Abstract
Phospholipase C-β (PLC-β) isozymes are key effectors in G protein-coupled signaling pathways. Previously, we showed that PLC-β1 and PLC-β3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+-stimulated PLC-β activities using an in vitro reconstitution assay. LY294002, a specific PI 3-kinase inhibitor, significantly inhibited 10 min of agonist-stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 sec of agonist-stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3-kinase catalytic subunit, increased 90 sec of oxytocin-stimulated IP3 accumulation. Receptor-ligand binding assays indicated that LY294002 did not affect G protein-coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC-β activity. When coexpressed with p110CAAX, fluorescence-tagged PLC-β3 was increasingly localized to the plasma membrane. Additional observations suggest that the PH domain of PLC-β is not important for p110CAAX-induced membrane association.
Acknowledgments
This work was supported by a grant from the NIH R01-GM61244 (to T.M.F.) and was made possible, in part, by the Confocal Microscopy Facility of the Environmental Health Sciences Center at Oregon State University, with funding from the National Institute of Environmental Health Sciences, National Institutes of Health grants P30 ES00210 and 1S10RR107903-01. The authors thank Dr. Andrew Henderson (Pennsylvania State University) for providing p110CAAX plasmid and Dr. Craig Montell (Johns Hopkins University) for providing GFP-Akt-PH and GFP-Akt-PH (R25C) plasmids.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.