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Previous Article | Next Article 
Molecular and Cellular Biology, April 2004, p. 3485-3496, Vol. 24, No. 8
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.8.3485-3496.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Pathology and New York University Cancer Institute,1 Departments of Medicine, Pharmacology and Cell Biology, New York University School of Medicine, New York, New York 100162
Received 28 January 2003/ Returned for modification 1 April 2003/ Accepted 17 December 2003
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ABSTRACT |
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- and RasGRP1-dependent fashion. The specificity of N-Ras signaling downstream of low-grade TCR stimulation was dependent on the monoacylation of the hypervariable membrane targeting sequence. Our data show that, in contrast to fibroblasts stimulated with growth factors in which all three Ras isoforms become activated and signaling occurs at both the plasma membrane and Golgi apparatus, Golgi-associated N-Ras is the critical Ras isoform and intracellular pool for low-grade TCR signaling in Jurkat T cells. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammals have three functional ras genes, H-ras, K-ras, and N-ras, the products of which have very similar structures (26). The K-ras gene contains two alternative fourth coding exons, giving rise to two splice variants, K-Ras4A and K-Ras4B. Since the K-Ras4A alternative accounts for less than 10% of total K-ras mRNA, we will refer to K-Ras4B as the K-Ras isoform. At the amino acid level, Ras isoforms are identical for the first 80 amino acids, exhibit 85% identity for the next 80 residues, and display only 15% amino acid conservation within the C-terminal 25 amino acids (3, 6). The C-terminal hypervariable region directs the posttranslational modifications of the primary ras gene products that determine their subcellular localization (18, 19).
Ras proteins play an important role in the signaling pathways that activate cytokine gene induction and in the control of T-cell development (17). Since the activation of Ras upon T-cell stimulation was first demonstrated (12), a critical role of Ras in antigen receptor signaling in lymphocytes has been appreciated (1, 16, 20). In fact, the loss of Ras function prevents the proliferation, cytokine production, and lymphocyte development induced by the recognition of the antigen (39, 43).
A number of functional differences between the Ras isoforms have been reported (26). For example, different ras genes have been found mutated in different tumor types (5, 33), and mice deficient in the different Ras isoforms exhibit different developmental phenotypes (14, 21, 24, 44). Despite these differences, the specific function(s), if any, of the various Ras isoforms is poorly understood. However, a number of studies have pointed out the importance of N-Ras in T-cell function. Firstly, activating mutations of N-Ras are frequently found in human and mouse hematopoietic tumors (5, 27, 33, 38, 42). More recently, by using an N-Ras-deficient mouse model, we have shown that N-Ras is an important component of the T-cell signaling network and its function (29). The functional consequences of the absence of N-Ras in T cells include deficient CD8+ selection, a decreased thymocyte proliferation, a significant reduction in the production of interleukin-2 upon thymocyte activation, and an increased sensitivity to influenza infection in vivo.
The purpose of this work was to determine the mechanism(s) underlying the specific role of N-Ras in T-cell function. Our results show that, although all three Ras isoforms are expressed in human T cells, N-Ras is the only isoform activated following low-grade stimulation of the T-cell receptors (TCR) in Jurkat T cells. Moreover, N-Ras activation takes place exclusively on the Golgi apparatus as a consequence of signaling through phospholipase C1 (PLC1) and RasGRP1.
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MATERIALS AND METHODS |
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1-deficient mutant (J gamma 1) are derived from a human acute T-cell leukemia and were obtained from the American Type Culture Collection. CEM (CCRF-CEM) and Karpas (KARPAS-299) cell lines are derived from a human T-cell acute lymphoblastic leukemia and a human T-cell non-Hodgkin lymphoma, respectively. HEK293 cells, which are a permanent line of primary human embryonic kidney cells, were also obtained from the American Type Culture Collection. All the cells were kept at logarithmic growth in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U of penicillin G and streptomycin each per ml. The majority of the transfection assays were performed by lipofection with DMRIE-C (Gibco BRL) (for Jurkat) or Superfect (Gibco BRL) (for COS-1) and the conditions recommended by the manufacturer. In the indicated cases, Amaxa technology was used to transfect Jurkat T cells according to the manufacturer's recommendations.
