Predominant contribution of DGKζ over DGKα in the control of PKC/PDK-1-regulated functions in T cells.

Abstract

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.