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Expression of a catalytically inactive form of diacylglycerol kinase induces sustained signaling through RasGRP¹

DAVID R. JONES^*, MIGUEL ANGEL SANJUÁN^*, JAMES C. STONE and ISABEL MÉRIDA^*2

^* Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, E-28049 Madrid, Spain; and
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

²Correspondence: Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Cantoblanco, E-28049 Madrid, Spain. E-mail: imerida{at}cnb.uam.es

SPECIFIC AIMS

The objective of this study was to examine how diacylglycerol kinase (DGK) modulates agonist-stimulated diacylglycerol (DAG) accumulation and the subsequent activation of downstream pathways in human T lymphocytes. We studied the effects of expressing a catalytically inactive DGK mutant (DGK-kd) on RasGRP translocation, a transmembrane signaling event dependent on DAG production.

PRINCIPAL FINDINGS

1. Membrane translocation of catalytically inactive DGK severely impedes membrane translocation of endogenous DGK and its lipid kinase activity
Stimulation of G-protein-coupled muscarinic type 1 receptor (HM-R1) ectopically expressed in Jurkat cells activates phospholipase C (PLCß) to generate inositol phosphates and DAG. The generation of these two second messengers has been shown to reproduce several TCR responses, validating this cell line (J-HM1-2.2) as an appropriate model for the study of functional T cell responses elicited by phosphatidylinositol turnover. We examined early signaling responses in J-HM1-2.2 cells stably expressing green fluorescent protein (GFP) (J-HM1-2.2-GFP) and a GFP-tagged kinase-dead DGK mutant (J-HM1-2.2-GFP-DGK-kd).

To examine the effect of GFP-DGK-kd on translocation of endogenous DGK to the plasma membrane, we analyzed expression of both proteins in membrane extracts of carbachol-treated cells. Concurring with our previous studies, DGK was found transiently at the membrane of carbachol-stimulated cells (Fig. 1 A) and GFP-DGK-kd remained at the membrane for as long as 1 h after carbachol addition (Fig. 1B ). Membrane levels of endogenous enzyme were lower in cells expressing GFP-DGK-kd than with nontransfected cells, confirming the transdominant negative capacity of GFP-DGK-kd. To confirm that DGK membrane translocation correlated with PtdOH generation in response to carbachol, we performed an in vitro phospholipid radiolabeling assay using [³²P]ATP and membrane extracts. PtdOH generation in carbachol-stimulated J-HM1-2.2-GFP cell membrane fractions was rapid (maximum at 5 min) and transient (Fig. 1C ). This increase was not observed in J-HM1-2.2-GFP-DGK-kd or R59949-pretreated J-HM1-2.2-GFP cells, suggesting that carbachol-induced PtdOH generation was prevented by GFP-DGK-kd expression as well as by pharmacological inhibition of the endogenous enzyme (Fig. 1C ). Together, these experiments confirm that blocking endogenous DGK translocation by expression of a transdominant negative form of this enzyme prevents DAG phosphorylation, allowing greater, more sustained production of this lipid within the plasma membrane.

View larger version (22K): [in this window] [in a new window]   Figure 1. Carbachol-stimulated DGK and GFP-DGK translocation in J-HM1-2.2-GFP and J-HM1-2.2-GFP-DGK-kd cells regulates in vitro PtdOH production in cell membranes. A) J-HM1-2.2-GFP and B) J-HM1-2.2-GFP-DGK-kd cells were stimulated with 50 µM carbachol, followed by rapid freezing of cell pellets. Membranes were prepared by high-speed centrifugation. Proteins were separated by SDS-PAGE, then transferred to nitrocellulose membranes; after blocking, membranes were probed with anti-DGK antibody. Representative Western blots are shown. C) J-HM1-2.2-GFP (pretreated with 0.1% v/v DMSO or 10 µM R59949 for 30 min) and J-HM1-2.2-GFP-DGK-kd cells were carbachol stimulated as in panel A, followed by rapid freezing of cell pellets. Membranes were prepared by high-speed centrifugation, followed by brief incubation with [32P]ATP and MgCl2. After total lipid extraction, radiolabeled phospholipids were separated by TLC and radioactivity incorporated into PtdOH was quantified by PhosphorImaging. A representative experiment is shown of 3 performed.

