PI-3 kinase effectors in insulin-responsive systems

胰岛素反应系统中的 PI-3 激酶效应器

基本信息

批准号：
7459063
负责人：
Silvia Corvera
金额：
$ 29.38万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-08-05 至 2011-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7459063
关键词：
1-Phosphatidylinositol 3-Kinase 3T3-L1 Cells Adipocytes Affinity Amino Acid Motifs Binding Biochemical Biological Caenorhabditis elegans Cell membrane Cells Cholera Toxin Class Classification Clathrin Cultured Cells Defect Disease EGF gene Early Endosome Endocytosis Endocytosis Inhibition Endosomes Event Funding GLUT4 gene Goals Homologous Gene Human Genome Hybrids Image Impairment In Transferrin Insulin Insulin Receptor Kinetics Laboratories Life Mammalian Cell Mass Spectrum Analysis Membrane Protein Traffic Microscopy Molecular Names Nature Non-Insulin-Dependent Diabetes Mellitus Pathway interactions Phosphatidylinositols Phosphotransferases Play Process Protein-Serine-Threonine Kinases Proteins Proteomics RNA Interference Recycling Reporting Research Personnel Role Signal Transduction Signal Transduction Pathway Site Small Interfering RNA Specificity Structure System Techniques Testing Time Transferrin Transferrin Receptor Transforming Growth Factor beta Vesicle Work base cancer type coated pit glucose transport glucose uptake human MADHIP protein in vivo insulin signaling new technology novel protein function receptor recycling tool trafficking transcription factor uptake

项目摘要

DESCRIPTION (provided by applicant): Ptdlns(3)-kinases play essential roles in signal transduction and membrane trafficking, and act by catalyzing the formation of 3'-phosphoinositides. Impairment in their function leads to diseases such as cancer and type 2 diabetes, and thus identifying the direct effectors of 3'-phosphoinositides is crucial to our progress in ameliorating these diseases. Work from our laboratory demonstrated that a specific protein motif, the FYVE domain, binds with high affinity and specificity to Ptdlns(3)P, a 3'-phosphoinositide highly enriched in endosomes (Patki et al. (1998), Nature 394: 433). Approximately 40 proteins in the human genome contain FYVE domains, implicating them as candidates for important functions in signal transduction and membrane trafficking. Thus for example, the FYVE-domain containing protein SARA is required for TGFp signaling to Smad2, which occurs in early endosomes (Hayes et al. (2003) J. Cell. Biol. 158(7): 1239-49. To accelerate the identification of the functions of FYVE domain-containing proteins, we have used siRNA screens in C. elegans. Using a strain that reveals endocytosis defects, we have identified a highly conserved, previously uncharacterized protein essential for endocytosis, called WDFY2. Its mammalian homologue is expressed ubiquitously, and its depletion from cultured cells results in a pronounced inhibition of transferrin uptake. Moreover, WDFY2 is found on vesicles that reside within 100 nm from the plasma membrane, and which lack typical early endocytic markers, thus defining a previously unrecognized class of early endosomes. Furthermore, depletion of WDFY2 inhibits insulin- stimulated glucose uptake in 3T3-L1 adipocytes to an extent similar to that seen upon depletion of the serine-threonine kinase Akt2. A direct interaction between WDFY2 and Akt has been observed, suggesting that WDFY2 may have a unique, conserved dual role in signal transduction and membrane trafficking. Because of its potentially central role in these essential processes in mammalian cells, in this competing proposal we will focus on understanding the function and mechanism of action of WDFY2. We seek to: 1) Define the molecular composition of WDFY2-enriched endosomes using immuno-isolation techniques and mass spectrometry. 2) Define the mechanism by which depletion of WDFY2 leads to inhibition of endocytosis, by analyzing the kinetics of internalization and recycling of the transferrin receptor. 3) Define the function of WDFY2 in insulin-stimulated glucose uptake in adipocytes, by testing whether insulin activation of Akt2 is impaired, or whether GLUT4 trafficking is impaired in the absence of WDFY2. 4) Define the mechanism of action of WDFY2 by identifying proteins that specifically interact with its WD-40 motifs using proteomic and 2-hybrid approaches.

描述（由申请人提供）：PTDLNS（3） - 激酶在信号转导和膜运输中起着至关重要的作用，并通过催化3'-磷酸肌醇的形成来起作用。其功能障碍会导致癌症和2型糖尿病等疾病，从而识别3'-磷酸肌醇的直接效应因素对于我们改善这些疾病的进展至关重要。我们实验室的工作表明，特定蛋白基序FYVE结构域具有高亲和力和特异性（3）p的特异性（3）P，A 3'-磷酸固醇高度富集在内体中（Patki等人（Patki等人（1998），自然394：433）。人类基因组中约有40种蛋白质包含FYVE结构域，这意味着它们是信号转导和膜运输中重要功能的候选者。因此，例如，TGFP信号向Smad2发出含有蛋白质的fyve域需要，这发生在早期内体内（Hayes等人（2003年）J。Cell。158（7）：1239-49。缺陷，我们已经确定了一种称为内吞作用的高度保守的蛋白质，称为WDFY2。内吞标记，因此，将先前未识别的早期内体定义，WDFY2的消耗抑制了3T3-L1脂肪细胞中胰岛素刺激的葡萄糖吸收的程度，其程度与丝氨酸 - 苏氨酸 - 硫代基因酶酶耗尽相似的程度。已经观察到WDFY2和AKT之间的直接相互作用，这表明WDFY2在信号转导和膜运输中可能具有独特的，保守的双重作用。由于其在哺乳动物细胞中这些基本过程中的潜在核心作用，因此在这项竞争性建议中，我们将重点放在理解WDFY2作用的功能和机理上。我们寻求：1）使用免疫隔离技术和质谱法定义富含WDFY2富含内体的分子组成。 2）定义WDFY2耗竭导致内吞作用的机制，通过分析内在化和回收的动力学的转铁蛋白受体的动力学。 3）通过测试是否会在没有WDFY2的情况下测试胰岛素激活是否受到损害，或者是否会损害WDFY2在脂肪细胞中胰岛素刺激的葡萄糖摄取的功能，是否会受到损害。 4）通过鉴定使用蛋白质组学和2杂交方法专门与其WD-40基序相互作用的蛋白质来定义WDFY2的作用机理。