Ras Activation in Jurkat T cells following Low-Grade Stimulation of the T-Cell Receptor Is Specific to N-Ras and Occurs Only on the Golgi Apparatus

Department of Pathology and New York University Cancer Institute, New York, New York 10016, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 05/2004; 24(8):3485-96. DOI: 10.1128/MCB.24.8.3485-3496.2004
Source: PubMed


Ras activation is critical for T-cell development and function, but the specific roles of the different Ras isoforms in T-lymphocyte function are poorly understood. We recently reported T-cell receptor (TCR) activation of ectopically expressed H-Ras on the the Golgi apparatus of T cells. Here we studied the isoform and subcellular compartment specificity of Ras signaling in Jurkat T cells. H-Ras was expressed at much lower levels than the other Ras isoforms in Jurkat and several other T-cell lines. Glutathione S-transferase-Ras-binding domain (RBD) pulldown assays revealed that, although high-grade TCR stimulation and phorbol ester activated both N-Ras and K-Ras, low-grade stimulation of the TCR resulted in specific activation of N-Ras. Surprisingly, whereas ectopically expressed H-Ras cocapped with the TCRs in lipid microdomains of the Jurkat plasma membrane, N-Ras did not. Live-cell imaging of Jurkat cells expressing green fluorescent protein-RBD, a fluorescent reporter of GTP-bound Ras, revealed that N-Ras activation occurs exclusively on the Golgi apparatus in a phospholipase Cgamma- 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.

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Available from: Ignacio Perez de Castro
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    • "Conversely, a doubly-palmitoylated N-Ras mutant (N-RasL184C, a.k.a. N-Ras-PalmH) could neither be activated at, nor signal from Golgi membranes (Perez de Castro et al., 2004). These results supported the hypothesis that the palmitoylation state of the Ras isoform determines its capacity to be functionally activated at Golgi membranes of T-cells. "
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    ABSTRACT: Despite a high degree of structural homology and shared exchange factors, effectors and GTPase activating proteins, a large body of evidence suggests functional heterogeneity among Ras isoforms. One aspect of Ras biology that may explain this heterogeneity is the differential subcellular localizations driven by the C-terminal hypervariable regions of Ras proteins. Spatial heterogeneity has been documented at the level of organelles: palmitoylated Ras isoforms (H-Ras and NRas) localize on the Golgi apparatus whereas K-Ras4B does not. We tested the hypothesis that spatial heterogeneity also exists at the sub-organelle level by studying the localization of differentially palmitoylated Ras isoforms within the Golgi apparatus. Using confocal, live cell fluorescent imaging and immunogold electron microscopy we found that, whereas the doubly palmitoylated H-Ras is distributed throughout the Golgi stacks, the singly palmitoylated N-Ras is polarized with a relative paucity of expression on the trans Golgi. Using palmitoylation mutants we show that the different sub-Golgi distributions of the Ras proteins are a consequence of their differential degree of palmitoylation. Thus, the acylation state of Ras proteins controls not only their distribution between the Golgi apparatus and the plasma membrane but also their distribution within the Golgi stacks. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Mar 2015 · Journal of Cellular Physiology
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    • "There are two of previous results from our group that are worth discussing in light of the results of the current study. The first is the observation that endogenous H-ras is expressed at much lower levels than endogenous N-ras in Jurkat T-cells and in other T-cell lines [22]. Earlier studies from our group have also demonstrated that H-ras mRNA is expressed at lower levels in both the spleen and thymus than mRNA for N-ras [1]. "
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    ABSTRACT: It has been recently shown that N-ras plays a preferential role in immune cell development and function; specifically: N-ras, but not H-ras or K-ras, could be activated at and signal from the Golgi membrane of immune cells following a low level T-cell receptor stimulus. The goal of our studies was to test the hypothesis that N-ras and H-ras played distinct roles in immune cells at the level of the transcriptome. First, we showed via mRNA expression profiling that there were over four hundred genes that were uniquely differentially regulated either by N-ras or H-ras, which provided strong evidence in favor of the hypothesis that N-ras and H-ras have distinct functions in immune cells. We next characterized the genes that were differentially regulated by N-ras in T cells following a low-level T-cell receptor stimulus. Of the large pool of candidate genes that were differentially regulated by N-ras downstream of TCR ligation, four genes were verified in qRT-PCR-based validation experiments (Dntt, Slc9a6, Chst1, and Lars2). Finally, although there was little overlap between individual genes that were regulated by N-ras in unstimulated thymocytes and stimulated CD4(+) T-cells, there was a nearly complete correspondence between the signaling pathways that were regulated by N-ras in these two immune cell types.
    Full-text · Article · Sep 2013 · PLoS ONE
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    • "Several lines of evidence suggest that N-ras is a particularly important Ras isoform in T cell lines (Perez de Castro et al., 2004), thymocytes, and for polarization of mature CD4 + T lymphocytes . To test whether N-ras is necessary for mature CD8 + T lymphocyte activation after antigen stimulation, we generated N-ras / mice expressing the transgenic OT-I TCR specific for OVA peptide 257 SIINFEKL 264 presented by H-2K b . "
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    ABSTRACT: Signals from the TCR that specifically contribute to effector versus memory CD8(+) T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras-deficient CD8(+) T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)-AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras-deficient CD8(+) T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8(+) T cell memory fate.
    Full-text · Article · Jun 2013 · Journal of Experimental Medicine
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