Phosphatidylinositol 4-phosphate Hydrolysis in Spatiotemporal Cell Signaling

描述(申请人提供)：由G蛋白偶联受体和受体/非受体酪氨酸激酶激活的一个主要的细胞信号转导途径是激活磷脂酰肌醇特异性磷脂酶C(PI-PLC)，刺激磷脂酰肌醇4，5-二磷酸(PIP2)的水解，产生肌醇1，4，5-三磷酸(IP3)和二酰甘油(DAG)。IP3控制体内钙释放，DAG调节蛋白激酶C。我们的实验室最近在心肌细胞中发现了PI-PLC活性的替代底物， 磷脂酰肌醇4-磷酸(PI4P)。PLC对PI4P的水解可产生生物惰性的1，4-二磷酸肌醇(IP2)和DAG。在这个应用中，我们建议研究PI4P作为长期和局部DAG来源的更广泛的作用，这些DAG对于维持分隔的和长期激活的蛋白激酶C和D(PKC和PKD)活性至关重要。蛋白激酶C的激活参与了数百种慢性生理过程。例如，在“PubMed”上搜索蛋白激酶C与癌症，可以找到近10,000篇参考文献。PKD的激活既受PKC的直接调控，又与DAG直接结合，近年来在细胞生理学和病理学中发挥着重要作用。因此，这项提案中概述的实验有可能揭示一种全新的基本信号机制，可能对调节广泛的细胞生理和病理生理产生潜在的影响。这些问题将以以下具体目标来解决：1：PI4P信号通路在细胞中的流行：我们已经在心肌细胞中清楚地证明，内皮素刺激细胞会导致核周高尔基体中PI4P依赖于PI-PLC的耗竭。这些实验的目的是描述和扩展这些观察结果，以确定PI4P水解酶是否在细胞信号转导中起着重要作用。2：PI4P水解酶的调控机制。我们的初步数据提供了强有力的证据表明，激动剂调控的PI4P水解是长期IP甚至DAG产生的主要贡献者，但参与这一过程的信号通路和酶似乎因受体信号机制和细胞类型的不同而不同。在这里，我们将确定调节PI4P水解酶的信号通路的分子参与者。3.PI4P在胰腺癌细胞信号转导、生长、凋亡和高尔基体功能中的作用：PKC激活参与包括肿瘤在内的多种细胞过程和疾病。在已建立的胰腺癌体外模型中，PANC-1细胞、神经降压素能有效刺激依赖PKC和PKD的DNA合成和细胞增殖，抑制PKD抑制胰腺癌细胞的体外生长。在这里，我们将研究PI4P在激活这一有丝分裂信号通路的关键成分中的作用，并将确定其在胰腺细胞有丝分裂中的作用。

DESCRIPTION (provided by applicant): A major cellular signal transduction pathway activated by G protein-coupled receptors and receptor/non- receptor tyrosine kinases is the activation of phosphoinositide-specific phospholipase C (PI-PLC), to stimulate phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and produce inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). IP3 controls calcium release from internal stores and DAG regulates protein kinase C. Our laboratory has recently discovered an alternate substrate for PI-PLC activity in cardiac cells, phosphatidylinositol 4-phosphate (PI4P). Hydrolysis of PI4P by PLC produces inositol 1,4 bisphosphate (IP2) which is biologically inert, and DAG. In this application we propose to investigate the broader role for PI4P hydrolysis as source for long-term and localized DAG critical for maintaining compartmentalized and chronically activated protein kinase C and D (PKC and PKD) activities. Protein Kinase C activation is implicated in hundreds of chronic physiological processes. For example, a "PubMed" search of protein kinase C and cancer reveal close to 10,000 references. PKD activation, directly regulated by both PKC, and direct binding of DAG, has recently emerged as a key player in cell physiology and pathology. Thus the experiments outlined in this proposal have the potential to uncover an entirely new fundamental signaling mechanism with potential implications for regulation of a wide range of cell physiologies and pathophysiologies. These issues will be addressed with the following specific aims: 1: Prevalence of the PI4P signaling pathway in cells: We have clearly demonstrated in cardiac myocytes that stimulation of cells with endothelin leads to PI-PLC- dependent depletion of PI4P in the perinuclear Golgi apparatus. The goal of these experiments is to characterize and extend these observations to determine if PI4P hydrolysis plays a prominent role in cell signaling in general. 2: Mechanism for regulation of PI4P hydrolysis. Our preliminary data provide strong evidence for agonist regulated PI4P hydrolysis as a major contributor to long term IP and perhaps DAG production but the signaling pathways and enzymes involved in this process appear to be different depending on the receptor signaling mechanism and cell type. Here we will identify the molecular participants in the signaling pathways that regulate PI4P hydrolysis. 3: Role of PI4P in pancreatic cancer cell signaling, growth, apoptosis and Golgi function: PKC activation is involved in many cellular processes and diseases including cancer. In an established in vitro model of pancreatic cancer, PANC-1 cells, neurotensin potently stimulated PKC and PKD dependent DNA synthesis and cell proliferation and PKD inhibition prevents pancreatic cancer cell growth in vitro. Here we will examine the role of PI4P hydrolysis in activation of key components of this mitogenic signaling pathway and will determine its role in pancreatic cell mitogenesis.