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to the Membrane: A Novel Mechanism for Ras Attenuation1
* Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, Madrid, Spain; and
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada
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Abstract |
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, using a transgenic mouse model in which injection of an antigenic peptide activates the majority of peripheral T cells. We demonstrate that DGK, highly expressed in resting T lymphocytes, is subject to complex control at the mRNA and protein levels during in vivo T cell activation. Subcellular fractionation of T lymphocytes shortly after in vivo engagement of the TCR shows rapid translocation of cytosolic DGK to the membrane fraction. At early time points, DGK translocation to the membrane correlates with rapid translocation of Ras guanyl nucleotide-releasing protein (RasGRP), a nucleotide exchange activator for Ras that associates to the membrane through a diacylglycerol-binding domain. To demonstrate a causal relationship between DGK activity and RasGRP relocation to the membrane, we determined RasGRP translocation kinetics in a T cell line transiently transfected with constitutive active and dominant-negative DGK mutants. We show that membrane localization of DGK is associated with a negative regulatory signal for Ras activation by reversing RasGRP translocation. This study is the first demonstration of in vivo regulation of DGK, and provides new insight into the functional role of a member of this family of lipid kinases in the regulation of the immune response. |
Introduction |
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DGK is a member of the type I DGK family, characterized by the presence in their N termini of two Ca2+-binding EF hands upstream of the two CRM and the C-terminal catalytic region. DGK is the major isoform in T lymphocytes, and experiments using T cell lines have shown DGK translocation to the plasma membrane following TCR engagement (4). DGK activation is also required for IL-2-regulated cell cycle entry (5). These studies point to DGK as a key component of the mechanisms that control T lymphocyte activation and proliferation, but to date no studies have analyzed the regulation of this isoform during T cell activation in vivo. In this study, we cloned the murine DGK cDNA and characterized expression and regulation of this protein throughout T cell activation using a well-established transgenic mouse model. The results indicate that cellular expression of this protein is subject to fine regulation following in vivo TCR engagement, possibly reflecting its essential role in different T cell activation stages. Moreover, in the transgenic mice, antigenic stimulation leads to rapid DGK membrane localization. Using this model, we also show for the first time the rapid relocalization in activated T lymphocytes of Ras guanylnucleotide-releasing protein (RasGRP)1, a guanylnucleotide exchange factor (GEF) for Ras. RasGRP1 relocates to the T cell membranes with a kinetic similar to that observed for DGK. Finally, using T cell lines and engineered mutant forms of DGK, we demonstrate that relocalization of this enzyme near the Ag receptor is an early event that regulates RasGRP localization in the plasma membrane. DGK thus acts as a negative modulator of Ras activation following TCR triggering. This study is the first demonstration of in vivo regulation of a DGK isoform and provides new insight into the functional role of a member of this family of lipid kinases.
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Materials and Methods |
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The Jurkat T cells used in these experiments were selected for high CD3 expression by cytofluorometric analysis to achieve homogeneity of response. Cells were grown in RPMI 1640 medium (Life Technologies, Rockville, MD) supplemented with 10% FBS (Life Technologies), 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, MEM nonessential amino acid solution (Life Technologies), and 100 U/ml each penicillin G and streptomycin. Cells in a logarithmic growth phase were transfected with the indicated amounts of plasmid DNAs by electroporation as described (4).
cDNA cloning
The primary murine DGK sequence was obtained by screening a 
ZAPII-cDNA library prepared using mRNA extracted from CMN-5 cells, a cell line derived from a thymic lymphoma (Clontech, Palo Alto, CA) (6). The library was screened using full-length porcine DGK cDNA (a gift of Dr. H. Kanoh (Sapporo, Japan)) as probe, labeled with Prime-it II (Stratagene, La Jolla, CA). Colonies (2 x 105) were screened and 19 positive clones were purified and sequenced. Three clones contained an open reading frame extending from DGK aa 150–729. The 5' end, absent in all clones sequenced, was obtained by RT-PCR with oligonucleotides based on expressed sequence tag sequences and murine thymus RNA. The PCR product was excised and subcloned in the pBlue vector containing the rest of the sequence.
