From the Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
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ABSTRACT |
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Members of the Ras superfamily of small molecular weight GTPases play diverse and critical roles in mediating cellular responses to extracellular stimuli, including mitogenesis, cytoskeletal maintenance and rearrangement, and integrin activation. In T lymphocytes, biochemical and genetic evidence demonstrate that Ras plays an essential role in coupling T cell receptor ligation to signaling cascades required for T cell proliferation and development. Recent observations that C3G, a guanine nucleotide exchange factor specific for the Ras-related GTPase Rap1, is recruited into tyrosine-phosphorylated protein signaling complexes in activated T cells have suggested that Rap1 may also play a role in T cell activation. Utilizing a recently developed technique for detection of endogenous, GTP-bound Rap1, we have found that Rap1, but not Rap2, is transiently activated following T cell receptor stimulation of normal human T lymphocytes. Increases in intracellular calcium is both necessary and sufficient to induce Rap1 activation. Remarkably, costimulation of T cells with mitogenic anti-CD28 antibody completely abolished T cell receptor-dependent activation of Rap1. This report demonstrates a potential role for Rap1 in T cell receptor signaling and suggests inactivation of Rap1 as a candidate target of CD28-dependent costimulatory signals required for T cell antigen responsiveness.
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INTRODUCTION |
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Biochemical, mutational, and genetic studies have demonstrated
that physical and functional coupling of the T cell receptor (TCR)1 to associated Src (Fyn
and Lck) and Syk (Syk, ZAP-70) family protein-tyrosine kinases (PTKs)
plays a critical role in both T cell activation and development
(reviewed in Refs. 1 and 2). The generation of lipid second messengers,
mobilization of calcium stores, and gene transcription critical for T
cell proliferation and cytokine secretion is entirely dependent on TCR-associated PTK activity (1, 3). In normal human T lymphocytes, ligation of the TCR alone is insufficient to induce proliferation and
cytokine secretion, and instead induces anergy, a long term unresponsiveness to antigen stimulation. Costimulation of T cells by
soluble interleukins (ILs) (e.g.. IL-2, -4, -7, or -15) or by ligation of the T cell accessory protein CD28 by antibodies or B7-1
and B7-2 ligands on antigen presenting cells avoids induction of
anergy and leads to a full proliferative T cell response (4, 5). The
mechanism(s) by which CD28 stimulation prevents anergy and cooperates
with TCR-derived signals to induce T cell proliferation are poorly
understood. CD28 can activate both Src and Tek family PTKs (reviewed in
Ref. 6). CD28-dependent PTK activity phosphorylates a
number of T cell signaling proteins previously shown to be
tyrosine-phosphorylated in response to TCR stimulation, including
phospholipase C-1, SLP-76, p36/38, and the proto-oncogene products
Vav and Cbl (1, 2, 6-8). Similarly, CD28 stimulation also leads to
activation of phosphatidylinositol 3-kinase and calcium mobilization in
T lymphocytes (reviewed in Ref. 6). However, CD28 stimulation also
generates second messengers distinct from TCR signaling, including
ceramide (9) and reactive oxygen intermediates (10), which may play a
role in the ability of CD28 stimulation to synergistically activate Jun
kinases and enhance IL-2 mRNA transcription and stability in
CD3-stimulated T cells (4, 6).
In T cells, stimulation by either anti-CD3 or anti-CD28 antibodies results in recruitment of the Ras nucleotide exchange factor Sos to tyrosine-phosphorylated Shc and p36/38 proteins via the adaptor protein Grb2 (7, 8, 11, 12). Subsequent conversion of Ras to its active, GTP-bound form (13) allows membrane localization and activation of Raf, which initiates a cascade of serine/threonine kinase activation, culminating in activation of transcription factors, gene transcription, and mitogenesis (14). Studies in transgenic mice and fetal thymic organ cultures have confirmed that an intact Ras signaling pathway is required for proper T cell development and function (15-19), and blocks in Ras signaling pathways are found in anergized T cells (20, 21).
