Synaptic removal of diacylglycerol by DGKζ and PSD‐95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKζ is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD‐95. Overexpression of DGKζ in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD‐95 binding. Conversely, DGKζ knockdown reduces spine density. Mice deficient in DGKζ expression show reduced spine density and excitatory synaptic transmission. Time‐lapse imaging indicates that DGKζ is required for spine maintenance but not formation. We propose that PSD‐95 targets DGKζ to synaptic DAG‐producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.     

Immunolocalization of Drosophila eye-specific diacylgylcerol kinase,rdgA, which is essential for the maintenance of the photoreceptor

The Drosophila retinal degeneration A (rdgA) mutant has photoreceptor cells that degenerate within a week after eclosion. The degeneration starts with the disruption of the subrhabdomeric cisternae (SRC), which are the organelles essential for the transport of phospholipids to the photoreceptive membranes. Our previous biochemical and molecular studies suggested that the rdgA gene encodes an eye-specific diacylglycerol kinase (DGK). In this study, we show that retinal degeneration is prevented by the introduction of the eye-DGK gene in the rdgA mutant genome, suggesting that the DGK activity is crucial for the maintenance of the photoreceptor. Furthermore, by immunohistochemical analysis, we have demonstrated that the rdgA protein is predominantly associated with the SRC, suggesting that the conversion from diacylglycerol (DG) to phosphatidic acid (PA) most actively occurs in SRC. The analysis of the eyes of mutants homozygous for rdgA and eye-protein kinase C mutations indicates that retinal degeneration is caused by the deficiency of PA rather than excessive accumulation of DG. From these data, we conclude that the production of PA in the SRC membranes is essential for the maintenance of the photoreceptor. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 695–706, 1997     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Diacylglycerol kinase α-selective inhibitors induce apoptosis and reduce viability of melanoma and several other cancer cell lines

Diacylglycerol (DG) kinase (DGK), which phosphorylates DG to generate phosphatidic acid (PA), consists of ten isozymes (α–к). Recently, we identified a novel small molecule inhibitor, CU-3, that selectively inhibits the activity of the α isozyme. In addition, we newly obtained Compound A, which selectively and strongly inhibits type I DGKs (α, β, and γ). In the present study, we demonstrated that both CU-3 and Compound A induced apoptosis (caspase 3/7 activity and DNA fragmentation) and viability reduction of AKI melanoma cells. Liquid chromatography-mass spectrometry revealed that the production of 32:0- and 34:0-PA species was commonly attenuated by CU-3 and Compound A, suggesting that lower levels of these PA molecular species are involved in the apoptosis induction and viability reduction of AKI cells. We determined the effects of the DGKα inhibitors on several other cancer cell lines derived from refractory cancers. In addition to melanoma, the DGKα inhibitors enhanced caspase 3/7 activity and reduced the viability of hepatocellular carcinoma, glioblastoma, and pancreatic cancer cells, but not breast adenocarcinoma cells. Interestingly, Western blot analysis indicated that the DGKα expression levels were positively correlated with the sensitivity to the DGK inhibitors. Because both CU-3 and Compound A induced interleukin-2 production by T cells, it is believed that these two compounds can enhance cancer immunity. Taken together, our results suggest that DGKα inhibitors are promising anticancer drugs.     

Protein kinase C does not regulate diacylglycerol metabolism in aortic smooth muscle cells

Summary The effect of a reduction in protein kinase C activity on the metabolism of exogenous [³H]diC8 by freshly isolated smooth muscle cells from rabbit aorta and cultured A10 smooth muscle cells was determined. The metabolism of [³H]diC8 by both smooth muscle cell preparations was predominantly by hydrolysis to yield monoC8 and glycerol (lipase pathway); very little radioactivity was incorporated into phospholipids. Diacylglycerol lipase activity measured in vitro with A10 cell homogenates was much greater than diacylglycerol kinase activity. The addition of the protein kinase C inhibitor H-7 to incubations of isolated aortic smooth muscle cells and cultured A10 cells had no significant effect on the metabolism of [³H]diC8. Protein kinase C activity in cultured A10 cells preincubated for 20 h with a phorbol ester was reduced to 14% of control as a consequence of down-regulation, but diC8 metabolism was not changed. Therefore, protein kinase C does not regulate the metabolism of diacylglycerols in aortic smooth muscle cells.Abbreviations IP₃ inositol 1,4,5-trisphosphate - DG diacylglycerol - MG monoacylglycerol - PL phospholipid(s) - diC8 dioctanoylglycerol - H-7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - monoC8 monooctanoylglycerol - PS phosphatidylserine - PDBu phorbol 12,13-dibutyrate