Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGKα, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGKα-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH2-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acid–mediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGKα exerts on diacylglycerol-derived cell responses. This study is the first to describe DGKα as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.

Multivesicular bodies (MVBs) are endocytic compartments that contain intraluminal vesicles formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these vesicles contain pro-apoptotic Fas ligand (FasL), which may be secreted as 'lethal exosomes' upon fusion of MVBs with the plasma membrane. Diacylglycerol kinase a (DGKa) regulate the secretion of exosomes, but it is unclear how this control is mediated. T-lymphocyte activation increases the number of MVBs that contain FasL. DGKa is recruited to MVBs and to exosomes in which it has a double function. DGKa kinase activity exerts a negative role in the formation of mature MVBs, as we demonstrate by the use of an inhibitor. Downmodulation of DGKa protein resulted in inhibition of both the polarisation of MVBs towards immune synapse and exosome secretion. The subcellular location of DGKa together with its complex role in the formation and polarised traffic of MVBs support the notion that DGKa is a key regulator of the polarised secretion of exosomes. 2 FasL induces crosslinking of the Fas death receptor on the target cell and apoptosis.3 In CTLs, FasL is located at the limiting membrane of secretory lysosomes containing perforin/granzyme with MVB structure.2 Upon T-cell receptor activation (TCR), MVBs undergo fusion with the PM, and relocalisation of FasL to the PM occurs. 2In addition to this transport of FasL, another mechanism for the delivery of FasL co-exists in CTLs. 4 This mechanism is because of the fact that FasL can be sorted from the limiting membrane of the MVBs to the ILVs by inward budding. 4,5 Upon cell activation, the fusion of preexisting MVBs with the PM results in the release of exosomes containing FasL. These authors contributed equally to this work.

Fas ligand (FasL) mediates both apoptotic and inflammatory responses in the immune system. FasL function critically depends on the different forms of FasL; soluble Fas ligand lacking the transmembrane and cytoplasmic domains is a poor mediator of apoptosis, whereas fulllength, membrane-associated FasL (mFasL) is pro-apoptotic. mFasL can be released from T lymphocytes, via the secretion of mFasL-bearing exosomes. mFasL in exosomes retains its activity in triggering Fas-dependent apoptosis, providing an alternative mechanism of cell death that does not necessarily imply cell-to-cell contact. Diacylglycerol kinase ␣ (DGK␣), a diacylglycerol (DAG)-consuming enzyme, is involved in the attenuation of DAG-derived responses initiated at the plasma membrane that lead to T lymphocyte activation. Here we studied the role of DGK␣ on activation-induced cell death on a T cell line and primary T lymphoblasts. The inhibition of DGK␣ increases the secretion of lethal exosomes bearing mFas ligand and subsequent apoptosis. On the contrary, the overactivation of the DGK␣ pathway inhibits exosome secretion and subsequent apoptosis. DGK␣ was found associated with the trans-Golgi network and late endosomal compartments. Our results support the hypothesis that the DGK␣ effect on apoptosis occurs via the regulation of the release of lethal exosomes by the exocytic pathway, and point out that the spatial orchestration of the different pools of DAG (plasma membrane and Golgi membranes) by DGK␣ is crucial for the control of cell activation and also for the regulation of the secretion of lethal exosomes, which in turn controls cell death.

Multivesicular bodies (MVB) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, ILVs are secreted as Fas ligand-bearing, pro-apoptotic exosomes following T cell receptor (TCR)-induced fusion of MVB with the plasma membrane at the immune synapse (IS). In this study we show that protein kinase C δ (PKCδ), a novel PKC isotype activated by diacylglycerol (DAG), regulates TCR-controlled MVB polarization toward the IS and exosome secretion. Concomitantly, we demonstrate that PKCδ-interfered T lymphocytes are defective in activation-induced cell death. Using a DAG sensor based on the C1 DAG-binding domain of PKCδ and a GFP-PKCδ chimera, we reveal that T lymphocyte activation enhances DAG levels at the MVB endomembranes which mediates the association of PKCδ to MVB. Spatiotemporal reorganization of F-actin at the IS is inhibited in PKCδ-interfered T lymphocytes. Therefore, we propose PKCδ as a DAG effector that regulates the actin reorganization necessary for MVB traffic and exosome secretion.

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Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic. Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types. 1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process. 2,3 Two main pathways are involved in MVB maturation. 4,5 In addition to the ESCRT (endosomal complex required for traffic) proteins, 6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA), 7 ceramides 8 and diacylglycerol (DAG) 9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activationinduced cell death (AICD), antigen presentation and intercellular miRNA exchange. [10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,16-21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes. 22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG. 23,24 The secretory vesicle pathway involves several DAGc...

T-lymphocyte activation via the antigen receptor complex (TCR) results in accumulation of p2l1 in the active GTP-bound state. Stimulation of protein kinase C (PKC) can also activate p2l'as, and it has been proposed that the TCR effect on p21r' occurs as a consequence of TCR regulation of PKC. To test the role of PKC in TCR regulation of p2l', a permeabilized cell system was used to examine TCR regulation of p2l1 under conditions in which TCR activation of PKC was blocked, first by using a PKC pseudosubstrate peptide inhibitor and second by using ionic conditions that prevent phosphatidyl inositol hydrolysis and hence diacylglycerol production and PKC stimulation. The data show that TCR-induced p2l1 activation is not mediated exclusively by PKC. Thus, in the absence of PKC stimulation, the TCR was still able to induce accumulation of p2l'-GTP complexes, and this stimulation correlated with an inactivation of p2l' GTPase-activating proteins. The protein tyrosine kinase inhibitor herbimycin could prevent the non-PKCmediated, TCR-induced stimulation of p2l'. These data indicate that two mechanisms for p21l'S regulation coexist in T cells: one PKC mediated and one not. The TCR can apparently couple to p2l1 via a non-PKC-controlled route that may involve tyrosine kinases.The three ras proto-oncogenes, Ha-, Ki-, and N-ras, encode 21,000-molecular-weight guanine nucleotide-binding proteins that cause cell transformation when constitutively activated by point mutation (3). The activity of p2lras is normally regulated by a cycle of binding GTP to give the biologically active form of the protein followed by hydrolysis of bound GTP to GDP (22). The GDP-bound form of p2lras is inactive and is reactivated by exchange of bound GDP for free cytosolic GTP. Recent studies have shown that in T lymphocytes, triggering of the T-cell antigen receptor (TCR), the adhesion molecule CD2, or the interleukin 2 (IL-2) receptor caused a very rapid stimulation of p2lras, as measured by its conversion from the GDP-to the GTP-bound state in activated cells (9,11,12). Ras proteins can also be regulated in fibroblasts by signals generated by the receptors for platelet-derived growth factor, epidermal growth factor, and insulin (5,29,30). In mast cells, p2lras is activated by the cytokines IL-3 and granulocyte macrophage colony-stimulating factor (31).In T lymphocytes, phorbol esters that activate protein kinase C (PKC) can mimic TCR triggering and induce accumulation of p21ras-GTP complexes (9). The TCR is known to stimulate PKC by a mechanism involving the generation of diacylglycerols (DAGs; the endogenous PKC activators) via phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids (PtdIns) (17,27). TCR-stimulated PKC could thus mediate the p2lras activation seen upon triggering of this receptor. The proposal that PKC functions as an upstream regulator of p2lras in T cells is in contrast to the previously described link between p2lras and PKC in fibroblasts, where, based on observations that "active" * Corresponding author.

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubuleorganizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1β phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1β phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1β re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1β regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization.