Mammalian diacylglycerol kinases: Molecular interactions and biological functions of selected isoforms

The mammalian diacylglycerol kinases (DGK) are a group of enzymes having important roles in regulating many biological processes. Both the product and the substrate of these enzymes, i.e. diacylglycerol and phosphatidic acid, are important lipid signalling molecules. Each DGK isoform appears to have a distinct biological function as a consequence of its location in the cell and/or the proteins with which it associates. This review discusses three of the more extensively studied forms of this enzyme, DGKα, DGK?, and DGKζ. DGKα has an important role in immune function and its activity is modulated by several mechanisms. DGK? has several unique features among which is its specificity for arachionoyl-containing substrates, suggesting its importance in phosphatidylinositol cycling. DGKζ is expressed in many tissues and also has several mechanisms to regulate its functions. It is localized in several subcellular organelles, including the nucleus. The current state of our understanding of the properties and functions of these proteins is reviewed.     

Diacylglycerol kinase alpha, from negative modulation of T cell activation to control of cancer progression

Like all other diacylglycerol kinase (DGK) family members, the best-characterized function of DGKα is that of a DAG receptor attenuator. This negative property of DGKα has been demonstrated clearly in T cell receptor (TCR) signaling. Studies in T lymphocytes demonstrate that attenuation of DGKα function is decisive in promoting the transition from an unresponsive/quiescent cell state, thus ensuring proliferation. DGKα might also exert this quiescence-induced function in other cell systems. Microarray studies point to DGKα as a p53 target gene, indicating that this enzyme participates in a cell program that protects against malignant transformation. Contrary to its negative regulation of DAG-mediated cell cycle progression, DGKα is required for growth factor receptor actions that result in cell proliferation, invasiveness and motility. DGKα participation in these mechanisms probably involves its phosphatidic acid (PA)-producing capacity. The dual role of DGKα in cell cycle progression suggests that this enzyme is part of a complex network that might be altered in cancerous states.     

Diacylglycerol kinase ζ: at the crossroads of lipid signaling and protein complex organization

Diacylglycerol (DAG) and phosphatidic acid (PA) are lipids with unique functions as metabolic intermediates, basic membrane constituents, and second-signal components. Diacylglycerol kinases (DGK) regulate the levels of these two lipids, catalyzing the interconversion of one to the other. The DGK family of enzymes is composed of 10 isoforms, grouped into five subfamilies based on the presence of distinct regulatory domains. From its initial characterization as a type IV DGK to the generation of mouse models showing its importance in cardiac dysfunction and immune pathologies, diacylglycerol kinase ζ (DGKζ) has proved an excellent example of the critical role of lipid-metabolizing enzymes in the control of cell responses. Although the mechanism that regulates this enzyme is not well known, many studies demonstrate its subtle regulation and its strategic function in specific signaling and as part of adaptor protein complexes. These data suggest that DGKζ offers new opportunities for therapeutic manipulation of lipid metabolism.     

Diacylglycerol kinase gamma interacts with and activates beta2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor

Diacylglycerol kinase (DGK)gamma was shown to act as an upstream suppressor of Rac1. Here we report that, in COS7 cells stimulated with epidermal growth factor (EGF), DGKgamma specifically interacts and co-localizes at the plasma membrane with beta2-chimaerin, a GTPase-activating protein (GAP) for Rac. Moreover, DGKgamma enhanced EGF-dependent translocation of beta2-chimaerin to the plasma membrane. Interestingly, DGKgamma markedly augmented EGF-dependent GAP activity of beta2-chimaerin through its catalytic action. These results indicate that DGKgamma is a novel regulator of beta2-chimaerin, and thus suggest that beta2-chimaerin is an effector molecule, linking DGKgamma functionally with Rac1.     

Study of arachidonoyl specificity in two enzymes of the PI cycle

We identified a conserved pattern of residues L-X_(3-4)-R-X₍₂₎-L-X₍₄₎-G, in which -X_(n)- is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGK?) and phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K Iα). DGK? is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the motif mentioned above. Mutations of the essential residues in this motif result in the loss of arachidonoyl specificity. Furthermore, DGKα can be converted to an enzyme having this motif by substituting only one residue. When DGKα was mutated so that it gained the motif, the enzyme also gained some specificity for arachidonoyl-containing diacylglycerol. This motif is present also in an isoform of phosphatidylinositol-4-phosphate-5-kinase that we demonstrated had arachidonoyl specificity for its substrate. Single residue mutations within the identified motif of this isoform result in the loss of activity against an arachidonoyl substrate. The importance of acyl chain specificity for the phosphatidic acid activation of phosphatidylinositol-4-phosphate-5-kinase is also shown. We demonstrate that the acyl chain dependence of this phosphatidic acid activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain in enzymes DGKε and PIP5K Iα.     

mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Resistance to anti-estrogen therapy is a major clinical concern in treatment of breast cancer. Estrogen-independent phosphorylation of estrogen receptor α, specifically on Ser167, is one of the contributing causes to development of resistance, and a prognostic marker for the disease. Here, we dissect the signaling pathways responsible for Ser167 phosphorylation. We report that the mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. This cooperation may be targeted in breast cancer treatment by a combination of mTOR and MAPK inhibitors.     

Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

Phospholipid metabolism and nuclear function: Roles of the lipin family of phosphatidic acid phosphatases

Phospholipids play important roles in nuclear function as dynamic building blocks for the biogenesis of the nuclear membrane, as well as signals by which the nucleus communicates with other organelles, and regulate a variety of nuclear events. The mechanisms underlying the nuclear roles of phospholipids remain poorly understood. Lipins represent a family of phosphatidic acid (PA) phosphatases that are conserved from yeasts to humans and perform essential functions in lipid metabolism. Several studies have identified key roles for lipins and their regulators in nuclear envelope organization, gene expression and the maintenance of lipid homeostasis in yeast and metazoans. This review discusses recent advances in understanding the roles of lipins in nuclear structure and function. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Identification of the orphan GPCR, P2Y(10) receptor as the sphingosine-1-phosphate and lysophosphatidic acid receptor

Phylogenetic analysis of transmembrane regions of GPCRs using PHYLIP indicated that the orphan receptor P2Y₁₀ receptor was classified into the cluster consisting nucleotide and lipid receptors. Based on the results, we studied the abilities of nucleotides and lipids to activate the P2Y₁₀ receptors. As a result, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) evoked intracellular Ca²⁺ increases in the CHO cells stably expressing the P2Y₁₀ fused with a G_16α protein. These Ca²⁺ responses were inhibited by S1P receptor and LPA receptor antagonists. The introduction of siRNA designed for P2Y₁₀ receptor into the P2Y₁₀-CHO cells effectively blocked both S1P- and LPA-induced Ca²⁺ increases. RT-PCR analysis showed that the mouse P2Y₁₀ was expressed in reproductive organs, brain, lung and skeletal muscle, suggesting the receptor plays physiological roles throughout the whole body. In conclusion, the P2Y₁₀ receptor is the first receptor identified as a dual lysophospholipid receptor.     

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.     

Phosphatidic acid regulation of phosphatidylinositol 4-phosphate 5-kinases

Phosphatidic acid (PA) production by receptor-stimulated phospholipase D is believed to play an important role in the regulation of cell function. The second messenger function of PA remains to be elucidated. PA can bind and affect the activities of different enzymes and here we summarise the current status of activation of Type I phosphatidylinositol 4-phosphate 5-kinase by PA. Type 1 phosphatidylinositol 4-phosphate 5-kinase is also regulated by ARF proteins as is phospholipase D and we discuss the contributions of ARF and PA towards phosphatidylinositol(4,5)bisphosphate synthesis at the plasma membrane.     

Synthesis of Some O,O-Dialkyl N-[4-(N-heteroarylsulfamoyl)-phenyl]phosphoramidothioates as Potential Fungicides

Fourteen new O, O-dialkyl N-[4-(N-heteroarylsulfamoyl)phosphoramidothioate having thiazoles and oxadiazoles as heteroaryl moieties have been synthesised. They have been tested against two species of fungi and their fungicidal activities have been compared with those of two commercial fungicides, viz., Dithane M-45 and Bavistin.     

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.     

Ceramide 1-phosphate/ceramide, a switch between life and death

Ceramide is a well-characterized sphingolipid metabolite and second messenger that participates in numerous biological processes. In addition to serving as a precursor to complex sphingolipids, ceramide is a potent signaling molecule capable of regulating vital cellular functions. Perhaps its major role in signal transduction is to induce cell cycle arrest, and promote apoptosis. In contrast, little is known about the metabolic or signaling pathways that are regulated by the phosphorylated form of ceramide. It was first demonstrated that ceramide-1-phosphate (C1P) had mitogenic properties, and more recently it has been described as potent inhibitor of apoptosis and inducer of cell survival. C1P and ceramide are antagonistic molecules that can be interconverted in cells by kinase and phosphatase activities. An appropriate balance between the levels of these two metabolites seems to be crucial for cell and tissue homeostasis. Switching this balance towards accumulation of one or the other may result in metabolic dysfunction, or disease. Therefore, the activity of the enzymes that are involved in C1P and ceramide metabolism must be efficiently coordinated to ensure normal cell functioning.     

Phospholipase D- and phosphatidic acid-mediated signaling in plants

The phospholipase D (PLD) family in higher plants is composed of multiple members, and each of the Arabidopsis PLDs characterized displays distinguishable properties in activity regulation and/or lipid preferences. The molecular and biochemical heterogeneities of the plant PLDs play important roles in the timing, location, and amount of phosphatidic acid (PA) produced. PLD-catalyzed production of PA has been shown to play important roles in plant growth, development, and response to various stresses, including drought, salinity, freezing, and nutrient deficiency. PLD and PA affect cellular processes through different modes of action, including direct target protein binding and biophysical effects on cell membranes. Improved knowledge on the mechanism by which specific PLDs and PA mediate given plant responses will facilitate the understanding of the molecular processes that connect the stimulus perception on membranes to intracellular actions and physiological responses.     

