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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.

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

Ignacio Perez de Castro,1 Trever G. Bivona,2 Mark R. Philips,2 and Angel Pellicer1*

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

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 C{gamma}- 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.

* Corresponding author. Mailing address: Department of Pathology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-5342. Fax: (212) 263-8211. E-mail: pellia01{at}med.nyu.edu.

Molecular and Cellular Biology, April 2004, p. 3485-3496, Vol. 24, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.8.3485-3496.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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