Recent studies in B and T lymphocytes have suggested a novel signaling pathway in lymphocytes involving the Ras-related GTPase Rap1. Antigen receptor-dependent tyrosine phosphorylation of the proto-oncogene product Cbl induces Cbl association with SH2 domain-containing adaptor proteins, including Crk family proteins (22-24) and the p85 subunit of phosphatidylinositol 3-kinase (25-28). In these studies, it was observed that CrkL was constitutively associated via its SH3 domains to the guanine nucleotide exchange factor C3G, which has guanine nucleotide exchange activity specific for Rap1 (29, 30). As C3G could be detected in complex with tyrosine-phosphorylated Cbl (22), it was suggested that Rap1 may be activated as a consequence of TCR stimulation. Rap1 was originally identified by its ability to revert viral Kras oncogenic transformation in fibroblasts, yet potential roles and functions for Rap1 in physiological signaling pathways remain poorly understood (31, 32). Rap1 has an effector domain nearly identical to Ras and binds to many of the same effector proteins as Ras, including Raf-1, the catalytic subunit p110 of phosphatidylinositol 3-kinase and the Ral guanine nucleotide exchange factors in vitro, suggesting that Rap1 might antagonize Ras signaling by sequestering potential Ras effectors in inactive complexes (33-36). Recent observations that Rap1 can mediate cAMP-dependent B-Raf and MAP kinase activation in PC12 cells (37) and can enhance mitogenic signaling pathways in Swiss 3T3 responses to insulin (38) suggest that in certain cells, Rap1 could contribute positive signals to mitogenic responses.
In this study, we utilize a recently developed technique for detecting GTP-bound Rap proteins (39) and demonstrate that Rap1 is transiently activated in TCR-stimulated normal human T cell PHA blasts and the cytotoxic T cell clone D11 but not in the leukemic T cell line Jurkat. Although TCR-mediated Rap1 activation is calcium-dependent, either phorbol ester or calcium ionophore alone are sufficient to induce Rap1 activation. Remarkably, activation of Rap1 by cross-linking TCR/CD3 could be completely inhibited by costimulation through CD28, suggesting that Rap1 may play a role in discerning the presence of costimulation during TCR-dependent activation.
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MATERIALS AND METHODS |
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Cells-- The human T cell leukemia cell line Jurkat was maintained as described previously (25). Human T cell PHA blasts were derived from fresh citrated human blood obtained from healthy volunteers (Minidonor Blood System, Academic Hospital, Utrecht and the Red Cross Blood Bank, Utrecht). Enriched peripheral blood lymphocytes were obtained by centrifugation over Ficoll-Paque (Pharmacia Biotech, The Netherlands) and two successive rounds of monocyte depletion by plastic adhesion. Resulting cells were then stimulated for 48 h with PHA (1 µg/ml; Sigma) and recombinant IL-2 (20 units/ml; ICN Biomedicals, The Netherlands) in RPMI 1640 media (containing 10% fetal bovine serum (Integro B.V., Netherlands), 0.03% glutamine, 100 units/liter each penicillin and streptomycin) (all media components from Imperial, UK, unless otherwise noted), washed, and expanded for an additional 4-8 days in media containing 20 units/ml IL-2 alone. PHA blasts were removed from IL-2 24 h before experiments to obtain quiescent cells. Maintenance, culture, and stimulation of the D11 allogeneic cytotoxic CD8+ human T cell clone was as described previously (40).
Lymphocyte Activation-- Washed cells were resuspended in RPMI 1640 media and incubated for 30 min on ice in the absence or presence of activating monoclonal antibodies against CD3 (SpV T3b, a generous gift of Dr. Hergen Spits, Netherlands Cancer Institute, Amsterdam) and/or CD28 (15E8, a mitogenic antibody kindly provided by Dr. René van Lier, Red Cross Central Blood Bank, Amsterdam) (41). After centrifugation at 4 °C and two washes in cold RPMI 1640, cells were activated by resuspension in 37 °C RPMI 1640 containing 10 µg/ml cross-linking goat anti-mouse antibodies (Cappel). Cold lysis buffer containing 0.5% Triton X-100, 50 mM Tris, pH 7.6, 150 mM sodium chloride, and phosphatase and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM sodium orthovanadate, and 10 mM sodium fluoride) was added after 2 min, unless indicated otherwise in figure legends, and were cells lysed for 45 min at 4 °C.
