Correspondence to: Isabel Mérida, Dept. of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientifícas, Cantoblanco, E-28049 Madrid, Spain. Tel:34-91-585-4665 Fax:34-91-372-0493 E-mail:imerida{at}cnb.uam.es.
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Abstract |
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Diacylglycerol kinase (DGK) is suggested to attenuate diacylglycerol-induced cell responses through the phosphorylation of this second messenger to phosphatidic acid. Here, we show that DGK, an isoform highly expressed in T lymphocytes, translocates from cytosol to the plasma membrane in response to two different receptors known to elicit T cell activation responses: an ectopically expressed muscarinic type I receptor and the endogenous T cell receptor. Translocation in response to receptor stimulation is rapid, transient, and requires calcium and tyrosine kinase activation. DGK
-mediated phosphatidic acid generation allows dissociation of the enzyme from the plasma membrane and return to the cytosol, as demonstrated using a pharmacological inhibitor and a catalytically inactive version of the enzyme. The NH2-terminal domain of the protein is shown to be responsible for receptor-induced translocation and phosphatidic acidmediated membrane dissociation. After examining induction of the T cell activation marker CD69 in cells expressing a constitutively active form of the enzyme, we present evidence of the negative regulation that DGK
exerts on diacylglycerol-derived cell responses. This study is the first to describe DGK
as an integral component of the signaling cascades that link plasma membrane receptors to nuclear responses.
Key Words: diacylglycerol kinase, lymphocytes, T cell activation, signal transduction, green fluorescent protein
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Introduction |
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The magnitude and specificity of cellular responses are dictated by a delicate balance between positive and negative signals that are generated after receptor stimulation. Although the mechanisms that promote the intracellular generation of signals have been extensively analyzed, those leading to negative regulation of signal generation remain largely undefined. DAG kinase (DGK)1 generates phosphatidic acid (PA) through the phosphorylation of DAG. Since DAG is a well known lipid second messenger that is rapidly generated in response to receptor stimulation, DGK activation may be related directly to the termination of DAG-derived signals. Nine different DGK isoforms have thus far been cloned and their cDNA characterized. Alignment of the different DGK sequences has allowed identification of motifs that may be important for DGK function and/or regulation (
DGK belongs to the type I DGK family and is abundant in the cytosol of T lymphocytes (
as a modulator of receptor-derived cell responses. Our results indicate that, in response to carbachol stimulation of J-HM1-2.2 cells, DGK
translocates rapidly from the cytosol to the plasma membrane by a mechanism that requires the NH2 terminus of the enzyme. This domain is also responsible for the dissociation of the enzyme from the plasma membrane which takes place after PA generation. Using a pharmacological inhibitor of the enzyme and plasmids encoding green fluorescent protein (GFP) fused to the wild-type or a truncated DGK
, we demonstrate for the first time the role of DGK
in the negative regulation of receptor-derived signals. DGK
translocation is also induced by T cell receptor (TCR) cross-linking, suggesting that membrane localization of DGK
is a general response to receptor stimulation. Plasma membrane translocation of DGK
cannot be induced by addition of DAG analogues but is mimicked by intracellular calcium elevation together with activation of tyrosine kinases, suggesting that receptor-induced enzyme translocation of DGK
requires at least these two signals.
