PI-3 kinase effectors in insulin-responsive systems

描述(申请人提供)：Ptdlns(3)-激酶在信号转导和膜转运中发挥重要作用，并通过催化3‘-磷酸肌醇的形成而起作用。它们的功能受损会导致癌症和2型糖尿病等疾病，因此识别3‘-磷脂酰肌醇的直接效应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，一个特定的蛋白质基序，FYVE结构域，与Ptdlns(3)P具有高亲和力和特异性，Ptdlns(3)P是一种高度富含在内体中的3‘-磷酸肌醇(Patki等人)。(1998)，《自然》394：433)。在人类基因组中大约有40个蛋白质含有FYVE结构域，这意味着它们可能在信号转导和膜转运中发挥重要功能。因此，例如，包含FYVE结构域的蛋白SARA是TGFp向Smad2发送信号所必需的，这发生在早期的内体中(Hayes等人。(2003)J.Cell.比奥尔。158(7)：1239-49。为了加快FYVE结构域蛋白功能的鉴定，我们在线虫中使用了siRNA筛选。使用一种揭示内吞缺陷的菌株，我们已经鉴定出一种高度保守的、以前未确定的内吞作用必需的蛋白质，称为WDFY2。它在哺乳动物中的同源物普遍表达，其在培养细胞中的缺失导致转铁蛋白摄取的明显抑制。此外，WDFY2在距离质膜100 nm以内的囊泡上被发现，这些囊泡缺乏典型的早期内吞标记，因此定义了一类以前未被识别的早期内吞体类。此外，WDFY2的缺失抑制了胰岛素刺激的3T3-L1脂肪细胞的葡萄糖摄取，其程度与丝氨酸苏氨酸激酶Akt2缺失时相似。已观察到WDFY2与Akt之间的直接相互作用，提示WDFY2可能在信号转导和膜转运中具有独特的、保守的双重作用。由于WDFY2在哺乳动物细胞的这些基本过程中潜在的核心作用，在这个竞争性的提案中，我们将重点了解WDFY2的功能和作用机制。我们试图：1)使用免疫分离技术和质谱学来确定WDFY2富含内体的分子组成。2)通过分析转铁蛋白受体内化和循环的动力学，明确WDFY2缺失导致内吞作用抑制的机制。3)确定WDFY2在胰岛素刺激的脂肪细胞葡萄糖摄取中的作用，通过测试Akt2的胰岛素激活是否受损，或者在没有WDFY2的情况下GLUT4的运输是否受到损害。4)通过蛋白质组学和2-杂交方法鉴定与其WD-40基序特异相互作用的蛋白质，确定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.