PI4P Hydrolysis Represents a General Mechanism for DAG Generation and PKC/PKD Activation

University Of Rochester, Rochester NY

Investigators

Abstract

? DESCRIPTION (provided by applicant): Receptor stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is critical for many physiological processes, such as neurotransmission and proliferation. Recently our laboratory identified a novel pathway in neonatal rat ventricular cardiac myocytes (NRVMs), where agonist stimulation triggers PLC dependent hydrolysis of the PIP2 precursor phosphatidylinositol 4-phosphate (PI4P) leading to DAG production, nuclear PKD activation and cardiac hypertrophy. We have seen that this newly identified pathway exists in other cell types as well, such as Mouse Embryo Fibroblasts (MEFs) and PANC-1 cells upon endothelin-1 (ET-1) and neurotensin (NT), respectively. To determine if PLC dependent PI4P hydrolysis represents a general mechanism for DAG generation and PKC/PKD activation we examined PI4P depletion and PKD activation in NRVMs, MEFs and PANC-1 cells. In our studies we used a PI 4-kinase inhibitor, phenylarsine oxide (PAO) or expression of a Golgi specific 4-phosphatase, Sac1-K2A (Sac1) to deplete PI4P. PAO treatment in NRVMs and MEFs, showed a dramatic inhibition of the ET-1-dependent global PKD activation in NRVMs and MEFs but not NT-dependent PKD activation in PANC-1 cells. Sac1 expression showed reduced ET-1-dependent global PKD activation in NRVMs and MEFs but did not change the NT-dependent PKD activation in PANC-1 cells. These results suggest that though Golgi PI4P hydrolysis occurs in multiple cell types, NRVMs and MEFs use PI4P hydrolysis as a source for DAG to promote PKD activation. This shows that PI4P hydrolysis occurs upon receptor stimulation and provides a novel signaling mechanism with numerous implications in physiology and disease. My project seeks to understand the role of PI4P hydrolysis as a source of DAG in cells for better understanding of PLC- and DAG- dependent processes such as ion channel regulation and kinase activation, while providing a new potential target for diseases, such as cardiac hypertrophy and cancer.