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Materials and Methods |
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Reagents
Carrier-free [32P]orthophosphate and [-32P]ATP were purchased from Amersham Pharmacia Biotech. Silica gel thin layer chromotography plates (60Å, LK6D) were from Whatman. R59949, 1,2-bis(aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), U73343, U73122, and herbimycin were purchased from Calbiochem. 1,2-dioleoylglycerol, phorbol-12,13-dibutyrate (PDBu), carbachol, 1,2-dioctanoyl sn-glycerol (DiC8), ionomycin, orthovanadate, and poly-L-lysine were from Sigma-Aldrich. Radiolabeled standards for strong ion exchange high performance liquid chromatography (SAX-HPLC) were prepared as described previously (
Cell Culture
The J-HM1-2.2 cell line was generated by stable transfection of HM-1 in the human leukemic Jurkat T cell line (
Constructs and Transfection
The PEF-GFPC1 plasmid was generated by replacing the cytomegalovirus promoter in the GFP-C1 plasmid (CLONTECH Laboratories, Inc.) with the PEF-BOS promoter (donated by Dr. J.A. Garcia-Sanz, DIO/CNB, Madrid, Spain). Full-length DGK and
(1192)DGK
cDNAs subcloned into PSRE plasmids have been described previously (
to the membrane, the GFP-DGK
NH2 terminus was modified by adding the pYes myristoylation sequence using specific primers flanked for restriction sites. The annealed DNA fragment was fused in frame via the respective restriction sites to the NH2 terminus of GFP in the GFP-DGK
construct. For transfection, cells in logarithmic growth were transfected with 25 µg of the corresponding plasmid DNA by electroporation at 230 V/975 µF. Cells were analyzed between 24 and 48 h after transfection.
In Vivo Generation of PA
When J-HM1-2.2 cells reached a density of 106 cells/ml, they were washed twice in phosphate-free RPMI medium supplemented with 2 mM glutamine and further incubated for 90 min. Metabolic labeling was performed using 150 µCi/ml [32P]orthophosphate for 1 h. Cells were then challenged as indicated in the figure legends. When R59949 (10 µM) was used, a 10-min preincubation was used before addition of carbachol. After the indicated times, total cellular lipids were extracted and separated by SAX-HPLC (
Determination of DAG Levels
DAG generation was quantified according to a modification of the DAG kinase assay (
Measurement of DGK Activity
COS-1 cells were transfected with the indicated cDNA, harvested after 3648 h, washed twice with ice-cold PBS, and frozen at -70°C. The cells were thawed and lysed by nitrogen cavitation (10 min at 500 psi, 4°C) in a buffer containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 50 mM NaF, 2 mM Na3VO5, 1 mM PMSF, and 10 µg/ml each of leupeptin and aprotinin. Lysates were then centrifuged (800 g) and the supernatant was used to determine DGK activity, as described previously (
Analysis of CD69 Cell Surface Expression
Cells were preincubated with vehicle or the DGK inhibitor R59949 for 10 min, then stimulated with carbachol or PDBu as indicated. CD69 expression on the cell surface was analyzed using a PE-conjugated antihuman CD69 monoclonal antibody. Immunofluorescence intensity of the cells was determined by flow cytometry (EPICS-XL; Beckman Coulter). The CD69 expression level in transfected cells was analyzed 48 h after transfection, gating for GFP-positive and -negative cells.
Immunoblotting
Cells were lysed in 1 ml of lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM sodium pyrophosphate, 1 mM Na3VO4, 1% Nonidet P-40, 1 mM PMSF, and 10 µg/ml each aprotinin and leupeptin) for 30 min on ice. After centrifugation (20,000 g, 10 min, 4°C), supernatants were analyzed by SDS-PAGE. Electrophoresed samples were transferred to nitrocellulose and expression of the GFP-coupled constructs was analyzed using anti-GFP antibody and the ECL detection kit (Amersham Pharmacia Biotech).
