Diacylglycerol kinases as sources of phosphatidic acid

There are ten mammalian diacylglycerol kinases (DGKs) whose primary role is to terminate diacylglycerol (DAG) signaling. However, it is becoming increasingly apparent that DGKs also influence signaling events through their product, phosphatidic acid (PA). They do so in some cases by associating with proteins and then modifying their activity by generating PA. In other cases, DGKs broadly regulate signaling events by virtue of their ability to provide PA for the synthesis of phosphatidylinositols (PtdIns).     

Signaling roles of diacylglycerol kinases

Diacylglycerol kinases (DGKs) attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid (PA). The nine mammalian DGKs that have been identified are widely expressed, but each isoform has a unique tissue and subcellular distribution. Their kinase activity is regulated by mechanisms that modify their access to diacylglycerol, directly affect their kinase activity, or alter their ability to bind to other proteins. In many cases, these enzymes regulate the activity of proteins that are modulated by either diacylglycerol or PA. Experiments using cultured cells and model organisms have demonstrated that DGKs have prominent roles in neuronal transmission, lymphocyte signaling, and carcinogenesis.     

The hydrophobic segment of Arabidopsis thaliana cluster I diacylglycerol kinases is sufficient to target the proteins to cell membranes

Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for epsilon type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.     

Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.     

Properties and functions of diacylglycerol kinases

Diacylglycerol kinases (DGKs) phosphorylate the second-messenger diacylglycerol (DAG) to phosphatidic acid (PA). The family of DGKs is well conserved among most species. Nine mammalian isotypes have been identified, and are classified into five subgroups based on their primary structure. DGKs contain a conserved catalytic domain and an array of other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signalling pathways dependent on DAG and/or PA production. DGK is therefore believed to be activated at the (plasma) membrane where DAG is generated. Some isotypes are found associated with and/or regulated by small GTPases of the Rho family, presumably acting in cytoskeletal rearrangements. Others are (also) found in the nucleus, in association with other regulatory enzymes of the phosphoinositide cycle, and have an effect on cell cycle progression. Most DGK isotypes show high expression in the brain, often in distinct brain regions, suggesting that each individual isotype has a unique function.     

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (～2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.     

Diacylglycerol kinase zeta regulates phosphatidylinositol 4-phosphate 5-kinase Ialpha by a novel mechanism

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays an important role during actin polymerization and is produced by the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KI), which are activated by phosphatidic acid (PA). As diacylglycerol kinases (DGKs) generate PA by phosphorylating diacylglycerol (DAG), we investigated whether DGKs were involved in controlling PIP2 levels by regulating PIP5KI activity. Here we show that expression of DGKzeta significantly enhances PIP5KIalpha activity in thrombin-stimulated HEK293 cells, and DGK activity is required for this stimulation. We also observed that DGKzeta co-immunoprecipitated and co-localized with PIP5KIalpha, suggesting that they reside in a regulated signaling complex. To explore the role of DGKzeta in actin polymerization, we examined the subcellular distribution of DGKzeta, PIP5KIalpha and actin, and found that these proteins co-localized with actin in lamellipodial protrusions. Supporting that PIP5KIalpha regulation occurs at the sites of actin polymerization, we found that PIP2 also accumulated in the actin-rich regions of lamellipodia. Significantly, in wounding assays, DGKzeta, PIP5KIalpha and PIP2 accumulated at the leading edge of migrating A172 cells, where massive actin polymerization is known to occur. Combined, these data support a novel function for DGKzeta: by generating PA, it stimulates PIP5KIalpha activity to increase local PIP2, which regulates actin polymerization.     

