No abstract

DGKs (diacylglycerol kinases) are members of a unique and conserved family of intracellular lipid kinases that phosphorylate DAG (diacylglycerol), catalysing its conversion into PA (phosphatidic acid). This reaction leads to attenuation of DAG levels in the cell membrane, regulating a host of intracellular signalling proteins that have evolved the ability to bind this lipid. The product of the DGK reaction, PA, is also linked to the regulation of diverse functions, including cell growth, membrane trafficking, differentiation and migration. In multicellular eukaryotes, DGKs provide a link between lipid metabolism and signalling. Genetic experiments in Caenorhabditis elegans, Drosophila melanogaster and mice have started to unveil the role of members of this protein family as modulators of receptor-dependent responses in processes such as synaptic transmission and photoreceptor transduction, as well as acquired and innate immune responses. Recent discoveries provide new insights into the complex mechanisms controlling DGK activation and their participation in receptor-regulated processes. After more than 50 years of intense research, the DGK pathway emerges as a key player in the regulation of cell responses, offering new possibilities of therapeutic intervention in human pathologies, including cancer, heart disease, diabetes, brain afflictions and immune dysfunctions.

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.

6 Corresponding author p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

The delicate balance between endocytosis and recycling of the cell surface receptors (NMDAR and AMPAR) is essential for controlling their surface levels and degradation, and is regulated by numerous processes including lateral membrane diffusion, scaffolding protein interactions and posttranslational modifications. Generally the NMDARs undergo activity-dependent endocytosis within clathrin-coated vesicles. They then enter the endosomal system where they are either sorted into the degradative lysosomal pathway, or are replenished via endosomal recycling. Defects in endosomal trafficking therefore lead to perturbed homeostasis of NMDARs. Our recent findings provide a comprehensive understanding of how post-translational modifications of NMDAR define an extended electrostatic peptide code for cargo sorting and influence their interactions with the trafficking machinery. Currently, I am trying to understand the mechanistic basis of intracellular trafficking in NMDAR receptor homeostasis. In my talk, I will be discussing about some of our efforts in the basic studies of the structure and function of SNX27, a unique member of PX-FERM module, that control membrane trafficking. Additionally, I will highlight the novel role for phosphorylation of the NMDARs in promoting SNX27-retromer interactions, which may have significant implications for activity-dependent trafficking of NMDARs during synaptic potentiation.

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.

Proliferation of T lymphocytes is triggered by the interaction of interleukin 2 (IL-2) with its high affinity specific receptor that is expressed on the cell surface following T lymphocyte activation. Significant advances have recently been made in identifying the multiple signals that follow IL-2 receptor occupancy, although the exact mechanism responsible for IL-2-induced proliferation remains an enigma. It has been shown previously that unique species of phosphatidic acid are rapidly produced in vivo following IL-2 binding. It was then suggested that, in contrast to other eukaryotic growth factor systems, phosphatidic acid was at least in part generated through IL-2-induced diacylglycerol (DG) kinase activation. In the present study we demonstrate IL-2-dependent activation of the alpha isoform of DG kinase. Confocal microscopy studies reveal that the enzyme is located in the cytosol and nuclei of resting T cells. Interleukin 2 stimulation induces translocation of the enzyme to the perinuclear region. Furthermore, our results indicate that inhibition of the alpha isoform of DG kinase has a profound effect on IL-2-induced T cell growth. Studies on cell cycle distribution demonstrate that the inhibition of IL-2-induced phosphatidic acid production induces arrest in late G1 phase of IL-2 dependent cells. Altogether, these results link previous observations of interleukin 2 and phosphatidic acid production to activation of an specific isoform of DG kinase and suggest that activation of this enzyme is part of a novel signaling cascade that utilizes phosphatidic acid as an effector molecule.