Immunofluorescence Microscopy
Cells were harvested 36 h after electroporation with the corresponding plasmids, washed, and allowed to attach to poly-L-lysinecoated coverslips (1 h, room temperature). Cells were treated as indicated (in corresponding figure legends). Cells were fixed with 4% (wt/vol) paraformaldehyde in PBS, washed twice with PBS, and analyzed by confocal microscopy (TCS-NT; Leica). For visualization of endogenous DGK, cells were stained with anti-DGK
antibody and with Cy3-goat antirabbit IgG. Samples were fixed and permeabilized with 2% BSA plus 0.1% Triton X-100 in PBS. To visualize the nuclei, cells were incubated with TOPRO-3 or Sylver Green (1 µg/ml, 10 min) (Molecular Probes). For TCR and anti-CD28 stimulation, slides were precoated with antibodies at a final concentration of 10 µg/ml. After coating, slides were washed with PBS, and the cells were seeded and centrifuged (80 g, 20 s). At the end of the times indicated, cells were fixed with 4% (wt/vol) paraformaldehyde in PBS, washed twice with PBS, and analyzed by confocal microscopy (TCS-NT; Leica).
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Results |
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DGK Activity Regulates Expression of T Cell Activation Markers
Carbachol stimulation of the Jurkat-derived J-HM1-2.2 cell line, which ectopically expresses the muscarinic type 1 receptor, has been shown previously to induce a rapid elevation of inositol polyphosphates (20% of total) was converted to PBut (data not shown). This indicates that the majority of PA is generated via DGK. DGK-regulated PA generation in response to carbachol was demonstrated further using the specific type I DGK inhibitor R59949 (
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Since the rapid PA increase appeared to be mediated by DGK activation, the DGK requirement for DAG transcriptionally regulated events was determined. In J-HM1-2.2 cells, carbachol is a potent inducer of the early activation marker CD69 (
Localization of GFP-tagged DGK in Response to Carbachol
Carbachol stimulates PA generation, and inhibition of DGK activity results in increased DAG generation and CD69 expression. These findings point to a model in which DGK participates in the early signaling generated by the receptor. Jurkat cells express high DGK levels, which have been shown to be mostly cytosolic by subcellular fractionation (
redistribution, we generated a plasmid encoding the cDNA for DGK
fused to GFP (Fig 2 a). The GFP-tagged DGK
fluorescence in unstimulated cells corresponded to a cytosolic enzyme, as described for endogenous DGK
(
(Fig 2 b, middle); nonetheless, R59949 completely prevented dissociation of the enzyme from the plasma membrane after carbachol addition. When cells were pretreated with the inhibitor, carbachol-induced DGK translocation was complete and lasted for
1 h after receptor stimulation (Fig 2 b, middle).
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Inhibition of DGK activity prevented enzyme dissociation from the plasma membrane. To determine whether this effect was directly related to enzyme activity, we examined the membrane localization of a catalytically inactive DGK fused to GFP. Rapid translocation of the inactive mutant was detected in response to carbachol (Fig 2 b, bottom). Nevertheless, as was the case after inhibitor treatment, the kinase-dead DGK
remained at the membrane at all the times examined.
GFP-DGK Behaves Similarly to Endogenous DGK
To determine whether GFP-tagged DGK responded to carbachol stimulation as did the endogenous enzyme, we analyzed the cells using a specific antibody shown to recognize endogenous DGK
in T lymphocytes (
redistribution to the plasma membrane. These results confirm that transfected GFP-tagged DGK
behaves as does the endogenous enzyme, providing a useful tool to examine protein translocation in response to receptor stimulation.
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The DGK NH2 -terminal Domain Contains a Negative Regulatory Site
The DGK NH2-terminal domain contains a pair of EF hand motifs found in a large number of functionally diverse Ca2+-binding proteins (
translocation, we generated a plasmid encoding the GFP protein fused to a DGK
truncation mutant, in which the first 192 NH2-terminal amino acids, including the EF hand domain, are deleted. DGK
bearing a deletion of the EF hand domain is described to have higher activity than the wild-type enzyme (
(1-192)DGK
was constitutively associated with the plasma membrane, even in the absence of carbachol (Fig 4 d, top). This suggests that the NH2-terminal domain, deleted in this construct, exerts a negative regulatory role, maintaining the enzyme in the cytosol in the absence of receptor signaling and also allowing its dissociation from the plasma membrane after PA generation. Constitutive membrane localization of the GFP
(1-192)DGK
construct is not exclusive of T cells, since a similar distribution was detected after transfection of the pre-B murine cell line Ba/F3 (Fig 4c and Fig d, bottom). To further evaluate the effect of expression of the different constructs on DGK activity, cells were sorted after transfection to enrich the GFP-positive population. PA formation was measured both in basal conditions and after 2 min of carbachol addition. We consistently found a threefold increase in PA levels in cells expressing wild-type DGK
compared with control cells. When the same experiment was performed in cells expressing the GFP
(1-192)DGK
, PA elevation was more than fivefold compared with that measured in GFP-transfected cells (data not shown).