In vivo antigenic activation
F5-TCR mice are double V4 and V
11 transgenic mice, able to recognize the influenza virus A nucleoprotein peptide (NP366–374) in the context of H2-Db. This TCR is positively selected on the H-2b haplotype; the majority of mature T cells are MHC class I-restricted CD8+V4+V11+ cells (7).
F5-TCR, or F5-TCR Rag1-/- mice (8) (kindly provided by Dr. D. Kioussis) were maintained in homozygosis on a C57BL/6 background. For in vivo T cell activation experiments, mice were injected i.p. with 200 µl of PBS (0 h) or 75 nmol of antigenic peptide NH2-ASNENMDAM-COOH (Isogen Bioscience, Maarsen, The Netherlands) in 200 µl of PBS. At different times after injection, animals were killed by cervical dislocation, lymph nodes were collected and minced, and single-cell suspensions were obtained. T cells from F5-TCR transgenics were negatively selected. Cell suspensions were incubated with anti-CD4 Ab (30 min; 4°C), washed, and incubated (30 min; 4°C) with anti-rat IgG and anti-mouse IgG coupled to Dynabeads (1 bead/10 cells and 1 bead/4 cells, respectively), followed by magnetic separation; the resulting T cell population was >85% CD8+V4+V11+. F5-TCR Rag1-/- mice were used for short-term experiments (15 and 30 min after in vivo peptide injection), as the nucleated cell suspensions obtained after mincing lymph nodes contained >85% CD8+V4+V11+ cells, with no requirement for further T cell purification.
Flow cytometry
Cells were stained (20 min; 4°C) with anti-TCRV11-(PE) and anti-CD25-(FITC) or -CD69-(FITC) Ab (BD PharMingen, San Diego, CA). Flow cytometry and multiparameter analyses were performed on an Epics XL flow cytometer (Coulter, Miami, FL). Dead cells, <7% in all cases, were excluded from the analysis by gating on forward and side light scatter.
RNA preparation and analysis
RNA was prepared from cells using the guanidinium-isothiocyanate/acid phenol method. After electrophoresis on denaturing 1.2% formaldehyde-agarose gels, RNA samples were transferred to nylon membranes (GeneScreen; NEN, Boston, MA), and rRNA distribution was visualized by methylene blue staining. Northern blots were then hybridized with an antisense [-32P]CTP-labeled mRNA mouse DGK-specific probe transcribed from a T7 promoter, followed by a [-32P]dCTP-labeled random-primed rPL32 cDNA probe. Hybridization temperatures were 65 and 42°C for cRNA and cDNA probes, respectively, both in the presence of 50% formamide. After washing, filters were exposed and quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Background was subtracted from each sample using the local median algorithm from the ImageQuant program (version 1.2; Molecular Dynamics).
Plasmids and transfection
The PEF-EGFP-DGK, PEF-EGFP-DGK-DN (previously reported as kinase-dead), and PEF-EGFP-
EF-DGK constructs have been described (4). PEF-RasGRP was prepared by excision of the rat RasGRP1 cDNA cloned into the retroviral vector pBabepuro with BamHI and XbaI and subcloned into pEF Bos-CX.
DGK assay
Transfected cells were frozen on dry ice and lysed by sonication in DGK buffer assay (50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.25 M saccharose, 1 mM DTT, 1 mM EDTA, 5 mM EGTA, and the protease inhibitors PMSF, leupeptin, and aprotinin). Cell lysates were centrifuged (20,000 xg) and the supernatant was used for DGK reaction, as described (9). Enzyme activity was determined by measuring 32P incorporation into PA in the presence of DAG. Protein (20 µg) from whole lysates or immunoprecipitates of tagged proteins was used as an enzyme source. Other assay constituents were 0.2 µg/ml DAG (18:1/18:1; Avanti Polar Lipids, Alabaster, AL), 5 mM Mg2Cl, and 20 mM [
-32P]ATP (20 Ci/mmol). The stock solution of pure DAG in chloroform was dried under nitrogen, resuspended in 10 mM Tris, and sonicated before adding to the phosphorylation mix. Standard phosphorylation assays were performed (10 min; 37°C) in a final volume of 50 µl. The reaction was terminated by adding 100 µl of 1 M HCl, and lipids were extracted with 20 µl of CHCl3/MeOH (1:1, v/v). Organic layers were recovered, dried, dissolved in 20 µl of CHCl3, and applied to silica gel TLC plates with dioleoyl-PA as a standard. Plates were developed with a CHCl3/MeOH/4 M NH4OH solvent system (9:7:2, v/v/v). Dried plates were autoradiographed, and bands corresponding to PA were quantified by autoradiogram scanning.