Nuclear expression of diacylglycerol kinases: possible involvement in DNA replication

The existence of intranuclear lipid-dependent signal transduction systems has been demonstrated by several independent groups. Remarkably, intranuclear lipid-dependent signal transduction pathways are regulated independently from their membrane/cytosolic counterparts. A sizable body of evidence suggests that nuclear lipid signaling controls critical biological functions such as cell proliferation, differentiation, and apoptosis. Diacylglycerol (DG) is a fundamental lipid second messenger which is produced in the nucleus. Since the levels of nuclear DG fluctuate during the cell cycle progression, it has been suggested that this lipid second messenger has important regulatory roles. Most likely, nuclear DG serves as a chemoattractant for some isoforms of protein kinase C that migrate to the nucleus in response to a variety of agonists. The nucleus also contains diacylglycerol kinases (DGKs), i.e. the enzymes that, by converting DG into phosphatidic acid (PA), terminate DG-dependent events. This review aims at highlighting the different isozymes of DGKs present within the nucleus as well as at discussing their potential functions with particular emphasis placed on DNA replication.     

Regulation and roles of neuronal diacylglycerol kinases: a lipid perspective

Diacylglycerol kinases (DGKs) are a class of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG) to phosphatidic acid (PtdOH), resulting in the coordinate regulation of these two lipid second messengers. This regulation is particularly important in the nervous system where it is now well-established that DAG and PtdOH serve very important roles in modulating a variety of neurological functions. There are currently 10 identified mammalian DGKs, organized into five classes or "Types" based upon similarities in their primary sequences. A number of studies have identified eight of these isoforms in various regions of the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-?, and DGK-θ. Further studies have provided compelling evidence supporting roles for these enzymes in neuronal spine density, myelination, synaptic activity, neuronal plasticity, epileptogenesis and neurotransmitter release. The physiological regulation of these enzymes is less clear. Like all interfacial enzymes, DGKs metabolize their hydrophobic substrate (DAG) at a membrane-aqueous interface. Therefore, these enzymes can be regulated by alterations in their subcellular localization, enzymatic activity, and/or membrane association. In this review, we summarize what is currently understood about the localization and regulation of the neuronal DGKs in the mammalian CNS.     

Inhibitors of 2,3-Oxidosqualene Cyclase as Tools for Studying the Mechanism and Function of the Enzyme

Diacylglycerol kinases (DGKs) are a class of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG) to phosphatidic acid (PtdOH), resulting in the coordinate regulation of these two lipid second messengers. This regulation is particularly important in the nervous system where it is now well-established that DAG and PtdOH serve very important roles in modulating a variety of neurological functions. There are currently 10 identified mammalian DGKs, organized into five classes or “Types” based upon similarities in their primary sequences. A number of studies have identified eight of these isoforms in various regions of the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-?, and DGK-θ. Further studies have provided compelling evidence supporting roles for these enzymes in neuronal spine density, myelination, synaptic activity, neuronal plasticity, epileptogenesis and neurotransmitter release. The physiological regulation of these enzymes is less clear. Like all interfacial enzymes, DGKs metabolize their hydrophobic substrate (DAG) at a membrane-aqueous interface. Therefore, these enzymes can be regulated by alterations in their subcellular localization, enzymatic activity, and/or membrane association. In this review, we summarize what is currently understood about the localization and regulation of the neuronal DGKs in the mammalian CNS.     

Phosphatidic acid: an emerging plant lipid second messenger

Evidence is accumulating that phosphatidic acid is a second messenger. Its level increases within minutes of a wide variety of stress treatments including ethylene, wounding, pathogen elicitors, osmotic and oxidative stress, and abscisic acid. Enhanced signal levels are rapidly attenuated by phosphorylating phosphatidic acid to diacylglycerol pyrophosphate. Phosphatidic acid is the product of two signalling pathways, those of phospholipases C and D, the former in combination with diacylglycerol kinase. Families of these genes are now being cloned from plants. Several downstream targets of phosphatidic acid have been identified, including protein kinases and ion channels.     

