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.     

Role of the hydrophobic segment of diacylglycerol kinase epsilon

Diacylglycerol kinase epsilon (DGKepsilon) is unique among mammalian DGK isoforms in having a segment of hydrophobic amino acids. We have evaluated the contributions of this segment to the membrane interactions and functions of this protein. To test the role of the hydrophobic segment, we have compared the properties of DGKepsilon with those of a truncated form of the protein (DGKDeltaepsilon) lacking the 40 N-terminal amino acids, which includes the hydrophobic segment. The proteins were expressed in COS-7 cells from a gene for human DGKepsilon or from a gene for a truncated form (DGKDeltaepsilon), both of which had a FLAG tag at the amino terminus. Full-length FLAG-DGKepsilon and truncated FLAG-DGKDeltaepsilon were both more specific for 1-stearoyl-2-arachidonoyl-sn-glycerol than for 1,2-dioleoyl-sn-glycerol. 1-Stearoyl-2-linoleoyl-sn-glycerol exhibited intermediate specificity for both forms of the enzyme. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increases the catalytic rate constant for all three substrates and may suggest a role in the negative regulation of this enzyme. A full-length DGKepsilon with a C-terminal His tag exhibited substrate specificity similar to that of the other two forms of the enzyme, indicating that the nature and position of the epitope tag did not strongly affect this property. Using an ultracentrifugation floatation assay, we showed that at neutral pH DGKDeltaepsilon is extracted with 1.5 M KCl while DGKepsilon remains essentially fully membrane bound. The full-length protein had a weak tendency to oligomerize in the presence of weak detergents. DGKepsilon was monomeric on SDS-PAGE but exhibited partial dimerization with low concentrations of perfluorooctanoic acid. The major conclusions of this work are that the hydrophobic domain of DGKepsilon does not contribute to substrate specificity but plays a role in permanently sequestering the enzyme to a membrane.     

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.     

Role of the diacylglycerol kinase alpha-conserved domains in membrane targeting in intact T cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.     

Differential activation of protein kinase C isozymes by short chain phosphatidylserines and phosphatidylcholines

To investigate the importance of the physical state of phospholipids for activation of protein kinase C, we have used short chain phospholipids, which, depending on their concentration, can exist as either monomers or micelles. We previously reported that short chain phosphatidylcholines (PC) can activate protein kinase C at concentrations that correlate with the critical micelle concentration of the activating lipid (Walker, J. M., and Sando, J. J. (1988) J. Biol. Chem. 263, 4537-4540). We have now expanded this work to short chain phosphatidylserine (PS) systems in order to examine the role of Ca2(+)-phospholipid interactions in the activation process. Short chain PS were synthesized from corresponding PC and purified by reverse-phase high pressure liquid chromatography. Use of the short chain system has revealed significant differences in the activation of type II and type III protein kinase C isozymes. The type II isozyme required Ca2+ in the presence of long chain PS vesicles; in the presence of the short chain phospholipid micelles (PC or PS), most of the activity was Ca2+ independent. Addition of diacylglycerol caused a small increase in type II activity in all phospholipid systems. In contrast, type III protein kinase C was Ca(+)-dependent in all of the lipid systems. The concentration of Ca2+ required to activate type III protein kinase C was independent of the phospholipid type despite large differences in the ability of these lipids to bind Ca2+. This isozyme required diacylglycerol only in the PC micelle system or with vesicles composed of long chain saturated PS. The presence of short chain PS micelles or long chain PS with unsaturated fatty acyl chains rendered this Ca2(+)-dependent protein kinase C virtually diacylglycerol independent. These results are consistent with a model in which type II protein kinase C requires Ca2+ primarily for membrane association, a requirement which is bypassed with the micelle system, whereas type III protein kinase C has an additional Ca2+ requirement for activity that does not involve Ca2(+)-phospholipid interactions.     