Ras activation assay
Jurkat cells were transfected with the corresponding plasmids and after 36 h were washed, placed on ice, and stimulated with anti-CD3 (OKT3) plus anti-CD28 (BD PharMingen) Abs. Cells were incubated at 37°C for the times indicated, centrifuged, and the pellet was lysed in 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.25% SDS, 10% glycerol, and 25 mM NaF plus protease inhibitors. After centrifugation to remove nuclei, 90% of each supernatant was incubated with a GST fusion protein of the Raf-1 Ras-binding domain-agarose (Upstate Biotechnology, Lake Placid, NY) to precipitate active Ras (GTP-bound Ras). Bead-associated Ras was detected with an anti-pan-Ras Ab (Oncogene Research Products, Cambridge, MA) by immunoblotting (10). The remaining 10% of each lysate was probed with the same anti-Ras Ab to compare active with total Ras in each condition.
Subcellular fractionation
T lymphocytes were harvested, washed in PBS, and resuspended in TES buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.25 M sucrose) with protein inhibitors and lysed by 10 passages of the cell suspension through a 25-gauge needle. Nuclei were eliminated by centrifugation (20,000 x g; 4°C). Supernatants were centrifuged (100,000 x g; 1 h), and pellets were resuspended in TES buffer supplemented with 1% Nonidet P-40, and sonicated (1 min). Total cell lysates, cytosol, and membrane fractions were resolved in SDS-PAGE and analyzed by Western blot. Anti-DGK Ab was raised against bacterially expressed rat DGK (residues 2–357), as previously described (11); anti-transferrin receptor (clone H68.4) was from Zymed (San Francisco, CA); anti-p56lck (clone 3A5) was from Upstate Biotechnology; and anti- actin (clone AC-15) was from Sigma-Aldrich (St. Louis, MO).
Western blot
To determine expression of transfected proteins, 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 x g; 10 min; 4°C), supernatants were analyzed by SDS-PAGE. Electrophoresed samples were transferred to nitrocellulose, and protein expression was analyzed using indicated Abs and ECL (Amersham, Arlington Heights, IL).
Immunofluorescence microscopy
Cells were harvested 36 h after electroporation with plasmids, washed, and allowed to attach to poly-L-lysine-coated coverslips (1 h; room temperature). For anti-CD3/anti-CD28 stimulation, slides were precoated with Abs (BD PharMingen) at a final concentration of 10 µg/ml. Samples were washed with PBS, fixed with 4% paraformaldehyde in PBS (4°C; 20 min) and blocked with 2% BSA in PBS (2 h). All subsequent incubations and washes used a 0.5% BSA/0.1% Triton X-100 in PBS buffer; cells were incubated (37°C; 1 h) in a humidified chamber with anti-HA (Covance, Richmond, CA) and anti-PDI (StressGen Biotechnologies, Victoria, British Columbia, Canada) Abs. After two washes, cells were incubated under the same conditions with Cy2 anti-mouse and Cy3 anti-rabbit (Jackson ImmunoResearch, West Grove, PA) secondary Abs. For double localization of RasGRP/green fluorescent protein (GFP) constructs after transfection, cells were incubated (30 min) with an anti-RasGRP Ab (clone M199C; Santa Cruz Biotechnology, Santa Cruz, CA) labeled with the Alexa Fluor 568 labeling kit (Molecular Probes, Eugene, OR) according to the manufacturer’s protocol. Nuclei were labeled with Topro-3 (Molecular Probes). Fluorescence was analyzed on a Leica confocal microscope (TCS-NT; Leica, Deerfield, IL) with associated software.
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Results |
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Mouse DGK cDNA was cloned by screening a murine thymoma cDNA library. The deduced 730-aa sequence (GenBank no. O88673) corresponds to a protein with a calculated Mr of 82.7 kDa, with 92% sequence similarity to the 80-kDa rat DGK (11), and 81% identity to the human and porcine counterpart (12, 13). Like all type-I DGKs, this protein contains two EF hand motifs and two CRM, zinc finger-like sequences upstream of the C-terminal half, which shows considerable similarity with the C-terminal portion of all known DGK isoforms.
Using probes corresponding to the murine sequence, DGK mRNA expression was analyzed by Northern blot hybridization in several mouse tissues. This revealed variable amounts of DGK mRNA in spleen, skeletal muscle, lung, and testis, whereas it was undetectable in brain, liver, and kidney (Fig. 1A). Equivalent RNA loading in each lane was confirmed by subsequent hybridization of the filter with an actin-specific probe (Fig. 1A). RT-PCR using specific DGK primers confirmed expression of this mRNA in the spleen and revealed expression in the thymus (Fig. 1B). As a control, hypoxanthine-guanine phosphoribosyltransferase was amplified from the same cDNAs (Fig. 1B).
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protein expression was then analyzed in Western blot of extracts from murine tissues using a polyclonal Ab raised against DGK of rat origin (11). The Ab recognized mouse DGK expressed in COS cells (Fig. 1C). When different mouse tissues were analyzed, a 80-kDa band was recognized by the Ab in spleen and thymus (Fig. 1D), suggesting that, as previously described for rat and pig orthologs (11, 14), DGK is highly expressed in those tissues. Despite similar or higher mRNA expression levels, DGK protein was undetectable in skeletal muscle and testis (Fig. 1, A and D). We did not detect expression of this protein or its encoding mRNA in whole brain tissue, despite reported high DGK protein expression levels in pig and rat oligodendrocytes (11); this may be due to the small proportion of oligodendrocytes in the whole brain sample analyzed.
These data confirm previous studies describing restricted tissue distribution of DGK mRNA and protein, and confirm tissue-specific genetic control for this isoform. The DGK protein expression in spleen and thymus reported in this study (Fig. 1D) is fully supported by previous reports describing DGK as a very abundant protein in pig thymus. The lack of correlation between mRNA and protein expression in other tissues such as testis (Fig. 1, A and D) is similar to that described for other DGK isoforms such as DGK
(15) and suggests tissue-specific differences in mRNA processing and/or protein stability for this isoform.
DGK expression following in vivo T cell activation
Although DGK is abundant in T lymphocytes, regulation of this gene during in vivo T cell activation has not been studied. To analyze this, we used the F5-TCR transgenic mouse model (7), in which i.p. injection of an antigenic peptide induces depletion of immature thymocytes and activates the majority of peripheral T cells.
In vivo T cell activation kinetics were analyzed in lymph node T cells at different times (0–24 h) after i.p. injection of peptide (75 nmol in 200 µl of PBS) or PBS as control (0 h). Following PBS injection, purified T cells did not express CD69 or CD25 activation markers. As early as 1 h after peptide injection, expression of the early activation marker CD69 increased >10-fold, reaching a plateau 4 h after peptide injection (Fig. 2A). Expression levels of CD25, the inducible subunit of the IL-2R, began to increase 4 h after peptide injection, reaching plateau levels at 8 h (Fig. 2A). Maximum expression for both CD69 and CD25 persisted for at least 16 h after Ag injection, a time point preceding the first cell division (not shown).
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mRNA expression in resting T cells, which decreased following activation, reaching 50% of resting cell levels 1 h after peptide injection. At 4–6 h, mRNA levels were <10% of those expressed in resting T cells (Fig. 2B). This decrease in DGK mRNA was followed by an increase, which reached plateau 12 h after peptide injection, with levels corresponding to 30–35% of DGK mRNA expression in resting T cells (Fig. 2B). Changes in protein expression, determined by Western blot over a similar time period, showed a steady decrease in DGK protein (Fig. 2C). Lowest expression levels were reached at 8 h after activation, and maintained for at least 20 h, with no concomitant changes in control -actin levels (Fig. 2C). DGK activity was also determined by measuring 32P incorporation into PA in the presence of DAG using 20 µg from whole lysates as source of the protein. Standard phosphorylation assays were performed for 10 min at 37°C and the reaction was terminated by adding 100 µl of 1 M HCl. Lipids were extracted and separated by TLC, and the band corresponding to PA was quantified by autoradiogram scanning. The phosphorylation assay indicated that DGK activity was lower at 4 h than at time 0, and decreased for the first 8 h to stabilize at 30% of initial levels (Fig. 2C).
