Phosphatidylinositol 4-Phosphate Hydrolysis in Spatiotemporal Cell Signaling

描述（由申请人提供）：由G蛋白偶联受体和受体/非受体酪氨酸激酶激活的主要细胞信号转导途径是激活磷酸肌醇特异性磷脂酶C（PI-PLC），以刺激磷脂酰肌醇4，5-二磷酸（PIP 2）水解并产生肌醇1，4，5三磷酸（IP 3）和二酰基甘油（DAG）。IP 3控制钙从内部储存的释放，DAG调节蛋白激酶C。我们的实验室最近发现了心脏细胞中PI-PLC活性的替代底物， 磷脂酰肌醇4-磷酸（PI 4P）。通过PLC水解PI 4P产生生物惰性的肌醇1，4二磷酸（IP 2）和DAG。在本申请中，我们建议研究PI 4P水解作为长期和局部DAG的来源的更广泛的作用，这些DAG对于维持区室化和慢性活化的蛋白激酶C和D（PKC和PKD）活性至关重要。蛋白激酶C的激活与数百种慢性生理过程有关。例如，“PubMed”对蛋白激酶C和癌症的搜索显示了近10，000篇参考文献。PKD的活化，直接受PKC和DAG的直接结合调节，最近已经成为细胞生理学和病理学中的关键参与者。因此，本提案中概述的实验有可能揭示一种全新的基本信号传导机制，对广泛的细胞生理学和病理生理学的调节具有潜在影响。这些问题将通过以下具体目标来解决：1：细胞中PI 4P信号传导途径的流行：我们已经清楚地证明，在心肌细胞中，内皮素刺激细胞导致核周高尔基体中PI 4P的PI-PLC依赖性耗尽。这些实验的目的是表征和扩展这些观察结果，以确定PI 4P水解是否在细胞信号传导中起重要作用。2：调节PI 4P水解的机制。我们的初步数据为激动剂调节的PI 4P水解作为长期IP和可能的DAG产生的主要贡献者提供了强有力的证据，但该过程中涉及的信号传导途径和酶似乎取决于受体信号传导机制和细胞类型而不同。在这里，我们将确定参与调节PI 4P水解的信号通路的分子。第三章：PI 4 P在胰腺癌细胞信号传导、生长、细胞凋亡和高尔基体功能中的作用：PKC激活参与许多细胞过程和疾病，包括癌症。在已建立的胰腺癌体外模型中，PANC-1细胞、神经降压素有效地刺激PKC和PKD依赖性DNA合成和细胞增殖，并且PKD抑制在体外防止胰腺癌细胞生长。在这里，我们将研究PI 4P水解的作用，在激活的关键组成部分，这一有丝分裂信号通路，并将确定其在胰腺细胞有丝分裂的作用。

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.