Determination of the topology of the hydrophobic segment of mammalian diacylglycerol kinase epsilon in a cell membrane and its relationship to predictions from modeling

The epsilon isoform of diacylglycerol kinase (DGKepsilon) is unique among mammalian DGKs in having a segment of hydrophobic amino acids comprising approximately residues 20 to 41. Several algorithms predict this segment to be a transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis of the conformational preference of the segment in a hydrophobic environment comprising residues 18 to 42 of DGKepsilon. We find that there are two distinct groups of stable conformations, one corresponding to a straight helix that would traverse the membrane and the second corresponding to a bent helix that would enter and leave the same side of the membrane. Furthermore, the calculations predict that substituting the Pro32 residue in the hydrophobic segment with an Ala will cause the hydrophobic segment to favor a TM orientation. We have expressed the P32A mutant of DGKepsilon, with a FLAG tag (an N-terminal 3xFLAG epitope tag) at the amino terminus, in COS-7 cells. We find that this mutation causes a large reduction in both k(cat) and K(m) while maintaining k(cat)/K(m) constant. Specificity of the P32A mutant for substrates with polyunsaturated acyl chains is retained. The P32A mutant also has higher affinity for membranes since it is more difficult to extract from the membrane with high salt concentration or high pH compared with the wild-type DGKepsilon. We also evaluated the topology of the proteins with confocal immunofluorescence microscopy using NIH 3T3 cells. We find that the FLAG tag at the amino terminus of the wild-type enzyme is not reactive with antibodies unless the cell membrane is permeabilized with detergent. We also demonstrate that at least a fraction of the wild-type DGKepsilon is present in the plasma membrane and that comparable amounts of the wild-type and P32A mutant proteins are in the plasma membrane fraction. This indicates that in these cells the hydrophobic segment of the wild-type DGKepsilon is not TM but takes up a bent conformation. In contrast, the FLAG tag at the amino terminus of the P32A mutant is exposed to antibody both before and after membrane permeabilization. This modeling approach thus provides an explanation, not provided by simple predictive algorithms, for the observed topology of this protein in cell membranes. The work also demonstrates that the wild-type DGKepsilon is a monotopic protein.     

Shaping up the membrane: diacylglycerol coordinates spatial orientation of signaling

Diacylglycerol signals by binding and activating C1 domain-containing proteins expressed principally in neuronal and immune tissues. This restricted expression profile suggests that diacylglycerol-regulated signals are particularly relevant in cell-cell communication processes in which active endocytosis and exocytosis take place. Not surprisingly, various experimental approaches have demonstrated a crucial role for diacylglycerol effectors and metabolizing enzymes in the control of immune responses, neuron communication and phagocytosis. Current research delineates a scenario in which coordinated decoding of diacylglycerol signals is translated into complex biological responses such as neuronal plasticity, T cell development or cytolytic killing. Diacylglycerol functions reach maximal diversity in these highly specialized systems in which signal intensity directly regulates distinct biological outcomes. This review brings together the most recent studies, emphasizing the contribution of compartmentalized DAG metabolism to orientated signaling events.     

Diacylglycerol kinase: a key modulator of signal transduction?

Diacylglycerol kinase (DGK) plays a central role in the metabolism of diacylglycerol released as a second messenger in agonist-stimulated cells. The major purified form of the enzyme (80 kDa DGK) is highly abundant in lymphocyte cytosol and may become membrane-associated via phosphorylation by protein kinase C. In addition, there are several kinase subspecies immunologically distinct from the 80 kDa enzyme, which differ markedly in their responses to several compounds such as sphingosine and R59022. Thus, further work on each enzyme species is needed to define the function of DGK in stimulated cells.     

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.     

Phospholipid synthesis,diacylglycerol compartmentation,and apoptosis

Apoptosis is a means by which organisms dispose of unwanted cells without inducing an inflammatory response. Alterations in apoptosis is a common process by which cells become cancerous. Paradoxically, many cancer chemotherapeutics preferentially kill cancer cells by inducing apoptosis. Diacylglycerol is a lipid second messenger that regulates cell growth and apoptosis and is produced during signal transduction by hydrolysis of membrane phospholipids. Protein kinase Cs are a family of diacyglycerol responsive enzymes that are recruited to cellular membranes as a consequence of diacylglycerol production where they phosphorylate specific target proteins responsible for regulating cell growth. In this review, we will first summarize our current understanding of the role of specific proteins kinase C isoforms in the induction of cell growth/apoptosis. Subsequently, we will discuss how insights gained in lipid-mediated regulation of protein kinase Cs promotes our understanding of the role specific family members play in regulating cell growth. Finally, other diacylglycerol binding proteins involved in regulating apoptosis will be discussed.     