2. Carbachol-stimulated activation of extracellular-related kinase (Erk) and membrane translocation of RasGRP is sustained in J-HM1-2.2-GFP-DGK-kd cells but transient in J-HM1-2.2-GFP cells
In T cells, a pathway directly linking DAG accumulation to Erk activation (via Ras) is well documented. As GFP-DGK-kd expression induced increased, sustained accumulation of DAG mass, we determined how this expression affected carbachol-stimulated Erk activation. In J-HM1-2.2-GFP cells, carbachol rapidly stimulated phosphorylation of Erk-1 and Erk-2. By 30 min poststimulation, Erk phosphorylation had returned to near basal levels. Phosphorylation of both Erk isoforms was higher in J-HM1-2.2-GFP-DGK-kd than in J-HM1-2.2-GFP cells. At 30 and 60 min after carbachol stimulation, both Erk isoforms were phosphorylated to an extent comparable to that seen at 5 and 15 min postcarbachol stimulation in J-HM1-2.2-GFP cells. As a control, we studied PDBu and diC8-DAG activation of this pathway in both cell types. The degree of Erk phosphorylation by these two pharmacological agents was comparable in both cell types at 15 min; this suggests that the differences observed between the cell lines after carbachol addition reflected different levels of receptor-derived DAG mass rather than distinct responses to the lipid messenger.

3. T cell receptor signaling through RasGRP and Erk activation is sustained in J-HM1-2.2-GFP-DGK-kd cells compared with J-HM1-2.2-GFP cells
We next studied kinetics of Erk phosphorylation and RasGRP translocation after triggering endogenous TCR in DGK-kd cells. J-HM1-2.2-GFP and J-HM1-2.2-GFP-DGK-kd cells were stimulated with a low and high dose of an anti-CD3 antibody (OKT3) for 5 and 60 min. A low dose of OKT3 caused rapid, transient phosphorylation of both Erk isoforms in J-HM1-2.2-GFP cells; when a higher dose was used, Erk phosphorylation was greater (Fig. 2 A). When J-HM1-2.2-GFP-DGK-kd cells were stimulated with low and high doses of OKT3, Erk phosphorylation was greater at 5 and 60 min compared with J-HM1-2.2-GFP cells (Fig. 2A ). The decline in Erk phosphorylation at 60 min post-TCR stimulation was less pronounced in J-HM1-2.2-GFP-DGK-kd cells than with J-HM1-2.2-GFP cells. Through its C1 domain, RasGRP binds to DAG in the membrane. We determined the time course of RasGRP translocation to the membrane in response to TCR stimulation in both cell types. Membrane association of RasGRP was transient in J-HM1-2.2-GFP cells (Fig. 2B ); in J-HM1-2.2-GFP-DGK-kd cells, OKT3 stimulation resulted in immediate membrane association of RasGRP, which was constant for up to 60 min poststimulation (Fig. 2B ).

View larger version (61K): [in this window] [in a new window]   Figure 2. OKT3-stimulated Erk activation and RasGRP membrane association in J-HM1-2.2-GFP-DGK-kd and J-HM1-2.2-GFP cells. A) J-HM1-2.2-GFP and J-HM1-2.2GFP-DGK-kd cells were stimulated with 0.2 or 5 µg OKT3. Cell lysates were prepared and proteins separated and transferred to nitrocellulose membranes. After blocking, membranes were probed with anti-phospho-p44/42 MAP kinase or anti-p44/42 MAP kinase antibody. B) J-HM1-2.2-GFP and J-HM1-2.2-GFP-DGK-kd cells were stimulated with 5 µg soluble OKT3. Cell membranes were prepared, separated, and transferred to nitrocellulose membranes. After blocking, membranes were probed with anti-RasGRP antibody. Representative Western blots are shown.

