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发挥重要作用，并通过催化3'-磷酸肌醇的形成发挥作用。其功能受损会导致癌症和 2 型糖尿病等疾病，因此识别 3'-磷酸肌醇的直接效应物对于我们在改善这些疾病方面取得进展至关重要。我们实验室的工作表明，特定的蛋白质基序（FYVE 结构域）以高亲和力和特异性与 Ptdlns(3)P（一种在内体中高度富集的 3'-磷酸肌醇）结合（Patki 等人（1998），Nature 394：433）。人类基因组中大约 40 种蛋白质含有 FYVE 结构域，这表明它们是信号转导和膜运输中重要功能的候选蛋白。因此，例如，含有 FYVE 结构域的蛋白 SARA 是 TGFp 信号传导至 Smad2 所必需的，这发生在早期内体中（Hayes 等人 (2003) J. Cell. Biol. 158(7): 1239-49。为了加速含 FYVE 结构域蛋白的功能的鉴定，我们在线虫中使用了 siRNA 筛选。使用揭示胞吞作用缺陷的菌株，我们鉴定了高度内吞作用所必需的保守的、以前未表征的蛋白质，称为 WDFY2，其哺乳动物同源物广泛表达，并且其从培养细胞中的消耗导致转铁蛋白摄取的显着抑制。此外，WDFY2 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，从而定义了一类以前未被识别的早期内体。 WDFY2 抑制 3T3-L1 脂肪细胞中胰岛素刺激的葡萄糖摄取，其程度与消耗丝氨酸-苏氨酸激酶 Akt2 时所见的程度相似。已观察到 WDFY2 和 Akt 之间的直接相互作用，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，因为它在哺乳动物细胞的这些基本过程中具有潜在的核心作用，因此在这项竞争性提案中，我们将重点了解其功能和作用机制。我们寻求： 1) 使用免疫分离技术和质谱法定义富含 WDFY2 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动力学，定义 WDFY2 耗尽导致内吞作用抑制的机制。 3) 通过测试 Akt2 的胰岛素激活是否受损，或者在 WDFY2 缺失的情况下 GLUT4 运输是否受损，定义 WDFY2 在脂肪细胞中胰岛素刺激的葡萄糖摄取中的功能。 4) 通过使用蛋白质组学和 2-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 WDFY2 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.