DGKα and DGKζ negatively regulate the DAG/RasGRP1/Ras pathway in T cells. Study of the specific contribution of each isoform to DAG metabolism during immune synapse formation by use of a combination of RNAi and videomicroscopy techniques identifies DGKζ as mainly responsible for DAG consumption at the immunological synapse.

No abstract

Diacylglycerol kinase (DGK)α converts diacylglycerol to phosphatidic acid. This lipid kinase sustains survival, migration and invasion of tumor cells, with no effect over untransformed cells, suggesting its potential as a cancer-specific target. Nonetheless the mechanisms that underlie DGKα specific contribution to cancer survival have not been elucidated. Using three-dimensional (3D) colon and breast cancer cell cultures, we demonstrate that DGKα upregulation is part of the transcriptional program that results in Src activation in these culture conditions. Pharmacological or genetic DGKα silencing impaired tumor growth in vivo confirming its function in malignant transformation. DGKα-mediated Src regulation contributed to limit the effect of Src inhibitors, and its transcriptional upregulation in response to PI3K/Akt inhibitors resulted in reduced toxicity. Src oncogenic properties and contribution to pharmacological resistance have been linked to its overactivation in cancer. DGKα participation in this central node helps to explain why its pharmacological inhibition or siRNA-mediated targeting specifically alters tumor viability with no effect on untransformed cells. Our results identify DGKα-mediated stabilization of Src activation as an important mechanism in tumor growth, and suggest that targeting this enzyme, alone or in combination with other inhibitors in wide clinical use, could constitute a treatment strategy for aggressive forms of cancer.

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...