PIP3-INDEPENDENT REGULATION OF AKT1 ACTIVITY IN T CELLS

描述（由申请人提供）：IP4是新发现的第二信使，我们认为它对T细胞的发育和激活至关重要。IP4如何传递T细胞受体信号诱导胸腺细胞成熟尚不清楚。它与膜脂PIP3的化学相似性表明它共同调节PIP3效应物，包括丝氨酸/苏氨酸激酶AKT。在这项研究中，我们提出AKT是IP4下游的一个重要效应通路，它有助于成熟CD4或CD8 T细胞和调节性T细胞的发育。我们假设IP4通过从膜上释放活性AKT和促进AKT与钙调蛋白结合来促进AKT的活性。钙调蛋白是一种Ca2+感知蛋白功能的调节剂。为了验证这一假设，我们提出了三个具体目标。首先，我们将通过竞争抑制分析分别表征AKT与IP4和PIP3的结合。其次，我们将确定IP4如何调节AKT的定位、激活和下游效应物的磷酸化。最后，我们将在体内研究钙调蛋白结合对AKT功能的重要性，并确定其对IP4的依赖性。我们希望通过阐明IP4调节AKT活性的机制，可以更好地理解发育信号如何精确和差异地诱导AKT激活以促进不同T细胞亚群的成熟。我们的长期目标是表征IP4如何传递来自T细胞表面受体的信号来指导特异性和保护性免疫反应。

DESCRIPTION (provided by applicant): IP4 is a newly discovered second messenger that we identified to be critical for T cell development and activation. How IP4 transmits T cell receptor signals to induce thymocyte maturation remains unclear. Its chemical similarity to the membrane lipid PIP3 suggests that it co-regulates PIP3-effectors, including the serine/threonine kinase AKT. In this grant, we propose that AKT is an important effector pathway downstream of IP4 that contributes to the development of mature CD4 or CD8 T cells and regulatory T cells. We hypothesize that IP4 promotes AKT activity through release of active AKT from the membrane and by facilitating AKT binding to Calmodulin, a Ca2+ sensing regulator of protein function. To test this hypothesis, we present three specific aims. In the first, we will characterize AKT binding to IP4 and PIP3 individually and by competitive inhibition analysis. Second, we will determine how IP4 regulates AKT localization, activation and phosphorylation of downstream effectors. Lastly, we will examine the importance of Calmodulin binding on AKT function in vivo and determine its dependence on IP4. We hope that elucidating the mechanism by which IP4 regulates AKT activity will allow a better understanding of how developmental signals precisely and differentially induce AKT activation to promote the maturation of different T cell subsets. Our long term objective is to characterize how IP4 relays signals from T cell surface receptors to direct specific and protective immune responses. PUBLIC HEALTH RELEVANCE: Itpks generate IP4, a novel second messenger that is required for T cell development and activation, and has also been genetically linked to autoimmunity. This proposal seeks to understand how IP4 regulation of the proto-oncogene AKT controls T cell function.