TCR-induced activation of Ras proceeds at the plasma membrane and requires palmitoylation of N-Ras.
Ras transmits manifold signals from the TCR at various crossroads in the life of a T cell. For example, selection programs in the thymus or the acquisition of a state of hypo-responsiveness known as anergy are just some of the T cell features known to be controlled by TCR-sparked signals that are intracellularly propagated by Ras. These findings raise the question of how Ras can transmit such a variety of signals leading to the shaping of equally many T cell traits. Because Ras proteins transit through endomembrane compartments on their way to the plasma membrane (PM), compartmentalized Ras activation at distinct subcellular sites represents a potential mechanism for signal diversification in TCR signaling. This hypothesis has been nurtured by studies in T cells engineered to overexpress Ras that reported distinct activation of Ras at the PM and Golgi. Contrary to this scenario, we report in this study that activation of endogenous Ras, imaged in live Jurkat T cells using novel affinity probes for Ras-GTP, proceeds only at the PM even upon enforced signal flux through the diacylglycerol/RasGRP1 pathway. Physiological engagement of the TCR at the immunological synapse in primary T cells caused focalized Ras-GTP accumulation also only at the PM. Analysis of palmitoylation-deficient Ras mutants, which are confined to endomembranes, confirmed that the TCR does not activate Ras in that compartment and revealed a critical function for palmitoylation in N-Ras/H-Ras activation. These findings identify the PM as the only site of TCR-driven Ras activation and document that endomembranes are not a signaling platform for Ras in T cells.
Rubio
Ignacio
I
Institute for Molecular Cell Biology, Centre for Molecular Biomedicine, University Hospital, Friedrich-Schiller-University Jena, Jena, Germany. ignacio.rubio@med.uni-jena.de
Grund
Stefan
S
Song
Shu-Ping
SP
Biskup
Christoph
C
Bandemer
Sabine
S
Fricke
Melanie
M
Förster
Martin
M
Graziani
Andrea
A
Wittig
Ute
U
Kliche
Stefanie
S
D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000276:immunology; D005456:Fluorescent Dyes / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D054878:Lipoylation / Q000235:genetics / Q000276:immunology*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D021381:Protein Transport / Q000235:genetics / Q000276:immunology; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism; D020663:ras Guanine Nucleotide Exchange Factors / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018631:ras Proteins / Q000096:biosynthesis / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D020662:Guanine Nucleotide Exchange Factors; C491789:Rasgrp1 protein, rat; D011948:Receptors, Antigen, T-Cell; D020663:ras Guanine Nucleotide Exchange Factors; D015689:Oncogene Protein p21(ras); D018631:ras Proteins