T Cell Activation In Vivo Targets Diacylglycerol Kinase α to the Membrane: A Novel Mechanism for Ras Attenuation

Cell culture

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 DGKα sequence was obtained by screening a λZAPII-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 DGKα cDNA (a gift of Dr. H. Kanoh (Sapporo, Japan)) as probe, labeled with Prime-it II (Stratagene, La Jolla, CA). Colonies (2 × 10⁵) were screened and 19 positive clones were purified and sequenced. Three clones contained an open reading frame extending from DGKα 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 Vα₄ and Vβ₁₁ transgenic mice, able to recognize the influenza virus A nucleoprotein peptide (NP_366–374) in the context of H2-D^b. This TCR is positively selected on the H-2^b haplotype; the majority of mature T cells are MHC class I-restricted CD8⁺Vα₄⁺Vβ₁₁⁺ 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⁺Vα₄⁺Vβ₁₁⁺. 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⁺Vα₄⁺Vβ₁₁⁺ cells, with no requirement for further T cell purification.

Flow cytometry

Cells were stained (20 min; 4°C) with anti-TCRVβ₁₁-(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 [α-³²P]CTP-labeled mRNA mouse DGKα-specific probe transcribed from a T₇ promoter, followed by a [α-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-DGKα, PEF-EGFP-DGK-DN (previously reported as kinase-dead), and PEF-EGFP-ΔEF-DGKα 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 ×g) 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 [γ-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 × g; 4°C). Supernatants were centrifuged (100,000 × 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-DGKα Ab was raised against bacterially expressed rat DGKα (residues 2–357), as previously described (11); anti-transferrin receptor (clone H68.4) was from Zymed (San Francisco, CA); anti-p56^lck (clone 3A5) was from Upstate Biotechnology; and anti-β 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 × 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.

Cloning and expression of murine DGKα

Mouse DGKα 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 M_r of 82.7 kDa, with 92% sequence similarity to the 80-kDa rat DGKα (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, DGKα mRNA expression was analyzed by Northern blot hybridization in several mouse tissues. This revealed variable amounts of DGKα mRNA in spleen, skeletal muscle, lung, and testis, whereas it was undetectable in brain, liver, and kidney (Fig. 1⇓A). Equivalent RNA loading in each lane was confirmed by subsequent hybridization of the filter with an actin-specific probe (Fig. 1⇓A). RT-PCR using specific DGKα primers confirmed expression of this mRNA in the spleen and revealed expression in the thymus (Fig. 1⇓B). As a control, hypoxanthine-guanine phosphoribosyltransferase was amplified from the same cDNAs (Fig. 1⇓B).

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FIGURE 1.

Tissue distribution of DGKα expression. A, Tissue distribution for DGKα mRNA was examined in a commercial Northern blot using ³²P-labeled cDNA probes from mouse DGKα and β-actin, respectively. B, Total RNA from spleen and thymus was reverse transcribed to cDNA and amplified with specific primers for mouse DGKα and hypoxanthine-guanine phosphoribosyltransferase as described in Materials and Methods. C, The DGKα Ab recognizes DGKα of mouse origin. COS cells were transfected with empty plasmid or a plasmid encoding mouse DGKα. After 48 h, cells were collected and protein expression was analyzed with an anti-DGKα Ab. D, Tissue distribution of DGKα protein. Mouse tissues were prepared and proteins (100 μg/lane) separated by SDS-PAGE and blotted. The blot was probed with an anti-DGKα Ab.