CONCLUSIONS AND SIGNIFICANCE

Several studies in recent years have begun to define the roles of DGK in regulating cell functions. Receptor-regulated membrane localization of DGK causes phosphorylation of DAG, modifying the balance of two lipids with second messenger functions, DAG and PtdOH. Here we extend these observations to demonstrate that membrane localization and activation of DGK act as a ‘switch-off’ signal for Ras activation via RasGRP in T lymphocytes. Furthermore, DGK-kd expression is sufficient to modify the time course of RasGRP translocation to the plasma membrane and to induce higher, more sustained Erk phosphorylation.

Our experiments indicate that not only TCR cross-linking, but also stimulation of a G-protein-coupled receptor (GPCR), probably through PLCß, induces RasGRP translocation to the membrane. This is the first demonstration of RasGRP regulation after stimulation of GPCR in T lymphocytes. Future studies should assess the role of DAG-based regulation of Ras activation in response to other GPCR such as chemokine receptors, potent activators of PLC and Ras with important functions in regulating T lymphocyte migration.

The relevance of Ras activation by RasGRP in T cell response regulation has recently been highlighted by analysis of RasGRP-deficient mice. A marked thymic phenotype is detected in these animals due to severe inhibition of positive selection of thymic cells. Although RasGRP deficiency caused no apparent defect in differentiation from double-negative to double-positive cells, single-positive cells were almost completely absent in thymus and peripheral lymphoid organs. Neither anti-CD3 nor phorbol ester stimulated the Ras-Erk pathway in thymocytes from RasGRP-deficient mice, suggesting that RasGRP is essential in TCR-mediated Ras activation. We studied DGK expression during thymic differentiation and found that the enzyme, absent in the double-negative population, is highly expressed in double-positive cells (S. Outram, and I. Merida, unpublished results). Experiments are under way to study the role of DGK in thymic differentiation. RasGRP-deficient thymocytes do not proliferate when stimulated with anti-CD3 plus anti-CD28 antibodies, with phorbol ester and a Ca²⁺-ionophore, or even with exogenous IL-2. This suggests that IL-2 receptor-mediated Ras activation may also be RasGRP dependent. Previous studies suggested that DGK activation is essential for correct IL-2-driven T cell proliferation, but no studies have reported the role of DGK in Ras activation by IL-2.

Our studies allow us to postulate a model whereby Ras activation in T lymphocytes is determined by positive and negative regulation of DAG levels. Receptor triggering induces DGK translocation to the membrane, where the enzyme removes the DAG generated after PLC activation. This fosters RasGRP dissociation from the membrane and allows termination of the Ras activation signal (Fig. 3 ). DGK and RasGRP both translocate from cytosol to the membrane in response to tyrosine kinase receptor or GPCR triggering, suggesting a role in regulation of Ras by multiple extracellular stimuli.

View larger version (35K): [in this window] [in a new window]   Figure 3. Ras activation is determined by positive and negative DAG regulation. PLC-mediated hydrolysis of PIP2 leads to DAG accumulation in the cell membrane, causing RasGRP translocation and activation through its binding to DAG. After increased GTP loading and Ras activation, the Raf/Mek/Erk pathway is activated. Receptor-mediated DGK translocation decreases DAG levels, recycling RasGRP to the cytosol and inactivating Ras.

In lymphocytes, Ras activation can be induced by tyrosine kinase-coupled receptors for antigen, coreceptors, and cytokines, as well as by GPCR for several chemokines. Correct immune system homeostasis probably requires multiple mechanisms to activate/inactivate Ras, thus keeping this important growth and differentiation factor under strict control. The contribution to Ras activation of GEF recruitment to the membranes via protein-protein complexes and/or lipid binding may therefore depend on the type of receptor triggered or perhaps on the cell differentiative/proliferative state. This study suggests that DGK is a good candidate for mediating negative regulation of the DAG-dependent route to Ras activation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0762fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0762fje

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