Plasmids, antibodies, and reagents. Yellow fluorescent protein (YFP)-Ras-binding domain (RBD), untagged Ras, green fluorescent protein (GFP)-Ras, and cyan fluorescent protein (CFP)-Ras vectors were previously described (8, 9). CFP-H-RasC184L and CFP-N-RasL184C palmitoylation mutants were generated by using a QuikChange site-directed mutagenesis kit (Stratagene). Human RasGRP cDNAs were amplified by PCR (primer sequences available upon request) and cloned in frame into the mammalian expression vectors pYFP-N1 (Clontech) and pcDNA3.1(+)/Neo (Invitrogen). All plasmids were verified by bidirectional sequencing. Antibodies used for Ras detection included agarose-conjugated anti-pan-Ras Y13-259 (Oncogene Research) and monoclonal antibodies for mouse N-Ras (F155), H-Ras (F235), and K-Ras (F234) (Santa Cruz Biotechnology). Mouse anti-human CD3 (UCHT1) and CD28 (5D10) (Ancell) were used for TCR-dependent activation, whereas phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Sigma-Aldrich) was used for TCR-independent activation of Jurkat cells.
Cell stimulation and imaging. For TCR-dependent stimulation, Jurkat cells were incubated with high (5 µg/ml) or low (1 µg/ml) doses of both mouse anti-human CD3 plus anti-CD28 antibodies. PMA (100 ng/ml) plus ionomycin (500 ng/ml) was used for TCR-independent stimulation. For the specific microlocalization of Ras proteins in the plasma membranes of T cells, Jurkat cells were first incubated with anti-CD3 and anti-CD28, washed with phosphate-buffered saline, and then incubated with Texas red-conjugated donkey anti-mouse immunoglobulin G (heavy plus light chains) (Jackson ImmunoResearch Laboratories, Inc). The stimulation of COS-1 cells was performed by adding 40 ng of epidermal growth factor (EGF)/ml to the media. For examination by fluorescence microscopy, the cells were plated in 35-mm dishes containing a glass coverslip-covered 15-mm cutout (MatTek). By using a Harvard Apparatus microincubator and dual confocal microscopy, the images were captured with a Zeiss 510 laser-scanning confocal microscope (LSM) with the manufacturer-specified filter sets for single and dual emissions. TIFF images were processed with Adobe Photoshop 6.0.
Protein expression and Ras activation. For the detection of Ras variants, the proteins were extracted in 200 µl of lysis buffer (10% glycerol, 1% Nonidet P-40, 50 mM Tris-HCl [pH 7.4], 200 mM NaCl, 2.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 10 µg of soybean trypsin inhibitor per ml, 0.1 µM aprotinin). Lysates were cleared by centrifugation, and the protein concentration was quantified by the Bradford method. Ras proteins from Jurkat cells (1 mg per sample) were also immunopurified by using an anti-pan-Ras immunoaffinity column. In all cases, the proteins were fractionated in triplicate in sodium dodecyl sulfate-15% polyacrylamide gels, and Ras variants were detected by using antibodies specific for each isoform.
For Ras activation experiments, 2 x 107 Jurkat T cells were serum starved at 37°C for 2 h and then incubated in the presence or absence of anti-CD3 plus anti-CD28 or PMA plus ionomycin for 10 min. To detect GTP-bound Ras, 75% of the cells were quickly sedimented and lysed in 400 µl of lysis buffer. Lysates were cleared by centrifugation, incubated for 2 h at 4°C with glutathione S-transferase (GST)-Raf-RBD fusion protein (a gift of J. L. Bos, Utrech University, Utrech, The Netherlands) coupled to glutathione agarose beads, and washed four times with lysis buffer. To detect total Ras, the remaining cells (25%) were lysed in 100 µl of Laemmli buffer. Finally, GTP-bound and total Ras proteins were detected by Western blotting with isoform-specific antibodies.
Active ERK1/2 proteins were detected in the primary lysates by using phospho-specific antibodies according to the manufacturer's recommendations (Promega). To normalize the activation of endogenous ERK1/2, lysates were also probed with anti-ERK1/2.
