LAUSR

Diacylglycerol kinase (DGK)‐mediated consumption of the diacylglycerol (DAG) generated in response to antigen recognition is an important mechanism to limit T‐cell function. Targeting DGK activity presents new opportunities for therapeutic manipulation of the immune response, but assessment of individual DGK functions is complex. T cells express two DGK isoforms, DGKα and DGKζ, and there are no isoform‐specific inhibitors. Here we used short interfering RNA‐mediated gene silencing in human T cells and DGKα‐ and DGKζ‐deficient mice to define DGK isoform‐specific regulation of key signaling pathways during T‐cell activation. Our results identify DGKζ as the predominant brake on basal/tonic conditions as well as on downstream T‐cell receptor/co‐stimulatory signals. DGKζ silencing triggers basal RasGTP activation and facilitates enhanced membrane stability of protein kinase C alpha as well as increased activity of AGC kinases. Downstream of T‐cell receptor/co‐stimulation, DGKζ silencing results in enhanced and maintained recruitment of PKC theta to the membrane, as well as phosphoinositide‐dependent protein kinase‐1 activation and scaffolding functions. Our studies identify a previously unrecognized DGKζ contribution as a negative regulator of the crosstalk between phospholipase C‐gamma‐ and phosphoinositide 3‐kinase‐regulated pathways. This DGKζ input helps to explain previous observations in DGK‐deficient mice and suggests that the development of isoform‐specific DGK inhibitors is of great interest for the manipulation of distinct aspects of T‐cell responses.