DGK translocates rapidly to the membrane after in vivo T cell activation
Stimulation of the TCR signaling pathway in T cell lines induces rapid DGK translocation from cytosol to the plasma membrane (4). To assess whether DGK translocation also takes place in vivo, membrane-associated DGK was analyzed at short times after antigenic peptide injection. The fraction of membrane-associated DGK in resting T cells (PBS-injected animals) was very low compared with that found in the cytosolic fraction (Fig. 3A). After Ag injection, a sharp increase was observed in membrane-bound DGK. Several controls were used to assess the validity of this model and to evaluate the efficiency of the fractionation procedure. To confirm the purity of membrane fractions, we determined expression of the transferrin receptor, a transmembraneprotein rapidly induced following T cell activation. The tyrosine kinase p56lck, located in membrane rafts in T cells, was found mainly in the membrane fraction. A shift in p56lck electrophoretic migration, indicative of protein phosphorylation, was detected as early as 15 min, further confirming that the early signaling events that drive T cell activation can be measured in this experimental model. RasGRP, a GEF for Ras that contains a DAG-binding domain has been proposed to translocate to the membrane during T cell activation in a DAG-dependent fashion (16). RasGRP was found mainly in the cytosol of resting cells and, following Ag injection, translocated to the membrane fraction with kinetics similar to that of DGK. DGK translocation to the membrane-associated fraction correlated with an increase in DGK activity that was linear at early time points (Fig. 3B). The rapid translocation of DGK to the membrane fraction, together with the increase in enzymatic activity that follows T cell activation in vivo, validate our earlier findings in Jurkat cells and imply immediate, direct DGK recruitment to the TCR signaling pathway.
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and RasGRP translocate to the plasma membrane following TCR/CD28 triggering
The previous experiments indicated that in vivo T cell activation is rapidly followed by RasGRP and DGK translocation to the membrane fraction. Subcellular fractionation does not allow examination of the exact localization of proteins in cells, so we decided to investigate subcellular distribution of these two protein by confocal microscopy. The Abs used for analysis of endogenous RasGRP do not work well in immunofluorescence in primary cells. Thus, we transfected Jurkat T cells line with a plasmid encoding RasGRP and examined relocalization of this protein after T cell activation. T cells were plated onto anti-CD3/anti-CD28-coated slides and RasGRP localization was visualized with Alexa-stained anti-mouse RasGRP to avoid interference with the coating Abs. As is the case in primary T cells, RasGRP was found in the cytosol of unstimulated cells and translocated to the plasma membrane as early as 5 min after TCR/CD28 triggering. By 60 min, most of the protein returned to the cytosol (Fig. 4A). This indicated that the transfected RasGRP behaved as endogenous protein. We next cotransfected this construct with a plasmid encoding DGK fused to enhanced GFP (EGFP). As previously shown (4), the EGFP-DGK fusion protein was cytosolic and translocated to the membrane as early as 5 min after receptor triggering. Previous studies in muscarinic receptor-expressing Jurkat T cells demonstrated that EGFP-DGK translocation in response to carbachol was rapid and transient (4). In this study, membrane translocation in response to anti-CD3/anti-CD28 is sustained because EGFP-DGK remained at the plasma membrane even at 60 min poststimulation. Coexpression of the EGFP-DGK fusion protein did not modify RasGRP translocation at early times because both proteins were found at the plasma membrane after 5 min. By 60 min, most of RasGRP was found at internal localizations. (Fig. 4B).
