Diacylglycerol kinase α regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes

Abstract

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.

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

“…DGKα limits exosome secretion in T lymphocytes. 9,14,28 This negative effect correlates with exosome secretion induced by addition of the cell-permeable DAG analog dioctanoyl glycerol. 14 We first assessed the role of PKD in exosome secretion by inhibiting its upstream activator PKC.…”

“…9 2 cells were co-transfected with DsRed2-CD63 and the constructs GFP-PKD1WT, GFP-PKD1KD (kinase-dead) or GFP-PKD1CA (constitutively active). Cells were preincubated alone or with R59949 (R59, 10 μM) and stimulated with CCh (500 μM) to induce exosome secretion.…”

“…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.…”

“…23,24 The secretory vesicle pathway involves several DAGcontrolled checkpoints at which DGKα may act; these include vesicle formation and fission at the trans-Golgi network (TGN), MVB maturation, as well as their transport, docking and fusion to the plasma membrane. 9,[16][17][18][19][20] The molecular components that regulate some of these trafficking processes include protein kinase D (PKD) family members. 21 PKD1 activity, for instance, regulates fission of transport vesicles from TGN via direct interaction with the pre-existing DAG pool at this site.…”

“…Protein kinase C (PKC) phosphorylation at the PKD activation loop further promotes PKD autophosphorylation and activation. 27 Based on our previous studies showing DGKα regulation of DAG in MVB formation and exosome secretion, 9,14,28 and the identification of PKD1/2 association to MVB, 14 we hypothesized that DGKα control of DAG mediates these events, at least in part, through PKD. Here we explored whether, in addition to its role in vesicle fission from TGN, 19 PKD regulates other steps in the DAG-controlled secretory traffic pathway.…”