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Membrane Localization of DGK Attenuates CD69 Expression
To examine the effect of DGK activity on carbachol-induced CD69 expression, J-HM1-2.2 cells were transfected with control GFP, GFP-DGK, or GFP
(1-192)DGK
. GFP expression allowed us to determine for each transfected cDNA CD69 surface expression in cells expressing high levels of the transfected proteins, compared with GFP-negative cells. Carbachol-induced CD69 expression was not altered in GFP-expressing cells compared with GFP-negative cells (Fig 5 a, top). A slight decrease on CD69 expression in the GFP population was detected when cells were transfected with the GFP-DGK
construct (Fig 5 a, middle). This indicates that the regulation exerted by carbachol on DGK
translocation to the plasma membrane remained effective. Nevertheless, when cells were transfected with the GFP
(1-192)DGK
construct, which has higher enzymatic activity and is constitutively located in the plasma membrane, CD69 expression was significantly reduced compared with that of untransfected cells (Fig 5 a, bottom). Moreover, there was an inverse correlation between GFP
(1-192)DGK
expression levels and CD69 cell surface protein levels (Fig 5 b). These results indicate that constitutive plasma membrane localization of DGK
attenuates CD69 expression in response to this ligand.
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To further demonstrate that membrane localization of DGK mediated the reduction of CD69 expression, a myristoylation signal sequence was added to the NH2-terminal end of the GFP-DGK
construct to induce constitutive association of this protein to the plasma membrane. After transfection of this construct, cells were stimulated with carbachol and CD69 expression in GFP-negative and -positive gated cells was assessed. As is shown in Fig 6 a, constitutive plasma membrane localization of DGK
severely reduces CD69 expression compared with the wild-type enzyme and behaves similarly to the truncated GFP-DGK
192 mutant. A comparison of CD69 levels in cells expressing DGK
versus those in cells expressing membrane-associated enzyme further confirms that membrane localization of DGK
has a dramatic effect on the downregulation of this activation marker. Finally, to demonstrate the relevance of DGK activity in the control of DAG-regulated responses, expression of CD69 was examined in cells expressing the GFP-tagged, kinase-dead version of the enzyme. As shown in Fig 6 a, CD69 expression after carbachol treatment was higher in GFP-positive cells compared with the GFP-negative ones. The effect on CD69 expression after transfection of the kinase-dead mutant was similar to that observed after pharmacological inhibition of DGK activity (Fig 1 e). These results suggest that overexpression of the kinase-dead mutant has a dominant negative effect over the wild-type enzyme.
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GFP-DGK Translocates to the Membrane after TCR/CD28 Cross-linking
We analyzed whether, in addition to carbachol, a more physiological stimulus could also induce DGK translocation. For this, we examined redistribution of GFP-tagged DGK
in J-HM1-2.2 cells after anti-CD3 and anti-CD28 antibody cross-linking, which resembles the physiological mechanism of T cell activation (
translocation after receptor triggering is a general response to both G protein and tyrosine kinasecoupled receptors, confirming the role of DGK
in the regulation of DAG-based signals.