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activity regulates Ras activation
The experiments with primary cells demonstrate a rapid relocalization of RasGRP and DGK to membrane fractions. Confocal microscopy analysis reveals that both proteins relocate following TCR triggering to the plasma membrane. RasGRP membrane localization depends on binding to DAG through a C1 domain in the protein (16), so we next decided to investigate whether modification of DAG membrane levels could directly modulate membrane localization of RasGRP. To this end, we made use of two DGK mutants, recently described (4), which exhibit transdominant-negative and constitutive active properties. Mutation of a single amino acid of the catalytic region of DGK renders this enzyme inactive and confers to this mutant transdominant-negative properties as has been shown by examining DAG-regulated events such as expression of the activation marker CD69 (4) or extracellular signal-regulated kinase phosphorylation (17). Analysis of the kinetics of RasGRP membrane translocation were examined by confocal microscopy after cotransfection with the dominant-negative DGK. In the presence of the catalytically inactive DGK form, RasGRP translocation after CD3/CD28 stimulation was more sustained, and the majority of protein was detected at the membrane even 60 min after activation (Fig. 5A). The next mutant assayed was a truncated DGK mutant that lacks the N-terminal domain containing the two EF hands (EGFP-EF-DGK). This mutant displays higher enzymatic activity and constitutive membrane localization when transfected in Jurkat T cells (4). Accordingly, expression of this construct markedly reduces activation responses such as CD69 expression (4). In the presence of EGFP-EF-DGK, only a minor fraction of RasGRP was found the plasma membrane at 5 min after stimulation (Fig. 5B, middle row), and the majority of the protein was found at internal localizations after 60 min (Fig. 5B, bottom row). Thus, the use of DGK mutants with either dominant-negative or constitutive active properties demonstrates that DGK-dependent modification of DAG membrane levels affected plasma membrane localization of the DAG-binding protein RasGRP.
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increased GTP-bound Ras levels compared with those in control cells, even in the absence of T cell activation as corresponds to partial RasGRP translocation. After TCR stimulation, Ras activation was higher at both time points, consistent with the confocal analysis showing enhanced RasGRP translocation. On the contrary, when cells were transfected with the constitutive active DGK form, Ras activation decreased dramatically at the two time points tested. These experiments further confirm that modification of DGK activity has a profound effect on the DAG-based signals proceeding from the TCR.
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Discussion |
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isolated from rat, human, and porcine sources is a very abundant protein in tissues of hematopoietic origin (11, 14). The data presented in this study confirm earlier studies describing restricted tissue distribution of DGK mRNA and protein, and demonstrate tissue-specific genetic control of this isoform. The lack of correlation between mRNA and protein expression in some tissues such as testis resembles that described for other DGK isoforms such as DGK (15), and suggests tissue-specific differences in mRNA processing and/or protein stability for this isoform. Differences in specific posttranscriptional regulation of the DGK gene in distinct tissues may reflect the diversity of receptor signaling pathways in which this enzyme is involved, and hence the variety of stimuli to which it must respond. Previous studies in our laboratory showed that DGK-mediated PA generation is required for IL-2-regulated G1-to-S phase transition in IL-2-dependent cell lines (5, 9). More recently, we showed that DGK participates in TCR signaling by phosphorylating the DAG generated during T cell activation, acting as a negative regulator of CD69 expression (4). The in vivo studies presented in this report demonstrate that DGK is expressed in resting T lymphocytes and that its mRNA expression levels are tightly controlled during the activation process, showing concomitant changes both in protein level and in activity. In T cell lines, DGK participates in TCR signaling by phosphorylating the DAG generated during T cell activation (4). This role for DGK in T cell activation is further confirmed in this study by the rapid DGK translocation to the membrane fraction, together with the increase in enzyme activity that follows T cell activation in vivo. Moreover, experiments using DGK constructs with dominant-negative and constitutive active properties suggest a functional role for this protein in regulating early TCR signals, such as the subcellular localization of the DAG-regulated molecule RasGRP.
