c Diacylglycerol kinase ␣ (DGK␣) regulates diacylglycerol levels, catalyzing its conversion into phosphatidic acid. The ␣ isoform is central to immune response regulation; it downmodulates Ras-dependent pathways and is necessary for establishment of the unresponsive state termed anergy. DGK␣ functions are regulated in part at the transcriptional level although the mechanisms involved remain poorly understood. Here, we analyzed the 5= end structure of the mouse DGK␣ gene and detected three binding sites for forkhead box O (FoxO) transcription factors, whose function was confirmed using luciferase reporter constructs. FoxO1 and FoxO3 bound to the 5= regulatory region of DGK␣ in quiescent T cells, as well as after interleukin-2 (IL-2) withdrawal in activated T cells. FoxO binding to this region was lost after complete T cell activation or IL-2 addition, events that correlated with FoxO phosphorylation and a sustained decrease in DGK␣ gene expression. These data strongly support a role for FoxO proteins in promoting high DGK␣ levels and indicate a mechanism by which DGK␣ function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGK␣ downregulation, IL-2, and anergy avoidance.T he diacylglycerol kinases (DGK) phosphorylate diacylglycerol (DAG) into phosphatidic acid (PA), modulating the levels of these two lipid second messengers, which have several key functions in cells. DAG propagates signals by membrane recruitment of cytosolic proteins containing C1 domains, such as protein kinase C and D (PKC and PKD, respectively), the Ras-guanine nucleotide exchange factor (GEF) RasGRP1, and the Rac-GTPaseactivating protein (GAP) chimaerins (3). DAG deregulation is linked to tumorigenesis, metastasis, diabetes, heart disease, and altered immune responses (9,13,45,53). PA binds and activates proteins involved in cell growth, survival, vesicular trafficking, and cytoskeletal remodeling, and its altered metabolism is also linked to disease onset (7,14,40). Interest in the DGK as key modulators of DAG and PA function has increased in recent years as better understanding of DGK regulatory mechanisms offers opportunities for the development of novel strategies to modulate lipid metabolism for therapeutic purposes (for reviews, see references 32 and 44).DGK function attracted special attention following the characterization of its role in T lymphocyte activation. Productive activation of T lymphocytes requires the integration of the pathways regulated by Ras/mitogen-activated protein kinase (MAPK)/AP1 and Ca 2ϩ /nuclear factor of activated T cells (NFAT). Failure to trigger an adequate balance of these signals, due, for example, to lack of costimulation, drives T cells into a nonresponsive state termed anergy, in which cells survive for long periods in the absence of proliferation (1). DGK␣ is a type I DGK particularly abundant in thymus and mature T lymphocytes (55), and early studies showed its function as a negative modulator of the Ras/ MAPK pathway. DGK␣ limits...

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.

SummaryConsiderable evidence indicates that diacylglycerol (DAG) generation at the immunological synapse (IS) determines T cell functions by regulating the duration and amplitude of Ras/ERK signals. The exact mechanism by which DAG regulates Ras/ERK activation downstream of the T cell receptor (TCR) nonetheless remains poorly understood. Here we characterize PKCa as a previously unrecognized component of the machinery that translates cell receptor occupancy into Ras/ERK-propagated signals. We show transient translocation of PKCa to the IS, mediated by DAG generation at the contact area. Diacylglycerol kinase (DGK)f negatively regulated PKCa translocation kinetics, whereas PKCa activity limited its own persistence at the IS. Coordinated activation of DGKf and PKCa in response to antigen recognition regulated the amplitude and duration of Ras/ERK activation; this in turn mediated early processes of T cell surface proteolysis such as L-selectin shedding. Analysis of DGKf-deficient mice further showed that increased DAG signaling is translated to downstream elements of this pathway, as reflected by enhanced PKCa-dependent L-selectin shedding. We propose that early activation of a DAG-PKCa axis contributes to the mechanisms by which antigen affinity translates into TCR biological responses.