Expression and localization of diacylglycerol kinase isozymes and enzymatic features in rat lung

Diacylglycerol kinase (DGK) catalyzes phosphorylation of diacylglycerol to generate phosphatidic acid, and both molecules are known to serve as second messengers as well as important intermediates for the synthesis of various lipids. In this study, we investigated the spatiotemporal expression patterns of DGK isozymes together with the developmental changes of the mRNA expression and enzymatic property in rat lung. Northern blot and RT-PCR analyses showed that mRNAs for DGKalpha, -epsilon, and -zeta were detected in the lung. By immunohistochemical examination, DGKalpha and -zeta were shown to be coexpressed in alveolar type II cells and macrophages. Interestingly, these isozymes were localized at distinct subcellular locations, i.e., DGKalpha in the cytoplasm and DGKzeta in the nucleus, suggesting different roles for these isozymes. In the developing lung, the expression for DGKalpha and -zeta was transiently elevated on embryonic day 21 (E21) to levels approximately two- to threefold higher than on postnatal day 0 (P0). On the other hand, the expression for DGKepsilon was inversely elevated approximately twofold on P0 compared with that on E21. These unique changes in the expression pattern during the perinatal period suggest that each isozyme may play a distinct role in the adaptation of the lung to air or oxygen breathing at birth.     

Type Ialpha phosphatidylinositol 4-phosphate 5-kinase is a putative target for increased intracellular phosphatidic acid

Despite the fact that phosphatidic acid (PtdOH) has been implicated as a lipid second messenger for nearly a decade, its intracellular targets have remained unclear. We sought to investigate how an increase in the level of PtdOH could modulate phosphatidylinositol 4-phosphate 5-kinase (PIPkin), an enzyme involved in phosphatidylinositol 4,5-bisphosphate synthesis. Transfection of porcine aortic endothelial (PAE) cells with haemagglutinin (HA)-tagged type Ialpha PIPkin followed by immunofluorescence confocal microscopy revealed the enzyme to be localised to the plasma membrane. When the transfected PAE cells were stimulated with lyso-PtdOH, increased PIPkin activity was found to be associated with HA immunoprecipitates in an in vitro assay. This PIPkin activation was found to be greatly reduced by prior treatment of the cells with 1-butanol, thereby implicating phospholipase D (PLD) as the in vivo generator of PtdOH. In order to determine if the PtdOH-dependent activation of type Ialpha PIPkin was dictated by a specific molecular composition of PtdOH, the wild type murine and porcine alpha isoforms of diacylglycerol kinase (DGK) were individually co-transfected along with type Ialpha PIPkin. Under these conditions an increase in type Ialpha PIPkin lipid kinase activity was found in HA immunoprecipitates in an in vitro assay. No increases in lipid kinase activity were observed when type Ialpha PIPkin was co-transfected with either the human DGKepsilon isoform or a kinase-dead mutant of the murine DGKalpha isoform. These results provide the first direct evidence for the unification of the production of saturated/monounsaturated PtdOH (through two different routes, PLD and DGK) and the in vivo activation of type Ialpha PIPkin by this lipid second messenger.     

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.     

Diacylglycerol stimulates phospholipase A2 from Swiss 3T3 fibroblasts

We recently demonstrated that diacylglycerol induced arachidonate release and prostaglandin E2 synthesis in 3T3 fibroblasts, and greatly augmented prostaglandin E2 synthesis in response to submaximal and maximal concentrations of bradykinin. We have now partially purified a phospholipase A2 from the cells. When phosphatidyl[3H]choline was used as substrate, several diacylglycerols augmented phospholipase A2 activity. Diacylglycerol was effective at concentrations as low as 30 nM. Protein kinase C inhibition did not affect diacylglycerol's stimulation of phospholipase A2. Diacylglycerol did not alter the calcium requirement for phospholipase A2 or its pH optimum. The present study demonstrates that the effect of diacylglycerol to augment arachidonate metabolism is at the level of phospholipase A2, itself.     

Evidence for a role of phosphatidylcholine-hydrolysing phospholipase C in the regulation of protein kinase C by ras and src oncogenes.

The products of ras and src oncogenes are thought to be important components in pathways regulating cell proliferation and differentiation. In fibroblasts transformed by these oncogenes, increased diacylglycerol levels have been found which most probably arise from activation of the turnover of phosphatidylcholine. Diacylglycerol is a key activator of protein kinase C whose role in cell growth and transformation has been proposed. We demonstrate here by using immunochemical techniques that transformation by ras or src oncogenes is associated with permanent translocation of protein kinase C to the cytoplasmic membrane. However, no down-regulation of the enzyme is observed despite its permanent activation in these transformants. Importantly, the lack of down-regulation observed in ras and src transformed cell lines is mimicked by chronic treatment of NIH 3T3 fibroblasts with exogenous Bacillus cereus phosphatidylcholine-hydrolysing phospholipase C, but not with phorbol myristate acetate or exogenous Bacillus thuringiensis phosphatidylinositol-hydrolysing phospholipase C. These results strongly suggest that diacylglycerol derived from phosphatidylcholine but not from phosphoinositide turnover is responsible for the atypical regulation of protein kinase C in cell lines transformed by ras and src oncogenes.     

