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'-磷酸肌苷的形成而起作用。它们的功能受损会导致癌症和2型糖尿病等疾病，因此确定3'-磷酸肌苷的直接效应对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结合具有高亲和力和特异性，Ptdlns(3)P是内体中高度富集的3'-磷酸肌肽（Patki et al. (1998), Nature 394: 433）。人类基因组中大约有40种蛋白质含有FYVE结构域，这意味着它们在信号转导和膜运输中具有重要功能。因此，例如，含有SARA蛋白的fyve结构域是TGFp向Smad2发出信号所必需的，而Smad2发生在早期核内体中(Hayes et al.（2003）。生物学报，58(7):1239- 1249。为了加速鉴定含FYVE结构域蛋白的功能，我们在秀丽隐杆线虫中使用了siRNA筛选。利用揭示内吞缺陷的菌株，我们已经确定了一种高度保守的，以前未表征的内吞作用所必需的蛋白质，称为WDFY2。它的哺乳动物同源物无处不在地表达，并且从培养细胞中去除它会导致明显的转铁蛋白摄取抑制。此外，WDFY2存在于距离质膜100 nm范围内的囊泡中，这些囊泡缺乏典型的早期内吞标记物，从而定义了以前未被识别的一类早期内体。此外，WDFY2的缺失抑制胰岛素刺激的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的缺失相似。研究发现WDFY2与Akt之间存在直接相互作用，这表明WDFY2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WDFY2在哺乳动物细胞的这些基本过程中具有潜在的核心作用，在这一竞争性提案中，我们将重点了解WDFY2的功能和作用机制。我们寻求：1)利用免疫分离技术和质谱技术确定富含wdfy2的核内体的分子组成。2)通过分析转铁蛋白受体内化和循环的动力学，明确WDFY2耗竭导致内吞作用抑制的机制。3)通过检测Akt2的胰岛素激活是否受损，或者在缺乏WDFY2的情况下GLUT4运输是否受损，确定WDFY2在胰岛素刺激脂肪细胞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WDFY2的WD-40基序特异性相互作用的蛋白，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.