Diacylglycerol kinase ζ controls diacylglycerol metabolism at the immunological synapse

Diacylglycerol (DAG) generation at the T cell immunological synapse (IS) determines the correct activation of antigen-specific immune responses. DAG kinases (DGKs) alpha and zeta act as negative regulators of DAG-mediated signals by catalyzing DAG conversion to phosphatidic acid (PA). Nonetheless, the specific input of each enzyme and their spatial regulation during IS formation remain uncharacterized. Here we report recruitment of endogenous DGK alpha and DGK zeta to the T cell receptor (TCR) complex following TCR/CD28 engagement. Specific DGK gene silencing shows that PA production at the activated complex depends mainly on DGK zeta, indicating functional differences between these proteins. DGK zeta kinase activity at the TCR is enhanced by phorbol-12-myristate-13-acetate cotreatment, suggesting DAG-mediated regulation of DGK zeta responsiveness. We used GFP-DGK zeta and -DGK alpha chimeras to assess translocation dynamics during IS formation. Only GFP-DGK zeta translocated rapidly to the plasma membrane at early stages of IS formation, independent of enzyme activity. Finally, use of a fluorescent DAG sensor confirmed rapid, sustained DAG accumulation at the IS and allowed us to directly correlate membrane translocation of active DGK zeta with DAG consumption at the IS. This study highlights a DGK zeta-specific function for local DAG metabolism at the IS and offers new clues to its mode of regulation.