Much is known about how T cell receptor (TCR) engagement leads to T cell activation; however, the mechanisms terminating TCR signaling remain less clear. Diacylglycerol, generated after TCR ligation, is essential in T cells. Its function must be controlled tightly to maintain normal T cell homeostasis. Previous studies have shown that diacylglycerol kinase zeta (DGKzeta), which converts diacylglycerol to phosphatidic acid, can inhibit TCR signaling. Here we show that DGKzeta-deficient T cells are hyperresponsive to TCR stimulation both ex vivo and in vivo. Furthermore, DGKzeta-deficient mice mounted a more robust immune response to lymphocytic choriomeningitis virus infection than did wild-type mice. These results demonstrate the importance of DGKzeta as a physiological negative regulator of TCR signaling and T cell activation.

. In peripheral lymphoid organs, engagement of the TCR by antigenic peptide presented by major histocompatibility complex molecules on antigen presenting cells initiates a signaling cascade that is required for T cell proliferation and effector function (1, 5). Abnormalities in signals delivered by the TCR may result in either hypo-or hyperactivation leading to undesirable outcomes such as immune deficiency (6 -9) or autoimmunity (10 -12). Engagement of the TCR initiates numerous second messenger cascades. The most proximal known biochemical signal is stimulation of protein tyrosine kinases with subsequent tyrosine phosphorylation of multiple substrates (5,13,14). Among these substrates are phospholipase C␥1 (PLC␥1) (15-17) and critical adapters including linker of activated T cells (18) and SLP-76 (SH2 domain containing leukocyte phosphoprotein of 76 kDa) (19). These proteins in association with another adapter protein, Grb2-related adapter molecule downstream of , are part of a larger multimolecular complex required for PLC␥1 to act efficiently on its substrate (23-28). Activated PLC␥1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate two second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (29). Inositol 1,4,5-trisphosphate induces an influx of Ca 2ϩ that activates the calcineurinnuclear factor of activated T cells (NFAT) signaling pathway (30). DAG allosterically activates both RasGRP (31, 32), a nucleotide exchange factor for Ras, and PKC (33), a serine (threonine) kinase, through associating with the C1 domains of both molecules. Activated RasGRP (34, 35) and PKC (36, 37) in turn stimulate the Ras-ERK-AP-1 and NFB pathways, respectively. Propagation of these signaling cascades promotes the transcription of numerous genes including those encoding cytokines critical for T cell development, activation, and proliferation. Deficiencies of RasGRP (35) and PKC (37) cause defects in positive selection in developing thymocytes and inefficient activation of peripheral T cells. Similarly, PLC␥1 deficiency (with consequent defects in phosphatidylinositol 4,5-bisphosphate-derived second messenger production) abrogates TCR-mediated cellular activation in the Jurkat T cell

SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa) is an adaptor molecule expressed in all hemopoietic cell lineages except mature B cells and is known to play critical roles in the function of T cells, mast cells, and platelets and in vascular differentiation. Although great progress has been achieved in our understanding of SLP-76 function, little is known about the mechanisms regulating its expression. In this study we report the initial characterization of essential elements that control SLP-76 transcription. We identify several DNase I-hypersensitive sites in the SLP-76 locus, with a prominent site located in its promoter region. This site exists in T cells and monocytic cells, but not in B cells or fibroblasts. Using transient transfection assays, we identify a 507-bp fragment containing the 5′-untranslated region of the first exon and the immediate upstream sequence that confers transcriptional activation in T cells and monocytic cells, but not in B cells. Analysis of the 5′ ends of SLP-76 transcripts reveals differential regulation of SLP-76 transcription initiation between T cells and monocytic cells. Mutational and gel-shift analyses further indicate a critical role within this region for a binding site for Ets family transcription factors. The present study provides the first data to address the mechanisms controlling SLP-76 transcription by providing evidence for several key cis-regulatory elements in the promoter region.