DGK mRNA expression is biphasic, with a first phase represented by an initial sharp decrease concomitant with the increase in CD69 expression. The initial decrease in DGK mRNA, protein levels, and enzymatic activity observed in in vivo-activated T cells may be interpreted as a direct consequence of sustained TCR cross-linking by the antigenic peptide, to ensure correct amplification of TCR signals. DGK mRNA was recently found to increase in anergized cells (18), suggesting that elevated DGK levels can participate in induction of anergy, a process in which Ag receptors are uncoupled from their downstream signaling pathways. The second phase of regulation of DGK gene expression begins 8–10 h after antigenic challenge and lasts for at least 20 h. During this phase, DGK mRNA increases and plateaus at 30–35% of initial levels; this coincides with maximum cell surface expression of high affinity IL-2R, and precedes cell cycling. DGK is essential for the IL-2-regulated G1-to-S phase transition in IL-2-dependent cell lines (5, 9). Thus, it is tempting to speculate that in vivo regulation of DGK expression and activity during this second phase is associated with IL-2R signaling rather than TCR signaling.
The in vivo studies showing tight control of DGK during T cell activation suggest that, as is the case for other signaling molecules in T lymphocytes (19, 20), DGK expression is coupled to cell cycle progression. The complex regulation of DGK during in vivo T cell activation is probably a consequence of the distinct role of this enzyme at different stages during T lymphocyte activation and proliferation. Experiments with T cell lines have shown that TCR regulation of DGK is Ca2+ dependent and results in protein translocation to the plasma membrane, where it attenuates the DAG produced by PLC activation (4). Accordingly, the in vivo experiments in this study show that cytosolic DGK translocates to the plasma membrane very rapidly, with kinetics similar to those of DAG-regulated proteins such as RasGRP. Experiments with nonlymphoid cells have suggested an additional mechanism for DGK regulation based on activation of Src-like tyrosine kinases (21). In T lymphocytes, we demonstrate that membrane translocation of DGK in vivo also correlates with the kinetics of p56lck activation, determined by the shift in electrophoretic migration. After activation, the DGK mRNA level decreases, to increase again before T cells enter cell cycle. In contrast to the TCR, IL-2 binding to its high affinity receptor does not generate Ca2+ flux (22) and, in T cell lines, induces DGK translocation to internal membranes (9). During T cell proliferation, IL-2-dependent activation of DGK is not required for attenuating PLC-generated DAG, but to generate PA by phosphorylating a pre-existing DAG pool (5, 23). According to the in vivo data, the TCR and IL-2R receptors thus use distinct mechanisms to recruit DGK to different subcellular locations to perform different functions. This may explain the requirement for two distinct steady-state DGK protein levels, achieved by mRNA and protein regulation. To our knowledge, this is the first description of differential regulation of DGK expression as a function of in vivo physiological stimulation at various stages of the cell cycle and/or activation program.
The precise regulation of DGK subcellular localization very early after in vivo TCR triggering suggests a critical role for this enzyme in DAG-regulated signaling pathways. The relevance of DAG generation in the initiation of the T cell response has been known for several years. The discovery of RasGRP, a GEF for Ras with a DAG-binding domain, helped explain the role of DAG in regulating Ras activation, one of the most striking enigmas in the field of T cell signaling. We demonstrate that, in vivo, RasGRP translocates to the membrane with kinetics similar to those of DGK. Experiments with Jurkat cells confirm that DGK translocates to the TCR activation site, where DAG-binding proteins such as RasGRP are located. DGK activity near the TCR attenuates DAG levels and results in the release of RasGRP from the plasma membrane, prompting termination of the Ras-regulated cascade. Our data in this study, as well as those of other recent studies (17), help define a model in which TCR-mediated regulation of DGK acts as a negative signal in Ras activation.
DGK is not the only DGK family member proposed to act as a negative modulator of TCR signaling. Very recently, overexpression of DGK
, a type IV DGK, in Jurkat cells has been reported to regulate DAG-dependent responses such as CD69 expression or Ras activation (24). In contrast to DGK, for which only constitutive active and/or dominant-negative enzyme forms modulate TCR responses, wild-type DGK overexpression is sufficient to modulate these signals. Differences in expression levels, activity, substrate preference, and/or subcellular localization of the endogenous proteins may be the reason for these results. We have shown that DGK is abundantly expressed as a cytosolic protein in Jurkat cells. Overexpression of wild-type enzyme only increased protein levels in cytosol, whereas the cells regulated protein translocation to the membrane. Only alteration of translocation kinetics by eliminating catalytic activity or by tagging the enzyme to the membrane modified the level of TCR-induced DAG.