Role of protein kinase C in transmembrane signaling

Many extracellular signals elicit Ca2+ mobilization and diacylglycerol formation in their target cells. Diacylglycerol is derived from the receptor-linked phosphoinositide turnover and serves as a second messenger for the activation of protein kinase C in the presence of Ca2+ and phosphatidylserine. Unique diacylglycerols such as 1-oleoyl-2-acetyl-glycerol, which activate intracellular protein kinase C when added to intact cells, have been synthesized. Tumor-promoting phorbol esters substitute for such diacylglycerols and directly activate protein kinase C in both intact cell and cell-free systems. Under appropriate conditions, the synthetic diacylglycerols and phorbol esters induce protein kinase C activation without Ca2+ mobilization, whereas Ca2+ ionophore A23187 induces Ca2+ mobilization without protein kinase C activation. Using these substances, we have obtained evidence that both protein C and Ca2+ are involved in and play a synergistic role in exocytosis, cell division, and other cellular functions. In this article, the role of protein kinase C in transmembrane signaling is discussed.     

Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone

The smooth muscle cells of resistance arteries depolarize and contract when intravascular pressure is elevated. This is a central characteristic of myogenic tone, which plays an important role in regulation of blood flow in many vascular beds. Pressure-induced vascular smooth muscle depolarization depends in part on the activation of cation channels. Here, we show that activation of these smooth muscle cation channels and pressure-induced depolarization are mediated by protein kinase C in cerebral resistance arteries. Diacylglycerol, phorbol myristate acetate, and cell swelling activate a cation current that we have previously shown is mediated by transient receptor potential channels. These currents, as well as the smooth muscle cell depolarizations of intact arteries induced by diacylglycerol, phorbol ester, and elevation of intravascular pressure, are nearly eliminated by protein kinase C inhibitors. These results suggest a major mechanism of myogenic tone involves mechanotransduction through phospholipase C, diacylglycerol production, and protein kinase C activation, which increase cation channel activity. The associated depolarization activates L-type calcium channels, leading to increased intracellular calcium and vasoconstriction.     

Diacylglycerol kinase-epsilon restores cardiac dysfunction under chronic pressure overload: a new specific regulator of Galpha(q) signaling cascade

Galpha(q) protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in cardiac hypertrophy. DAG kinase (DGK) catalyzes DAG phosphorylation and controls cellular DAG levels, thus acting as a regulator of GPCR signaling. It has been reported that DGKepsilon acts specifically on DAG produced by inositol cycling. In this study, we examined whether DGKepsilon prevents cardiac hypertrophy and progression to heart failure under chronic pressure overload. We generated transgenic mice with cardiac-specific overexpression of DGKepsilon (DGKepsilon-TG) using an alpha-myosin heavy chain promoter. There were no differences in cardiac morphology and function between wild-type (WT) and DGKepsilon-TG mice at the basal condition. Either continuous phenylephrine infusion or thoracic transverse aortic constriction (TAC) was performed in WT and DGKepsilon-TG mice. Increases in heart weight after phenylephrine infusion and TAC were abolished in DGKepsilon-TG mice compared with WT mice. Cardiac dysfunction after TAC was prevented in DGKepsilon-TG mice, and the survival rate after TAC was higher in DGKepsilon-TG mice than in WT mice. Phenylephrine- and TAC-induced DAG accumulation, the translocation of PKC isoforms, and the induction of fetal genes were blocked in DGKepsilon-TG mouse hearts. The upregulation of transient receptor potential channel (TRPC)-6 expression after TAC was attenuated in DGKepsilon-TG mice. In conclusion, these results demonstrate the first evidence that DGKepsilon restores cardiac dysfunction and improves survival under chronic pressure overload by controlling cellular DAG levels and TRPC-6 expression. DGKepsilon may be a novel therapeutic target to prevent cardiac hypertrophy and progression to heart failure.