In some experiments, cells were stimulated by resuspension in prewarmed media containing 100 ng/ml TPA (Sigma) or 1 µM ionomycin (calcium salt; Calbiochem). For experiments using pharmacological inhibitors, cells were incubated with BAPTA-AM (30 min), staurosporine (30 min), or herbimycin A (2 h) (all from Sigma) at 37 °C at the concentrations indicated in figure legends before washing and incubation with antibodies as described above.Antibodies and Fusion Proteins-- Monoclonal antibodies used were anti-Rap2A (Transduction Laboratories) and anti-phosphotyrosine 4G10 (Upstate Biotechnology). Anti-Rap1 antibodies were generated in our laboratory by immunization of rabbits with synthetic Rap1A carboxyl-terminal peptide (amino acids 100-114, CDLVRQINRKTPVEKK, cysteine added for coupling) (Eurogentec, Belgium) coupled to keyhole limpet hemocyanin (Pierce). Anti-MAP kinase ERK2 polyclonal antibodies have been previously described (42).
Plasmid-encoding glutathione S-transferase (GST) fused to amino acids of the Rap binding domain (RBD) of Ral-GDS and purification of GST-RBD fusion protein have been previously described (36).Immunoprecipitations, Binding Reactions, SDS-PAGE, and Immunoblotting-- Lysates were clarified for 10 min at 4 C° by centrifugation at 14,000 rpm. For GST-RBD binding reactions, lysate corresponding to 5-7.5 × 106 cell equivalents were incubated for 45 min at 4 °C with 5 µg of GST-RBD precoupled to glutathione-agarose beads (Pharmacia). Resolution of bound proteins by SDS-PAGE and detection by immunoblotting and ECL using horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin antibodies (New England BioLabs) were as described previously (39). For all Rap binding experiments, equivalent portions of cell lysates used for binding reactions were loaded on separate SDS-PAGE gels and analyzed for Rap1 content to confirm that equivalent amounts of Rap1 were available for binding (data not shown).
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RESULTS AND DISCUSSION |
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TCR Stimulation Transiently Activates Rap1 in T Lymphocytes-- Recent reports have suggested that Rap1 may be involved in B and T cell antigen receptor signaling in lymphocytes, as these studies reported recruitment of the Rap1-specific guanine nucleotide exchange factor C3G to tyrosine-phosphorylated Cbl, an early and prominent substrate of antigen receptor-associated PTKs, via the CrkL adaptor protein (22). To assess if TCR stimulation activated Rap1 in T lymphocytes, we utilized a recently developed novel technique using the RBD of Ral-GDS to precipitate GTP-bound Rap1 from cell lysates. GST-RBD fusion proteins bind with high affinity to GTP-bound Rap1 and Rap2 (Kd 10 nM) although binding negligibly to GDP-bound Rap proteins (Kd 10 µM) (36) and can be utilized as sensitive probes for detecting activated GTP-bound Rap1 proteins in cell lysates. Our laboratory has previously used this technique to report the thrombin-dependent activation of Rap1 in human platelets, where treatment with thrombin led to rapid, calcium-dependent activation of Rap1 (39).
We first compared levels of GTP-bound active Rap1 in normal human T cell PHA blasts before or after TCR/CD3 stimulation. T cells were incubated in the absence or presence of anti-CD3 antibody, followed by washing and activation by cross-linking antibody for 2 min. After lysis, endogenous GTP-bound Rap1 was precipitated with immobilized GST-RBD fusion protein and detected by anti-Rap1 immunoblotting (Fig. 1). Small amounts of activated Rap1 were detected in unstimulated T cells, and after TCR stimulation, a distinct increase in GTP-bound Rap1 was observed. Anti-Rap1 immunoblotting of whole cell lysates confirmed that equivalent amounts of Rap1 were available for binding in all samples (data not shown). The basal levels of GTP-bound Rap1 varied between experiments, but in each of four independent experiments, an estimated 2-5-fold increase in Rap1 GTP content was observed.