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Tyrosine Kinase Activation Is Required for GFP-DGK Translocation
Translocation of GFP-DGK occurs very rapidly in response to two alternative receptors known to activate either PLC
or PLCß, suggesting DAG- and/or calcium-dependent regulation of DGK
. PDBu and ionomycin treatment of GFP-DGK
transfected cells did not induce enzyme translocation (not shown), thereby excluding PKC- and/or calcium-dependent mechanisms. Unchanged subcellular localization of the transfected GFP-DGK
correlated with no increase in endogenous PA (Fig 1 b). This further confirmed that endogenous DGK
is not activated/translocated by PKC-dependent signals. We then evaluated whether GFP-DGK
translocation, although independent of PKC activation, was directly mediated through PLC-mediated generation of second messengers. Extracellular addition of DiC8, a DAG analogue with short fatty acid chains, did not induce GFP-DGK
translocation to the plasma membrane (Fig 8 a). No translocation was detected after ionomycin treatment, either alone (Fig 8 b) or in combination with DiC8 (Fig 8 c). The same results were obtained when the cells were pretreated with the DGK inhibitor R59949 to stabilize the plasma membrane localization of the enzyme, which would exclude the existence of very rapid and/or transient translocation after calcium and/or DAG increase (data not shown). GFP-DGK
translocation apparently required a receptor-derived signal independent of those generated after PLC activation. Cell surface receptors that lack intrinsic tyrosine kinase activity, such as the antigen TCR, are known to initiate their actions by recruiting nonreceptor tyrosine kinases (
has been shown to be a direct substrate of the EGF receptor in vivo (
. Induction of tyrosine kinase activity in the transfected cells had no apparent effect either alone (Fig 8 d) or combined with extracellular addition of DiC8 (Fig 8 e). Nonetheless, activation of tyrosine kinases together with an increase in intracellular calcium concentration clearly induced redistribution of the enzyme from the cytosol to the plasma membrane (Fig 8 f). In agreement with these data, carbachol-induced translocation of GFP-DGK
(Fig 9 a) was prevented by pretreatment of transfected cells with the PLC inhibitor U-73122 (Fig 9 b) or with the calcium chelator BAPTA (Fig 9 c), indicating that calcium signals, although not sufficient, are necessary for DGK
recruitment to the plasma membrane. Finally, enzyme translocation in response to carbachol was blocked by pretreatment of the cells with the tyrosine kinase inhibitor herbimycin A (Fig 9 d). This inhibitor is known to interfere with early T cell signaling events by inhibiting lck and ZAP-70 functions, although it does not prevent the carbachol-induced calcium increase (
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Discussion |
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DAG phosphorylation by DGK to generate PA has long been considered an intermediate step in the resynthesis of phosphatidylinositol, with no specific role in the transduction of receptor-derived signals. Nonetheless, several recent studies have suggested a role for DGK in the regulation of cell responses elicited by external stimuli. DGKs are encoded by a large gene family conserved throughout phylogeny. This suggests that, as shown for other enzymes involved in signaling such as PKC, PLC, or phosphatidylinositide kinases, this is an extended family that shares common enzymatic activity. The simplest explanation for the existence of multiple DGK isoforms is that of redundancy in their actions. Notwithstanding, the differences in the regulatory domains of the various subtypes, together with the dissimilar expression pattern in multiple tissues for each isoform, indicate that the various DGK isoforms serve distinct although related functions. DGK was the first enzyme of this family to be purified and have its cDNA cloned (
in the control of T cell responses was suggested by the activation of this enzyme in response to IL-2, the main T cell growth factor, and the demonstration that this activity is necessary for coordinated transition of T cells from late G1 to the S phase of the cell cycle (
correlated with receptor-regulated signals and suggested a functional role for this enzyme in T cell response control. Binding of IL-2 to its high affinity plasma membrane receptor does not, however, cause an appreciable increase in DAG levels. As we have shown, PA is generated rapidly in response to IL-2 through the phosphorylation of a preexisting DAG pool in the cells (
T lymphocytes express high DGK levels in cytosol, and the possibility that this enzyme has a role in the DAG downregulation generated in response to receptor stimulation remained to be investigated. In this study, we have demonstrated that DGK
translocates to the plasma membrane in response to two alternative receptor pathways known to promote T cell activation responses. DGK
translocation is induced through stimulation of an ectopically expressed, G proteincoupled muscarinic type 1 receptor that exerts its actions through activation of PLCß. Our results demonstrate that DGK
translocates to the plasma membrane in response to receptor stimulation and that membrane localization of DGK
is directly related to the attenuation of transcriptionally regulated DAG-derived responses, such as CD69 cell surface expression. Moreover, translocation takes place in response to a more physiological stimulus such as TCR cross-linking, where DAG is also generated from PI4,5P2 through activation of the PLC
isoform. All together, these results indicate that receptor-stimulated DGK
activation is a general feature responsible for regulation of the cellular DAG level generated through PI4,5P2 hydrolysis, a role that has been suggested but never directly demonstrated.
