Diacylglycerol kinase ξ regulates RhoA activation via a kinase-independent scaffolding mechanism

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase zeta (DGK zeta), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase C alpha (PKC alpha) selectively releases RhoA. Here we show DGK zeta is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGK zeta-deficient fibroblasts and rescued by wild-type DGK zeta or a catalytically inactive mutant. DGK zeta bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCa and RhoA. DGK zeta-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGK zeta-null cells. Collectively our findings suggest DGK zeta functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGK zeta is a critical factor linking changes in lipid signaling to actin reorganization.