Phosphatidic acid as a regulator of matrix metalloproteinase-9 expression via the TNF-alpha signaling pathway

Phosphatidic acid (PA) is implicated in pathophysiological processes associated with cellular signaling events and inflammation, which include the expressional regulation of numerous genes. Here, we show that PA stimulation increases matrix metalloproteinase-9 (MMP-9) expression in macrophages through tumor necrosis factor (TNF)-alpha signaling. We performed antibody array analysis on proteins from macrophages stimulated with PA. PA was found to induce the production of TNF-alpha, but not of TNF receptor (TNFR)1 and TNFR2 in a time-dependent manner and stimulated significant, though delayed, MMP-9 expression. PA induced the phosphorylations of both ERK1/2 and p38, but not of c-jun amino-terminal kinase. Moreover, only ERK1/2 inhibition by U0126 suppressed PA-induced TNF-alpha production and MMP-9 expression. Neutralizing TNF-alpha, TNFR1 or TNFR2 antibodies significantly suppressed PA-induced MMP-9 expression, suggesting that the production of TNF-alpha in response to PA preceded the expression of MMP-9. Moreover, lipopolysaccharide-induced PA also led to TNF-alpha release and resulted in MMP-9 expression. Taken together, these observations suggest that PA may play a role in MMP-9 regulation through ERKs/TNF-alpha/TNFRs-dependent signaling pathway.     

Generation of lysophosphatidylinositol by DDHD domain containing 1 (DDHD1): Possible involvement of phospholipase D/phosphatidic acid in the activation of DDHD1

GPR55 is a seven-transmembrane G-protein-coupled receptor that has been proposed as a novel type of cannabinoid receptor. Previously, we identified lysophosphatidylinositol (LPI), in particular 2-arachidonoyl-LPI, as an agonist for GPR55. In the present study, we examined whether intracellular phospholipase A1 (DDHD domain containing 1, or DDHD1), previously identified as phosphatidic acid (PA)-preferring PLA1 (PA-PLA1), is involved in the formation of 2-arachidonoyl-LPI. HEK293 cells expressing DDHD1 produced [³H]arachidonic acid-containing LPI after prelabeling with [³H]arachidonic acid and subsequent activation by ionomycin; the formation of [³H]LPI was inhibited by n-butanol and the overexpression of an inactive PLD1 mutant PLD1K898R. DDHD1 was translocated from the cytosol to membranes upon ionomycin treatment. A purified recombinant DDHD1 formed [³H]LPI when incubated with [³H]PI; the V_max and apparent K_m were 190 µmol/min/mg protein and 10 mol% PI, respectively. DDHD1 binds PA, and the addition of PA to DDHD1 increased the affinity for PI (K_m ; 3 mol%) and augmented the PI-PLA1 activity. DDHD1 activated by PA was returned to a basal state by its own PA-hydrolytic activity. These results implicate DDHD1 in the formation of 2-arachidonoyl-LPI and indicate that the process is modulated by PA released by phospholipase D. Similar observations for the production of arachidonic acid-containing LPI in neuroblastoma cells suggest the DDHD1-LPI-GPR55 axis to be involved in functions in the brain.     

Enrichment of phosphatidylinositols with specific acyl chains

There are six major species of phospholipids in eukaryotes, each of which plays unique structural and functional roles. One species, phosphatidylinositol (PI) only contributes about 2–10% of the total phospholipid pool. However, they are critical factors in the regulation of several fundamental processes such as in membrane dynamics and signal transduction pathways. Although numerous acyl species exist, PI species are enriched with one specific acyl chain composition at both sn−1 and sn−2 positions. Recent work has identified several enzymes that act on lipids to lead to the formation or interconversion of PI species that exhibit acyl chain specificity. These enzymes contribute to this lipid's enrichment with specific acyl chains. The nature of the acyl chains on signaling lipids has been shown to contribute to their specificity. Here we review some of the critical functions of PI and the multiple pathways in which PI can be produced and metabolized. We also discuss a common motif that may confer arachidonoyl specificity to several of the enzymes involved. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Phospholipids: “Greasing the wheels” of humoral immunity

Phospholipids are major structural components of all cellular membranes. In addition, certain phospholipids execute regulatory activities that affect cell behavior, function and fate in critically important physiological settings. The influence of phospholipids is especially obvious in the adaptive immune system, where these macromolecules mediate both intrinsic and extrinsic effects on B and T lymphocytes. This review article highlights the action of lysophospholipid sphingosine-1-phosphate as a lymphocyte chemoattractant, the function of phosphatidylinositol phosphates as signaling conduits in lymphocytes and the role of phospholipids as raw materials for membrane assembly and organelle biogenesis in activated B lymphocytes. Special emphasis is placed on the means by which these three processes push humoral immune responses forward. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.