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.
Graphical Abstract
Highlights
► WNK1 protein kinase and the Gq-phospholipase C-β pathway act synergistically in cells ► Maximal activity of either pathway requires the activity of the other ► WNK1 acts by stimulating PI4KIIIα to increase synthesis of the PLC-β substrate PIP2 ► IGF-1 potentiates PLC activity via this mechanism