MARCKS domain phosphorylation regulates the differential interaction of Diacylglycerol Kinase ζ with Rac1, RhoA and Syntrophin

Abstract

Cells can switch between Rac1, lamellipodia-based and RhoA, blebbing-based migration modes but the molecular mechanisms regulating this choice are not fully understood. Diacylglycerol kinase {zeta} (DGK{zeta}), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from RhoGDI. DGK{zeta} catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation. Instead, DGK{zeta} functions as a scaffold that stimulates RhoA release by enhancing RhoGDI phosphorylation by protein kinase C (PKC). Here, PKC-mediated phosphorylation of the DGK{zeta} MARCKS domain increased DGK{zeta} association with RhoA and decreased its interaction with Rac1. The same modification increased binding of the DGK{zeta} C-terminus to the 1-syntrophin PDZ domain. Expression of a phosphomimetic DGK{zeta} mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGK{zeta} expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the Rho-ROCK pathway. These events were independent of DGK{zeta} catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGK{zeta}-null cells required the PDZ-binding motif, suggesting syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism involving DGK{zeta} phosphorylation by PKC that favours RhoA-driven blebbing over Rac1-driven lamellipodia formation and macropinocytosis. These findings provide a mechanistic basis for the effect of PKC signaling on Rho GTPase activity and suggest PKC activity plays a role in the interconversion between Rac1 and RhoA signaling that underlies different migration modes.