DGKα protein expression was then analyzed in Western blot of extracts from murine tissues using a polyclonal Ab raised against DGKα of rat origin (11). The Ab recognized mouse DGKα expressed in COS cells (Fig. 1⇑C). When different mouse tissues were analyzed, a 80-kDa band was recognized by the Ab in spleen and thymus (Fig. 1⇑D), suggesting that, as previously described for rat and pig orthologs (11, 14), DGKα is highly expressed in those tissues. Despite similar or higher mRNA expression levels, DGKα 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 DGKα 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 DGKα mRNA and protein, and confirm tissue-specific genetic control for this isoform. The DGKα protein expression in spleen and thymus reported in this study (Fig. 1⇑D) is fully supported by previous reports describing DGKα 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 DGKη (15) and suggests tissue-specific differences in mRNA processing and/or protein stability for this isoform.

DGKα expression following in vivo T cell activation

Although DGKα 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. 2⇓A). 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. 2⇓A). 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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FIGURE 2.

DGKα regulation during in vivo T cell activation. A, F5-TCR mice received i.p. injections of antigenic peptide (75 nmol in 200 μl of PBS) or PBS in the controls; T cell activation kinetics were analyzed following expression of the activation markers CD69 (immediate early) and CD25 (early) in the transgenic cells. B, mRNA was prepared from the same samples, and DGKα expression levels were determined by Northern blot analysis. DGKα mRNA expression levels are represented on a semilogarithmic scale after correction for rPL32 ribosomal protein expression in the same sample. ○ and •, Two different experiments. Inset shows hybridization signals for DGKα and 18S rRNA levels at each time point measured. C, DGK activity and DGKα protein levels (inset) were analyzed by in vitro assay and in Western blot, respectively. Determination of enzyme activity was conducted by measuring ³²P incorporation into PA using [γ-³²P]ATP, Mg₂Cl, DAG (18:1/18:1), and 20 μg of lysates as enzyme source. Phosphorylation assays were performed during 10 min at 37°C and the reactions were terminated by adding 100 μl of 1 M HCl. Lipids were extracted and separated by TLC using dioleoyl-PA as a standard. Dried plates were autoradiographed, and bands corresponding to PA were quantified by autoradiogram scanning. Shown is a representative experiment of two performed with similar results. β-Actin was measured as a control (inset).

Northern blot analysis showed high DGKα 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. 2⇑B). This decrease in DGKα mRNA was followed by an increase, which reached plateau 12 h after peptide injection, with levels corresponding to 30–35% of DGKα mRNA expression in resting T cells (Fig. 2⇑B). Changes in protein expression, determined by Western blot over a similar time period, showed a steady decrease in DGKα protein (Fig. 2⇑C). Lowest expression levels were reached at 8 h after activation, and maintained for at least 20 h, with no concomitant changes in control β-actin levels (Fig. 2⇑C). 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. 2⇑C).

DGKα translocates rapidly to the membrane after in vivo T cell activation

Stimulation of the TCR signaling pathway in T cell lines induces rapid DGKα translocation from cytosol to the plasma membrane (4). To assess whether DGKα translocation also takes place in vivo, membrane-associated DGKα was analyzed at short times after antigenic peptide injection. The fraction of membrane-associated DGKα in resting T cells (PBS-injected animals) was very low compared with that found in the cytosolic fraction (Fig. 3⇓A). After Ag injection, a sharp increase was observed in membrane-bound DGKα. 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 p56^lck, located in membrane rafts in T cells, was found mainly in the membrane fraction. A shift in p56^lck 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 DGKα. DGKα translocation to the membrane-associated fraction correlated with an increase in DGK activity that was linear at early time points (Fig. 3⇓B). The rapid translocation of DGKα 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 DGKα recruitment to the TCR signaling pathway.

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FIGURE 3.

DGKα translocates to the membrane fraction after in vivo T cell activation. F5-TCR mice on a Rag^−/− background received i.p. injections of antigenic peptide, mice were killed at times indicated, and T cells were purified from lymph nodes. A, Membrane and cytosolic fractions were separated, and proteins (20 μg/lane) were analyzed by Western blot with specific Abs. B, DGK activity was determined in total cell lysates of triplicate samples.

