白重塑产生的质膜褶皱内CLASP2和GLUT4的检测。共聚焦成像表明，在胰岛素刺激的质膜脊上，CLASP2与GLUT4共定位。已知CLASP2将微管的远端引导到细胞皮层，并且已显示GLUT4沿着微管轨道行驶。为了支持CLASP2指导基于微管的GLUT4向细胞皮层降落区域的递送对胰岛素作用很重要的递送，siRNA介导的L6 Myotubes中CLASP2的敲低抑制了胰岛素刺激的GLUT4易位到质膜。此外，siRNA介导的3T3-L1脂肪细胞中的CLASP2敲低抑制胰岛素刺激的葡萄糖转运。修订后的提案介绍了来自先前提出的内源性CLASP2相互作用的新初步数据，来自3T3-L1脂肪细胞，其中鉴定出几种已知的CLASP2相互作用蛋白，从而验证了该方法。分析了小鼠组织，并在胰岛素敏感组织（例如肌肉和脂肪）中的同工型 - 特异性CLASP2蛋白表达模式。新的初步数据已经确定了L6肌管内的胰岛素调节的内源性CLASP2磷酸化，包括对抑制糖原合酶激酶3（GSK3）或PI 3-激酶（PI 3-K）敏感的磷酸化。在诸如细胞迁移之类的系统中，已知CLASP2受GSK3负调节。因此，我们建议检验总体假设，即CLASP2受到基底状态的GSK3-介导的磷酸化负面调节。通过PI 3-K通过胰岛素灭活GSK3，可以缓解CLASP2，从而使微管的远端位于细胞皮质上的特定CLASP2靶向着陆区域，从而将Glut4含有Glut4，其中含有与血浆膜的囊泡相邻的囊泡。