Diacylglycerol kinase α (DGKα) regulates diacylglycerol levels catalyzing its conversion into phosphatidic acid. a mechanism by which DGKα function is downregulated during productive T cell responses. Our study establishes a basis for a causal relationship between DGKα downregulation IL-2 and anergy avoidance. INTRODUCTION The 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 ZM 336372 (PKC and PKD respectively) the Ras-guanine nucleotide exchange factor (GEF) RasGRP1 and the Rac-GTPase-activating protein (GAP) Mouse monoclonal to CD3E 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 ZM 336372 (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 Ca2+/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). DGKα 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. DGKα limits DAG-mediated membrane localization and activation of the Ras GEF RasGRP1 following T cell receptor (TCR) triggering and is subjected to precise transcriptional regulation throughout T cell activation. Naive T cells express ZM 336372 high DGKα levels which diminish rapidly following T cell encounter with antigen-presenting cells (47). DGKα downregulation permits adequate DAG-mediated activation of the RasGRP1/Ras/MAPK/AP1 pathway essential for productive T cell responses. as an anergy-induced gene and its acute downregulation during T cell activation the basic mechanisms that regulate expression in T lymphocytes remain unknown. The earliest attempt to dissect expression is not regulated by NFAT regardless of the critical role of this transcription factor in the control of other anergy-induced genes (49). Here we report the initial characterization of the 5′-end structure of ZM 336372 the mouse DGKα (mDGKα) gene. Analysis of this region revealed several conserved binding sites for various transcription factors and suggests the presence of at least two putative alternative promoters. DGKα mRNA levels are high in quiescent lymphocytes but decrease after TCR activation. We observed that the magnitude and duration of this decrease correlated with the intensity of activation that they were enhanced by costimulation and that they were further maintained by interleukin-2 (IL-2) addition. Our data strongly support the concept that elevated expression in quiescent nonactivated cells is regulated by three FoxO-binding sites identified at the distal 5′ end region of the gene and conserved in mammals. Our studies identify a mechanism in T cells by which DGKα function is downstream of the AKT/FoxO axis. This mechanism provides a plausible explanation for the causal relation between “weak” TCR stimulation and anergy induction and the capacity of IL-2 to rescue anergic cells. MATERIALS AND METHODS Mice tissue preparation cell lines and cell culture. Mouse tissues were isolated from 6- to 12-week-old BALB/c or C57BL/6J mice according to protocols approved by the CNB/CSIC Ethics Committee on Animal Experimentation. C57BL/6 Y660) (Ambion) as a negative control and primer d located in exon 1 of the mDGKα transcript ({“type”:”entrez-nucleotide” attrs.