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

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, DGK alpha and DGK zeta, and there are no isoform-specific inhibitors. Here we used short interfering RNA-mediated gene silencing in human T cells and DGK alpha- and DGK zeta-deficient mice to define DGK isoform-specific regulation of key signaling pathways during T-cell activation. Our results identify DGK zeta as the predominant brake on basal/tonic conditions as well as on downstream T-cell receptor/co-stimulatory signals. DGK zeta 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, DGK zeta 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 DGK zeta contribution as a negative regulator of the crosstalk between phospholipase C-gamma- and phosphoinositide 3-kinase-regulated pathways. This DGK zeta 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.