In all cases, the immunoblots were revealed by using Supersignal West Femto maximum sensitivity substrate (Pierce). Scanned TIFF images were processed and bands were quantified with the Quantity One software (Bio-Rad).
Indirect immunofluorescence assays. Jurkat T cells were fixed with 4% paraformaldehyde and permeabilized and blocked with 0.5% Triton X-100-5% bovine serum albumin in phosphate-buffered saline. The cells were dual stained with monoclonal antibodies to N-Ras (F155; Santa Cruz Biotechnology) or RasGRP1 (M133; a gift of James C. Stone, University of Alberta, Alberta, Canada) and a polyclonal antiserum against human giantin (PRB-114; Covance Research) (each diluted 1:250), followed by Texas red-conjugated horse anti-mouse combined with fluorescein isothiocyanate-conjugated goat anti-rabbit antisera (Jackson ImmunoResearch). The cells were mounted with photobleach retardant medium (DAKO Corp.), and they were imaged with a Zeiss 510 inverted LSM.
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RESULTS |
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The apparent differential activation of N-Ras and K-Ras upon low-grade TCR stimulation may be due to different sensitivities of the method toward the different isoforms. However, two factors argue against this possibility. First, there is no evidence that GST-RBD shows any Ras isoform preference. Second, the K-Ras- versus N-Ras-specific antibodies used to analyze the pulldown were equally efficient at recognizing their cognate proteins (data not shown). Nevertheless, we directly tested the possibility that the difference was due to different sensitivities of detection by activating Jurkat T cells with either a high concentration (10 µg/ml) of anti-CD3 and anti-CD28 or a strong TCR-independent stimulus (PMA plus ionomycin). In both cases, K-Ras activation was readily detected in this cell type, and no differences were detected between its activation levels and those of N-Ras (Fig. 1C). Thus, the preferential activation of N-Ras upon low-grade TCR stimulation is not due to differential sensitivities of the assay but rather to an intrinsic difference among the isoforms that is likely to have physiologic consequences.
Although mammalian ras genes are expressed in all cell lineages and organs, some differences have been detected in the levels of expression of each of these genes in embryonic development and in various adult tissues (15, 25). For example, human leukemia cells and mouse primary thymocytes express substantially more N-Ras and K-Ras than H-Ras (25, 37). Since differential expression of Ras isoforms could explain why only N-Ras was activated in Jurkat cells upon TCR engagement, we determined the levels of expression of N-, K-, and H-Ras in human T cells. First, we determined the relative affinities of the isoform-specific anti-Ras antisera and discovered that, whereas the anti-K-Ras and anti-N-Ras reagents had very similar affinities for their cognate antigens, the anti-H-Ras antiserum was 10-fold more efficient (data not shown). Ras expression in Jurkat cells, two other human T-cell lines (Karpas and CEM), and a human epithelial cell line (HEK293) was analyzed by immunoblotting with these isoform-specific antibodies (40). HEK293 cells expressed all three Ras isoforms (Fig. 2A). In contrast, whereas all three T-cell lines expressed relatively high levels of K- and N-Ras, we did not detect H-Ras expression in these cells, despite the higher sensitivity afforded by the anti-H-Ras antibody (Fig. 2A). To increase the sensitivity of the assay, we immunoprecipitated all cellular Ras with a pan-Ras antibody and then probed the immunoprecipitates with isoform-specific antisera. By using this method, H-Ras was detected in Jurkat cells, although at a much lower level than were K-Ras and N-Ras (Fig. 2B).
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20%), recruitment was exclusively to the plasma membrane (Fig. 4A, f). Importantly, no membrane recruitment to any compartment was observed in cells coexpressing YFP-RBD and K-Ras following low-grade TCR stimulation (Fig. 4A, i). Thus, results with YFP-RBD membrane recruitment (Fig. 4A) were concordant with those obtained by GST-RBD pulldown (Fig. 1) and confirmed that, under conditions of low-intensity TCR stimulation, N-Ras was preferentially activated. Moreover, the latter assay demonstrated that the pool of activated N-Ras was restricted to the Golgi apparatus.