DGKα 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 DGKα 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. 4⇓A). This indicated that the transfected RasGRP behaved as endogenous protein. We next cotransfected this construct with a plasmid encoding DGKα fused to enhanced GFP (EGFP). As previously shown (4), the EGFP-DGKα 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-DGKα 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-DGKα remained at the plasma membrane even at 60 min poststimulation. Coexpression of the EGFP-DGKα 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. 4⇓B).

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FIGURE 4.

Effect of TCR/CD28 cross-linking on the subcellular redistribution of RasGRP and DGKα. A, Jurkat cells were transfected with a plasmid encoding rat RasGRP1; after 48 h, cells were seeded onto slides precoated with poly-l-lysine (time 0) or anti-CD3/anti-CD28 Abs (10 μg/ml). After incubation for the times indicated, cells were fixed and RasGRP1 was visualized by staining with an Alexa Fluor 568-labeled anti-RasGRP Ab that did not recognize the endogenous protein. B, Jurkat cells were cotransfected with plasmids encoding EGFP-DGKα and RasGRP1; after 48 h, cells were treated as in A and fixed. GFP fluorescence is shown in green (left) and RasGRP is shown in red (middle). The overlay is shown at the right.

DGKα activity regulates Ras activation

The experiments with primary cells demonstrate a rapid relocalization of RasGRP and DGKα 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 DGKα mutants, recently described (4), which exhibit transdominant-negative and constitutive active properties. Mutation of a single amino acid of the catalytic region of DGKα 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 DGKα. In the presence of the catalytically inactive DGKα 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. 5⇓A). The next mutant assayed was a truncated DGKα mutant that lacks the N-terminal domain containing the two EF hands (EGFP-ΔEF-DGKα). 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-ΔEF-DGKα, only a minor fraction of RasGRP was found the plasma membrane at 5 min after stimulation (Fig. 5⇓B, middle row), and the majority of the protein was found at internal localizations after 60 min (Fig. 5⇓B, bottom row). Thus, the use of DGKα mutants with either dominant-negative or constitutive active properties demonstrates that DGKα-dependent modification of DAG membrane levels affected plasma membrane localization of the DAG-binding protein RasGRP.

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FIGURE 5.

DGKα activity at the plasma membrane acts as a negative modulator of RasGRP translocation. A, Jurkat cells were cotransfected with plasmids encoding RasGRP1 and a catalytically inactive version of DGKα with dominant-negative properties, fused to GFP (GFP-DGK-DN). After 48 h, cells were stimulated and localization of the two proteins was determined by confocal microscopy as in Fig. 4⇑B. Jurkat cells were cotransfected with plasmids encoding for RasGRP and GFP-ΔEF-DGK, a constitutive version of DGK. At 48 h posttransfection, cells were treated as in A and fixed, and localization of both proteins was determined by confocal microscopy. GFP fluorescence is shown in green (left); RasGRP is shown in red (middle). The overlay is shown at the right.

We next examined whether these modifications of RasGRP translocation were reflected by changes in Ras activation. In Jurkat cells, TCR triggering was followed by a rapid increase in GTP-bound Ras levels, which diminished by 60 min (Fig. 6⇓). Transfection of the dominant-negative DGKα 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 DGKα form, Ras activation decreased dramatically at the two time points tested. These experiments further confirm that modification of DGKα activity has a profound effect on the DAG-based signals proceeding from the TCR.

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FIGURE 6.

DGKα activity at the plasma membrane acts as a negative modulator of Ras activation. Jurkat cells were transfected with vectors encoding GFP, GFP-DGK-DN, or GFP-ΔEF-DGK; after 48 h, cells were stimulated with anti-CD3 plus anti-CD28 for the indicated times, and Ras activation was determined as described (in Materials and Methods). In the upper panel, activated Ras (GTP-bound) was precipitated with Raf-1 BSD agarose and detected in Western blot with an anti-Ras Ab; in the lower panel, total Ras was determined in the same samples.