PI-3 kinase effectors in insulin-responsive systems

描述（由申请人提供）：Ptdlns（3）-激酶在信号转导和膜运输中起重要作用，并通过催化3 '-磷酸肌醇的形成发挥作用。它们的功能受损会导致癌症和2型糖尿病等疾病，因此鉴定3 '-磷酸肌醇的直接效应物对我们改善这些疾病的进展至关重要。我们实验室的工作证明，特异性蛋白基序FYVE结构域以高亲和力和特异性结合Ptdlns（3）P，Ptdlns（3）P是一种在核内体中高度富集的3 ′-磷酸肌醇（Patki等（1998），Nature 394：433）。人类基因组中约有40种蛋白质含有FYVE结构域，暗示它们是信号转导和膜运输中重要功能的候选者。因此，例如，含FYVE结构域的蛋白SARA是TGF β信号传导至Smad 2所需的，其发生在早期核内体中（Hayes等人（2003）J. Cell. 158（7）：1239-49。为了加快FYVE结构域蛋白功能的鉴定，我们在C.优雅使用一个菌株，揭示内吞缺陷，我们已经确定了一个高度保守的，以前未知的蛋白质的内吞作用，称为WDFY 2。它的哺乳动物同源物是普遍表达的，并且其从培养细胞中的耗尽导致转铁蛋白摄取的显著抑制。此外，WDFY 2存在于距离质膜100 nm以内的囊泡上，并且缺乏典型的早期内吞标记物，因此定义了先前未识别的早期内体类别。此外，WDFY 2的消耗抑制3 T3-L1脂肪细胞中胰岛素刺激的葡萄糖摄取的程度与丝氨酸-苏氨酸激酶Akt 2消耗时所见的程度相似。已经观察到WDFY 2和Akt之间的直接相互作用，表明WDFY 2可能在信号转导和膜运输中具有独特的、保守的双重作用。由于其在哺乳动物细胞中的这些基本过程中的潜在核心作用，在这个竞争性的建议中，我们将重点了解WDFY 2的功能和作用机制。我们寻求：1）使用免疫分离技术和质谱法确定WDFY 2富集的内体的分子组成。2)通过分析转铁蛋白受体的内化和再循环的动力学，定义WDFY 2耗竭导致内吞抑制的机制。3)通过检测胰岛素对Akt 2的激活是否受损，或者在缺乏WDFY 2的情况下GLUT 4运输是否受损，确定WDFY 2在胰岛素刺激的脂肪细胞葡萄糖摄取中的功能。4)通过使用蛋白质组学和双杂交方法鉴定与其WD-40基序特异性相互作用的蛋白质，定义WDFY 2的作用机制。

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.