The distinct behavior of the two proteins may also be attributed to the action of these enzymes on different signaling pathways and/or at different times during T cell activation. Using Jurkat cells ectopically expressing a muscarinic receptor, we showed that the two isoforms translocate to the plasma membrane by distinct mechanisms. DGK is a Ca2+-regulated enzyme that requires tyrosine kinases and calcium elevation to relocate to the plasma membrane (4). In contrast, translocation of DGK is a PKC-regulated mechanism for which phosphorylation of the DGK myristoylated alanine-rich C kinase substrate domain is essential for membrane localization (25). The translocation kinetics for the two enzymes are also very different; DGK translocation is rapid and transient (4), whereas DGK relocation is a more sustained event (25). Future experiments will examine the kinetics and subcellular DGK localization in resting vs activated cells, as well as regulation of mRNA and protein levels of this isoform; this will help elucidate the contribution of each isoform to the regulation of T cell responses.
Activation of signaling cascades following TCR engagement has received extraordinary attention in the last several years. Only very recently has the importance of negative modulation in the onset of T cell activation responses been highlighted. This study, together with previous reports from our group and other laboratories, are beginning to show the critical role of DGK family members in T cell response regulation. DGK relocation to the cell membrane after in vivo T cell activation with kinetics that parallel those of RasGRP, as well as the modulation of Ras signaling by DGK, point to this isoform as essential in early regulation of DAG-based signals during T cell activation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Isabel Mérida, Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail address: imerida{at}cnb.uam.es
3 Abbreviations used in this paper: DAG, diacylglycerol; PA, phosphatidic acid; PLC, phospholipase C; DGK, DAG kinase; CRM, cysteine-rich motif; RasGRP, Ras guanyl nucleotide-releasing protein; GEF, guanylnucleotide exchange factor; GFP, green fluorescent protein; EGFP, enhanced GFP.
Received for publication October 18, 2002. Accepted for publication January 8, 2003.
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E. Diaz-Guerra, R. Vernal, M. J. del Prete, A. Silva, and J. A. Garcia-Sanz CCL2 Inhibits the Apoptosis Program Induced by Growth Factor Deprivation, Rescuing Functional T Cells J. Immunol., December 1, 2007; 179(11): 7352 - 7357. [Abstract] [Full Text] [PDF]
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N. Beaulieu, B. Zahedi, R. E. Goulding, G. Tazmini, K. V. Anthony, S. L. Omeis, D. R. de Jong, and R. J. Kay Regulation of RasGRP1 by B Cell Antigen Receptor Requires Cooperativity between Three Domains Controlling Translocation to the Plasma Membrane Mol. Biol. Cell, August 1, 2007; 18(8): 3156 - 3168. [Abstract] [Full Text] [PDF]
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R. Alonso, M. C. Rodriguez, J. Pindado, E. Merino, I. Merida, and M. Izquierdo Diacylglycerol Kinase {alpha} Regulates the Secretion of Lethal Exosomes Bearing Fas Ligand during Activation-induced Cell Death of T Lymphocytes J. Biol. Chem., August 5, 2005; 280(31): 28439 - 28450. [Abstract] [Full Text] [PDF]
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A. Avila-Flores, T. Santos, E. Rincon, and I. Merida Modulation of the Mammalian Target of Rapamycin Pathway by Diacylglycerol Kinase-produced Phosphatidic Acid J. Biol. Chem., March 18, 2005; 280(11): 10091 - 10099. [Abstract] [Full Text] [PDF]
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S. Carrasco and I. Merida Diacylglycerol-dependent Binding Recruits PKC{theta} and RasGRP1 C1 Domains to Specific Subcellular Localizations in Living T Lymphocytes Mol. Biol. Cell, June 1, 2004; 15(6): 2932 - 2942. [Abstract] [Full Text] [PDF]
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R. A. Rambaratsingh, J. C. Stone, P. M. Blumberg, and P. S. Lorenzo RasGRP1 Represents a Novel Non-protein Kinase C Phorbol Ester Signaling Pathway in Mouse Epidermal Keratinocytes J. Biol. Chem., December 26, 2003; 278(52): 52792 - 52801. [Abstract] [Full Text] [PDF]
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and •, Two different experiments. Inset shows hybridization signals for DGK
