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PLC‐Dependent Hydrolysis of Phosphatidylinositol 4‐Phosphate is Responsible for the Majority of Total Inositol Phosphate Production and PKC/D Activation Downstream of Multiple GPCRs
Author(s) -
Rubio Rafael Gil,
Malik Sundeep,
Smrcka Alan V
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.816.3
Subject(s) - phospholipase c , phosphatidylinositol , inositol , g protein coupled receptor , microbiology and biotechnology , inositol trisphosphate , phosphatase , inositol phosphate , biology , diacylglycerol kinase , biochemistry , golgi apparatus , phosphatidic acid , protein kinase c , kinase , phosphorylation , receptor , signal transduction , cell , phospholipid , membrane
Receptor activation of phospholipase C (PLC) is important for many physiological processes, including neurotransmission and cell proliferation. PLC enzymes are 6 families of proteins that hydrolyze the plasma membrane lipid, phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) into inositol trisphosphate and diacylglycerol (DAG). Our group identified a novel pathway in cardiac cells, where G protein coupled receptor (GPCR) stimulation leads to PLC‐dependent hydrolysis of the PIP 2 ‐precursor, phosphatidylinositol 4‐phosphate (PI4P) on intracellular Golgi membranes. We proposed that this localized DAG production was essential for nuclear processes involved in cardiac hypertrophy. Our hypothesis is that receptor‐stimulated PI4P hydrolysis expands beyond cardiac cells as a mechanism for DAG production without calcium signaling. We measured PI4P in various cell types using PI4P specific binding domains fused to GFP and found that PI4P was depleted both at the Golgi and plasma membrane (PM) in response to GPCR‐stimulation. Since PI4P is the precursor of PIP 2 , one could argue that depletion of PI4P happens to replenish PIP 2 used by PLC and not as an actual substrate. To test this, we depleted PIP 2 prior to GPCR stimulation. We used the PM‐targeted FRB/FKBP‐5‐phosphatase rapamycin‐induced dimerization system. After rapamycin addition, the PI‐5‐phosphatase is recruited to the PM where it removes the 5 phosphate from the head group of PIP 2 . As predicted, depletion of PIP 2 did not inhibit GPCR‐stimulated PM or Golgi PI4P depletion, total IP production or PKD activation. In contrast, pharmacological inhibition of PI4‐kinases to deplete PI4P specifically at the PM, without affecting PIP 2 levels, completely blocked GPCR‐dependent total IP production and PKD activation. These data strongly suggest that PI4P hydrolysis is the major reaction catalyzed by PLCs in terms of phosphoinositide mass and is a key source of DAG for regulation of protein kinases. While we have identified PI4P hydrolysis in various cell types, it is not clear which PLC isoforms catalyze this reaction. PLC epsilon performs this function at the Golgi‐Nuclear Envelope interface, but all PLC isoforms hydrolyze PI4P in vitro and may participate in PI4P hydrolysis in different cell types. To determine the identity of PLC isoforms responsible for PI4P hydrolysis, we used CRISPR technology to knockout individual PLC isoforms and will assess their roles in total IP production and PKD activation. Ultimately, finding which PLC isoforms are involved may provide new and more specific therapeutic targets for diseases. Support or Funding Information Support from NIH, RO1 grants GM053536 and GM111735 (A.V.S.) and NRSA predoctoral grant F31 GM116557 (R.G.).