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Activation of Rap1 by TCR Stimulation is Calcium-dependent-- Previous studies in human platelets demonstrated that treatment of platelets with phorbol ester or calcium ionophore was sufficient to induce Rap1 activation, and activation of Rap1 by thrombin was completely dependent on calcium (39). To assess if CD3-dependent activation of Rap1 in T cells was similarly regulated, we examined the ability of phorbol ester and calcium ionophore, alone or in combination, to activate Rap1 in T cell PHA blasts. T cells treated with TPA demonstrated activation of Rap1 within 1 min (Fig. 3A, upper panel). Activation was maximal within 2-5 min of exposure to TPA, before dropping toward basal levels by 10 min. Maximal activation was estimated to be 2-3 times greater than that observed by CD3 stimulation (second lane). Treatment of PHA blasts with the calcium ionophore ionomycin also resulted in transient activation of Rap1 (Fig. 3A, middle panel). However, ionomycin-induced Rap1 activation was maximal after 1 min and returned quickly toward basal levels. Treatment with a combination of TPA plus ionomycin resulted in activation kinetics of Rap1 that appeared to represent a composite of the kinetics observed when TPA and ionomycin were administered individually (Fig. 3A, lower panel). In this case, Rap1 activation was highest 1 min after stimulation but remained well above basal levels for 5 min. Based on comparisons of GST-bound Rap1 with whole cell lysates representing 5% of protein used in binding reactions, treatment with TPA and/or ionomycin converted 15-20% of cellular Rap1 to its GTP-bound state. In contrast, stimulation of Jurkat with ionomycin or TPA alone or in combination failed to activate Rap1 (Fig. 3B).
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Costimulation through CD28 Prevents CD3-dependent Rap1 Activation-- It has recently been reported that ligation of the T cell accessory protein CD28, like TCR stimulation, also leads to tyrosine phosphorylation of the Cbl proto-oncogene (8), and we therefore anticipated that CD28 ligation might also induce Rap1 activation in T lymphocytes. Quiescent T cells were incubated with media alone or media containing anti-CD3, anti-CD28, or a combination of anti-CD3 and anti-CD28 antibodies. Stimulation by CD3 again increased the amount of GTP-bound Rap1 as compared with unstimulated T cells (Fig. 5A, left panel). In contrast, stimulation of CD28 failed to activate Rap1. Strikingly, costimulatory anti-CD28 antibodies, when combined with anti-CD3 antibodies, completely blocked CD3-dependent Rap1 activation. Indeed, levels of GTP-bound Rap1 were reduced below levels observed in unstimulated T cells. Identical results were observed in three independent experiments, excluding donor-specific effects. Stimulation through CD28, either alone or in combination with anti-CD3 antibodies, failed to modulate Rap2 activation (Fig. 5A, right panel). Time course analysis of Rap1 GTP loading after stimulation by anti-CD28 or anti-CD28 plus anti-CD3 antibodies revealed that the effect of CD28 on CD3-dependent Rap1 activation represented a complete block in Rap1 activation rather than a shift in activation kinetics (Fig. 5B). Treatment of T cells with anti-CD28 antibodies alone resulted in a slow decrease in GTP-bound Rap1, as compared with levels in unstimulated cells over a period of 10 min, to nearly undetectable levels (Fig. 5B, left panel). Costimulation through TCR and CD28 led to a more rapid decrease in levels of GTP-bound Rap1 (right panel). Under these conditions, GTP-bound Rap1 could no longer be detected 2 min after stimulation. Neither CD28 or TCR plus CD28 stimulation resulted in activation of Rap2 (data not shown). Thus, stimulation through CD28 initiates signals that result in a decrease in GTP-bound Rap1 and completely blocks CD3-dependent conversion of Rap1 to its active state.
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ACKNOWLEDGEMENTS |
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We acknowledge Miranda van Triest for expert technical assistance and Dr. René Medema for generating anti-Rap1 antiserum. We are greatly indebted to Drs. Hergen Spits (Netherlands Cancer Institute, Amsterdam), Paul Coffer, Rolf de Groot, Frank Staal (University Hospital, Utrecht), and René van Lier (Red Cross Central Blood Bank, Amsterdam) for contributing critical reagents. Lastly, we thank Dr. Hergen Spits and Drs. Boudewijn Burgering and Fried Zwartkruis in our laboratory for critical reading of this manuscript.
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FOOTNOTES |
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* This research was supported by an European Molecular Biology long term fellowship (to K. A. R.) and by funding from Onyx Pharmaceuticals (to J. L. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31 30 2538959;
Fax: 31 30 2539035; E-mail: k.a.reedquist{at}med.ruu.nl.
1 The abbreviations used are: TCR, T cell receptor; PTK, protein-tyrosine kinases; IL, interleukin; MAP, mitogen-activated protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; ERK, extracellular-regulated kinase; GST, glutathione S-transferase; RBD, Rap binding domain; Ral-GDS, Ral guanine nucleotide dissociation stimulator; PAGE, polyacrylamide gel electrophoresis.
2 K. A. Reedquist, unpublished observation.
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REFERENCES |
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