Nuclear transportation of diacylglycerol kinase gamma and its possible function in the nucleus

Diacylglycerol kinases (DGKs) convert diacylglycerol (DG) to phosphatidic acid, and both lipids are known to play important roles in lipid signal transduction. Thereby, DGKs are considered to be a one of the key players in lipid signaling, but its physiological function remains to be solved. In an effort to investigate one of nine subtypes, we found that DGKgamma came to be localized in the nucleus with time in all cell lines tested while seen only in the cytoplasm at the early stage of culture, indicating that DGKgamma is transported from the cytoplasm to the nucleus. The nuclear transportation of DGKgamma didn't necessarily need DGK activity, but its C1 domain was indispensable, suggesting that the C1 domain of DGKgamma acts as a nuclear transport signal. Furthermore, to address the function of DGKgamma in the nucleus, we produced stable cell lines of wild-type DGKgamma and mutants, including kinase negative, and investigated their cell size, growth rate, and cell cycle. The cells expressing the kinase-negative mutant of DGKgamma were larger in size and showed slower growth rate, and the S phase of the cells was extended. These findings implicate that nuclear DGKgamma regulates cell cycle.     

Diacylglycerol Kinase Inhibition and Vascular Function

Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP(3)), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.     

Analysis of a novel diacylglycerol kinase from Dictyostelium discoideum: DGKA

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).     

Nuclear diacylglycerol kinases: emerging downstream regulators in cell signaling networks

There exists an active lipid metabolism in the nucleus, which is regulated differentially from the lipid metabolism taking place elsewhere in the cell. Evidence has been accumulated that nuclear lipid metabolism is closely involved in a variety of cell responses, including proliferation, differentiation, and apoptosis. A fundamental lipid second messenger which is generated in the nucleus is diacylglycerol, that is mainly known for its role as an activator of some protein kinase C isoforms. Diacylglycerol kinases attenuate diacylglycerol signaling by converting this lipid to phosphatidic acid, which also has signaling functions. Ten mammalian diacylglycerol kinase isoforms have been cloned so far, and some of them are found also in the nucleus, either as resident proteins or after migration from cytoplasm in response to various agonists. Experiments using cultured cells have demonstrated that nuclear diacylglycerol kinases have prominent roles in cell cycle regulation and differentiation. In this review, the emerging roles played by diacylglycerol kinases in the nucleus, such as the control of G1/S phase transition, are discussed.     

Domains, amino acid residues, and new isoforms of Caenorhabditis elegans diacylglycerol kinase 1 (DGK-1) important for terminating diacylglycerol signaling in vivo

Diacylglycerol kinases (DGKs) inhibit diacylglycerol (DAG) signaling by phosphorylating DAG. DGK-1, the Caenorhabditis elegans ortholog of human neuronal DGK, inhibits neurotransmission to control behavior. DGK-1, like DGK, has three cysteine-rich domains (CRDs), a pleckstrin homology domain, and a kinase domain. To identify DGK domains and amino acid residues critical for terminating DAG signaling in vivo, we analyzed 20 dgk-1 mutants defective in DGK-1-controlled behaviors. We found by sequencing that the mutations included nine amino acid substitutions and seven premature stop codons that impair the physiological functions of DGK-1. All nine amino acid substitutions are in the second CRD, the third CRD, or the kinase domain. Thus, these domains are important for the termination of DAG signaling by DGK-1 in vivo. Seven of the substituted amino acid residues are present in all human DGKs and likely define key residues required for the function of all DGKs. An ATP-binding site mutation expected to inactivate the kinase domain retained very little physiological function, but we found two stop codon mutants predicted to truncate DGK-1 before its kinase domain that retained significantly more function. We detected novel splice forms of dgk-1 that can reconcile this apparent conflict, as they skip exons containing the stop codons to produce DGK-1 isoforms that contain the kinase domain. Two of these isoforms lack an intact pleckstrin homology domain and yet appear to have significant function. Additional novel isoform(s) account for all of the DGK-1 function necessary for one behavior, dopamine response.     

Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30

Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy.     

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.