Background: PLC-β signaling is generally thought to be mediated by allosteric activation by G proteins and Ca2+. Although availability of the phosphatidylinositol-4,5-biphosphate (PIP2) substrate is limiting in some cases, its production has not been shown to be independently regulated as a signaling mechanism. WNK1 protein kinase is known to regulate ion homeostasis and cause hypertension when expression is increased by gene mutations. However, its signaling functions remain largely elusive. Results: Using diacylglycerol-stimulated TRPC6 and inositol trisphosphate-mediated Ca2+ transients as cellular biosensors, we show that WNK1 stimulates PLC-β signaling in cells by promoting the synthesis of PIP2 via stimulation of phosphatidylinositol 4-kinase IIIα. WNK1 kinase activity is not required. Stimulation of PLC-β by WNK1 and by Gαq are synergistic; WNK1 activity is essential for regulation of PLC-β signaling by Gq-coupled receptors, and basal input from Gq is necessary for WNK1 signaling via PLC-β. WNK1 further amplifies PLC-β signaling when it is phosphorylated by Akt kinase in response to insulin-like growth factor. Conclusions: WNK1 is a novel regulator of PLC-β that acts by controlling substrate availability. WNK1 thereby coordinates signaling between G protein and Akt kinase pathways. Because PIP2 is itself a signaling molecule, regulation of PIP2 synthesis by WNK1 also allows the cell to initiate PLC signaling while independently controlling the effects of PIP2 on other targets. These findings describe a new signaling pathway for Akt-activating growth factors, a mechanism for G protein-growth factor crosstalk, and a means to independently control PLC signaling and PIP2 availability.
Bibliographical noteFunding Information:
This study was supported by the National Institutes of Health (GM30355 to E.M.R. and DK59530, DK85726, and DK-079328 to C.-L.H), the Welch Foundation (I-0982 to E.M.R.), and Genzyme (GRIP grant to C.-L.H.). We are particularly indebted to Barbara Barylko and Joseph Albanesi for purification of PI4KIIIβ and for continuing help and advice. We thank Melanie Cobb for anti-WNK1 antibody, Tamas Balla for GFP-PLC-δ1-PH, Melvin Simon for Gα q/11 -null mouse fibroblasts, and Anna Dugas for technical assistance.
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)