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.

Public Health Relevance

Hormone receptor driven cellular signaling pathways control basic physiology and can also drive pathophysiology's including heart disease, diabetes, autoimmune diseases and cancer. One of these pathways involved in the metabolism of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate is one of these key receptor driven pathways. Here we will investigate a novel branch of this pathway where phosphatidylinositol 4-phosphate is the key precursor in the pathway that may underlie many fundamental signaling pathways and related physiologies and diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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University of Rochester
Schools of Dentistry
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de Rubio, Rafael Gil; Ransom, Richard F; Malik, Sundeep et al. (2018) Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production. Sci Signal 11:
Kim, Kyun-Do; Bae, Seyeon; Capece, Tara et al. (2017) Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment. Nat Commun 8:15365
Smrcka, Alan V (2015) Regulation of phosphatidylinositol-specific phospholipase C at the nuclear envelope in cardiac myocytes. J Cardiovasc Pharmacol 65:203-10