Diacylglycerol kinase alpha produced phosphatidic acid controls myosin light chain kinase localization and activity in HGF stimulated epithelial cells.

Diacylglycerol kinases (DGKs) regulate lipid signaling by phosphorylating diacylglycerol to phosphatidic acid (PA). Membrane recruitment and activation of DGK-alpha (DGKA) is required for cell proliferation, migration and matrix invasion induced by growth factor, chemokines and oncogenes in epithelial and endothelial cells. Through a proteomic approach we identified 76 proteins associated to tyrosine phosphorylated DGKA in Kaposi sarcoma cells. Among these proteins we selected non-muscle myosin light chain kinase (MLCK), for further characterization. MLCK is a regulator of acto-myosin driven contraction, which phosphorylates myosin light chain (MLC). DGKA co-immunoprecipitates with both long (non-muscle) and short (smooth muscle) MLCK isoforms, consistently with the interaction observed with the proteomic strategy. Inhere we show that in epithelial cells: (i) HGF-induced activation of DGKA produces PA at the nascent ruffle; (ii) DGKA-mediated PA generation is necessary and sufficient to recruit MLCK at ruffling site were MLCK promotes ruffle extension; (iii) expression of a constitutively active and membrane-bound DGKA mutant induces ruffles formation by recruiting MLCK at plasma membrane and promoting MLCK-mediated MLC phosphorylation; (iv) expression of constitutively active MLCK is sufficient to lead to ruffle assembly independently from DGKA activity. Altogether those data indicate that PA produced by HGFinduced activation of DGKA, is a novel regulator of the localization and the activity of MLCK required for ruffle formation in epithelial cells