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To unambiguously identify the Golgi apparatus as the paranuclear structure on which N-Ras and H-Ras are activated upon TCR engagement, we used Golgi-specific markers in both the live-cell imaging and indirect immunofluorescence of fixed cells (Fig. 5). We cotransfected Jurkat cells with CFP-tagged N-Ras or H-Ras plus the Golgi marker galactosyl transferase (GalT) tagged with YFP. The paranuclear pool of N-Ras and H-Ras colocalized with GalT (Fig. 5A), confirming the Golgi localization of both Ras isoforms. We also analyzed the localization of endogenous N-Ras by indirect immunofluorescence with an N-Ras isoform-specific antibody and anti-giantin as a Golgi marker. Interestingly, the staining for endogenous N-Ras was stronger on the Golgi apparatus than on the plasma membrane (Fig. 5B). These data demonstrate that the Golgi localization of CFP-N-Ras reflects the localization of the endogenous protein in Jurkat cells.
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TCR stimulation in Jurkat cells activates N-Ras via PLC and RasGRP1.
It has recently been demonstrated that H-Ras activation on the Golgi apparatus of fibroblasts is mediated by a pathway dependent on PLC and RasGRP1 (4, 7). Similar results were obtained in Jurkat cells overexpressing H-Ras (4). Because the in vitro studies described above and previous in vivo studies of lymphocytes from N-Ras-deficient mice (29) suggest that Ras signaling in lymphocytes may preferentially involve N-Ras, we sought to extend the studies of PLC and RasGRP1 to N-Ras. Whereas N-Ras was expressed on both the plasma membrane and the Golgi apparatus in Jurkat cells deficient in PLC, stimulation of the TCR failed to induce the activation of N-Ras on any compartment (Fig. 7A). Similar results were obtained when wild-type Jurkat cells were pretreated with the PLC inhibitor U73122 (4). In contrast, when the TCR was bypassed by stimulation with PMA and ionomycin, YFP-RBD was recruited to the Golgi apparatus in PLC-deficient cells (Fig. 7A). The spatially resolved results obtained with YFP-RBD recruitment were recapitulated by GST-RBD pulldown assays. Low-grade stimulation of the TCR activated N-Ras in wild-type, but not PLC-deficient, Jurkat cells (Fig. 7B). In contrast, bypassing the TCR by stimulation with PMA plus ionomycin activated N-Ras in both types of cells (Fig. 7B). Thus, the activation of N-Ras on the Golgi apparatus of Jurkat cells following TCR stimulation depends on PLC.
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/Ca2+ plus diacylglycerol/RasGRP1-dependent pathway.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until recently, the three Ras isoforms have been considered redundant, and few biochemical differences have been described. The embryonic lethality of K-Ras deficiency (24), but not of N-Ras (44) or H-Ras (14) deficiencies, demonstrates conclusively that the functions of all Ras proteins are not entirely overlapping. Differential membrane trafficking of the various Ras isoforms is firmly established (9) and has led to a search for cell biological rather than biochemical differences among the isoforms. Recent studies have focused on isoform differences in localization in plasma membrane microdomains (32, 34) and in endomembrane signaling (8) as possible explanations.
TCR are believed to signal from plasma membrane microdomains known as lipid rafts that are enriched in signaling molecules, including the adaptor protein LAT and enzymes such as Lck and PLC (23). The enrichment of Ras in lipid rafts is somewhat controversial (31). In two recent studies of nonlymphoid cells, H-Ras was enriched in lipid rafts but K-Ras was excluded (32, 34). Our failure to observe K-Ras in clustered rafts on Jurkat cells is consistent with these studies. Interestingly, whereas inactive H-Ras was found to be enriched in lipid rafts, activated, GTP-bound H-Ras was excluded, suggesting a dynamic interaction with the microdomain (32). Our observation that H-Ras is not activated by TCR stimulation but that it is nevertheless colocalized with the receptor in lipid rafts is consistent with the lipid raft association of inactive H-Ras. In support of this interpretation, when GFP-H-Ras61L, a constitutively active mutant, was substituted in our system for GFP-H-Ras, the GTP-bound H-Ras protein failed to cocap with the TCR (I. Pérez de Castro, T. G. Bivona, A. Pellicer, and M. R. Philips, unpublished observation). Although in MDCK cells N-Ras has been colocalized with H-Ras in lipid rafts (28), its localization had not been previously analyzed in T cells. Our results demonstrate that N-Ras behaves like K-Ras in failing to partition into the T-cell membrane microdomains defined by TCR and CD8. From these data, we conclude that diacylation is required for Ras proteins to partition into lipid rafts of Jurkat T cells. Since the diacylated form of Ras, H-Ras, is expressed at very low levels in T cells and, even when overexpressed, is not activated downstream of the TCR, we further conclude that the lipid rafts of T-cell plasma membranes do not participate directly in Ras activation and cannot explain the preference for N-Ras over K-Ras in activation following low-grade TCR stimulation.
