Ras activation is critical for T-cell function (17
). Recently, we analyzed the role of N-Ras in T-cell signaling by using mice deficient in N-Ras (29
). Compared to wild-type littermates, mice lacking N-Ras were more sensitive to low-titer influenza virus infection. Moreover, N-Ras-deficient mice exhibited a defect in the selection of CD8-positive T cells, a process that is regulated by low-grade TCR signaling (45
). In the present work, we have complemented these in vivo genetic experiments with a cell biological analysis of Ras isoform expression and signaling in T cells. Our study demonstrates that, under conditions of low-grade TCR stimulation, N-Ras is preferentially activated in Jurkat T cells. This observation explains why other Ras isoforms cannot substitute for N-Ras in deficient animals and demonstrates a critical role for N-Ras in T-cell function.
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
) 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 PLCγ (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
). 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 PLCγ 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 PLCγ 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
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
). 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/PLCγ/RasGRP1/N-Ras pathway plays a pivotal role in low-grade TCR signaling.