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.
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.
Perez de Castro
Ignacio
I
Department of Pathology and New York University Cancer Institute, New York, New York 10016, USA.
Bivona
Trever G
TG
Philips
Mark R
MR
Pellicer
Angel
A
D000595:Amino Acid Sequence; D018106:CD28 Antigens / Q000378:metabolism; D017252:CD3 Complex / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D011905:Genes, ras; D006056:Golgi Apparatus / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D019169:Jurkat Cells; D051966:Phospholipase C gamma; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000378:metabolism* / Q000648:ultrastructure; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D018106:CD28 Antigens; D017252:CD3 Complex; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D020033:Protein Isoforms; C491790:RASGRP1 protein, human; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D018631:ras Proteins