Here, we show that the plasma membrane localization of DGK is tightly regulated by receptor-derived signals, where tyrosine kinase activation after receptor occupancy seems to be essential for receptor-induced translocation. Moreover, membrane localization is regulated by enzyme activity, suggesting that PA generation releases the enzyme from the membrane back to the cytosol. Experiments with the myristoylated form of the enzyme indicate that, although enzymatic activity regulates subcellular localization, the contrary is also true since targeting of the enzyme to the membrane appears to be sufficient to maintain the protein in an active conformation. Those results further confirm the close correlation between membrane localization and enzyme activity and indicate that the more important aspect when studying the physiological regulation of this enzyme by different stimuli should be that of its subcellular localization. This study also allows us to draw some important conclusions on the role of the different DGK
domains in the control of its subcellular distribution in response to receptor activation. We demonstrate the negative regulatory role of the DGK
NH2-terminal domain not only by examining enzymatic activity but also by analyzing the subcellular localization of the truncated protein. Deletion of the NH2-terminal domain increases enzymatic activity and induces constitutive localization of the enzyme to the plasma membrane in both Jurkat and Ba/F3 cells, suggesting that DGK
association with the plasma membrane does not require receptor-generated signals, including DAG generation. A model can be envisaged in which the DGK
NH2-terminal domain is necessary to prevent membrane interaction, maintaining the enzyme in a cytosolic/inactive conformation unless modified by receptor-derived signals to an active/membrane-bound conformation. The type I DGK NH2-terminal domain is characterized by two EF hand motifs, a helix-loop-helix structure, found in a large number of functionally unrelated calcium-regulated proteins (
purified from thymus cytosol binds calcium with an apparent Kd of 0.3 µM (
activation is that in which increases in cytosolic calcium that follow receptor ligation induce a "closed-to-open" conformational transition of the enzyme, allowing interaction of the COOH-terminal domain with the membrane. Our experiments nonetheless indicate that cytosolic calcium elevation, although necessary, is not sufficient even in combination with DAG to induce enzyme translocation. Our results suggest the existence of a more complex model in which activation of tyrosine kinases, triggered by receptor occupancy, would also participate in DGK
activation/translocation. These data concur with previous studies in which it was shown that DGK
was tyrosine phosphorylated after EGF stimulation (
association with the receptor. We do not detect tyrosine phosphorylation of the enzyme either in response to receptor stimulation or after truncation of the NH2-terminal domain, suggesting a more complex mechanism of enzyme regulation, possibly through interaction with some adaptor-like protein. Activation of src-family tyrosine kinases has very recently been described as necessary for hepatocyte growth factorinduced DGK
activation in endothelial cells (
activation by an undefined mechanism. As is the case in our studies, hepatocyte growth factorinduced DGK
activation does not induce DGK
phosphorylation on tyrosine residues.