Having failed to explain the isoform preference of Ras signaling in T cells on plasma membrane microdomains, we next investigated subcellular compartment-specific signaling. It has recently been demonstrated that, although H-Ras expressed ectopically in Jurkat cells was present on both the plasma membrane and Golgi apparatus, the signaling in response to high-grade TCR activation was restricted to the Golgi apparatus and was dependent on PLC and RasGRP1 (4). Moreover, we showed that the Ca2+-activated Ras GAP CAPRI blocked H-Ras activation on the plasma membrane (4). We have now shown that, like H-Ras, N-Ras was activated only on the Golgi apparatus following TCR stimulation. Unlike H-Ras, low-grade TCR stimulation was sufficient to activate N-Ras on the Golgi apparatus. N-Ras activation on the Golgi apparatus was also dependent on both PLC and RasGRP1. These results suggest that plasma membrane-associated Ras exchange factors such as Grb2/Sos are counterbalanced in T cells by CAPRI. These data are consistent with the finding that the mutation of the phospho-tyrosine docking sites for Grb2/Sos on LAT does not inhibit TCR-mediated Ras activation (46). However, since we observed the activation of K-Ras during high-grade stimulation of TCR, our results are consistent with a role for Grb2/Sos in the activation of K-Ras on the plasma membrane. Thus, the intensity of TCR stimulation controls not only the Ras isoform utilization but also the subcellular compartment from which the Ras signal is propagated.
Our analysis of palmitoylation mutants of N-Ras and H-Ras demonstrates that monoacylation regulates the ability of Golgi-associated Ras to become activated in response to low-grade TCR stimulation. One model that may explain this result is that in which monoacylation is required for the relevant Ras protein to partition into the proper microdomain of the trans-Golgi network membrane to be acted upon by RasGRP1. Alternatively, mono- versus diacylation may specify interactions between Ras and various guanine nucleotide exchange factors or GAPs. Indeed, the posttranslational modification of Ras influences not only subcellular localization but also interaction with regulators (35, 41).
Because working with primary lymphocytes presents obstacles, several T-cell lines have been extensively used for studying T-cell signaling and function. Although the results obtained with these cell lines require validation in primary T cells, increasing evidence supports the utility of Jurkat and other T-cell lines in elucidating signaling pathways. For example, the requirement for RasGRP1 in the activation of N-Ras on the Golgi apparatus of Jurkat cells is consistent with the severe impairment in Ras signaling observed in murine T cells deficient in this exchange factor (11). Indeed, RasGRP1 has been strongly associated with Ras activation in T cells, thymocyte development, and TCR signaling (11, 13). Importantly, it was recently reported that RasGRP1 plays a critical role in T-cell development, homeostasis, and differentiation by transducing low-grade TCR signals (30). Moreover, N-Ras-deficient mice are also defective in some T-cell functions mediated by low-grade stimuli (29). Thus, the striking similarities between the T-cell phenotypes of RasGRP1- and N-Ras-deficient mice can be explained by the elimination of elements of a common pathway. These data strongly support the idea that the TCR/PLC/RasGRP1/N-Ras pathway plays a pivotal role in low-grade TCR signaling.
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ACKNOWLEDGMENTS |
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This work was supported by grants AI36224 and GM55279 (to M.R.P.), grants CA36327 and CA50434 (to A.P.) from the National Institutes of Health, the New York State Breast Cancer Research Program, and the Burroughs Welcome Fund (to M.R.P.), and by a General Clinical Research Center grant from NIH NCRR (M01RR00096) awarded to the New York University School of Medicine.
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FOOTNOTES |
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