Plasma membrane translocation of GFP-DGK in response to receptor stimulation is rapid and reversible. Nevertheless, a considerable amount of enzyme is still plasma membrane bound even at 1 h after receptor stimulation. This indicates that receptor-mediated translocation is not sustained, as has been found for the in vivo translocation of certain PKC isoforms in response to physiological stimuli (
is no longer reversible in the presence of the DGK inhibitor R59949. Similar results are obtained with a catalytically inactive form of the enzyme, suggesting that local PA generation is necessary for enzyme release from the plasma membrane. This implies the existence of a feedback mechanism in which the enzymatic reaction product releases the enzyme from the membrane. The truncated enzyme form, which has higher activity than the wild-type, is insensitive to this feedback regulatory mechanism since it is constitutively located in the plasma membrane. This indicates that the deleted NH2-terminal domain, which allows translocation in response to receptor-derived signals, is also responsible for enzyme dissociation from the plasma membrane in response to high local PA generation. In this regard, in vitro experiments demonstrate that the NH2-terminal domain of DGK
is responsible for the regulation of enzyme activity by PA and other acidic lipids (our unpublished results). Generation of PA is suggested to have a significant role in protein redistribution from cytosol to the membrane, as proposed for Rac release from RhoGDI (
membrane localization.
This study demonstrates that subcellular translocation is an important mechanism in the control of DGK. Plasma membrane localization DGK
is tightly regulated not only by receptor-derived signals but also by its own enzymatic activity. Studies by us and other authors have indicated the existence of different subcellular localizations for this and other isoforms, suggesting that DGKs may phosphorylate different DAG pools in distinct subcellular compartments (
, for instance, is found in the nucleus and translocates to the cytosol through a PKC-dependent mechanism (
DAG generated after receptor activation is responsible for the binding and subsequent activation of classical and novel PKCs and other proteins with phorbol estersensitive domains such as unc-13 and chimerins () expressed in human brain and retina has 49% identity with rdgA and maps to a known locus of inherited retinitis pigmentosa (
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The magnitude, duration, and frequency of activation of a signaling pathway exert a major influence on the cellular response, which in turn demands that attention be paid to the various control mechanisms that attenuate these signals. Our studies indicate that DGK is another player in the generation of the complex scaffold of signaling proteins that locate to the plasma membrane in response to receptor activation. A better knowledge of the role of DGK
in the termination and propagation of the signals generated after triggering of plasma membrane receptors will be the object of future studies.
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Footnotes |
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1 Abbreviations used in this paper: BAPTA, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; DGK, DAG kinase; DiC8, 1,2-dioctanoyl sn-glycerol; GFP, green fluorescent protein; IL, interleukin; PA, phosphatidic acid; PBut, phosphatidylbutanol; PDBu, 1,2-dioleoylglycerol, phorbol-12,13-dibutyrate; PE, phycoerythrin; PI4,5P2, phosphatidylinositol 4,5-bisphosphate; PLD, phospholipase D; SAX-HPLC, strong ion exchange high performance liquid chromatography; TCR, T cell receptor.
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Acknowledgements |
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We are grateful to F. Sakane and H. Kanoh for the gift of reagents. We also thank J.A. Garcia-Sanz, A. Bernad, M. Torres, and A. Carrera for reading and critical discussion of the manuscript, T. Casaseca for excellent technical assistance, and C. Mark for editorial assistance. We thank Dr. A. Weiss and Genentech, Inc. (South San Francisco, CA) for providing the J-HMI-2.2 cell line.
This work was partially supported by grants PM97-0132 from Dirección General de Enseñanza Superior e Investigación Cientifica and 08.3/0016.1/99 from the Comunidad Autónoma de Madrid to I. Merída. M.A. Sanjuán is a fellow of the Comunidad de Madrid. The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by the Pharmacia Corporation.
Submitted: 9 August 2000
Revised: 25 January 2001
Accepted: 14 February 2001
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References |
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