In TCR-Stimulated T-cells, N-ras Regulates Specific Genes and Signal Transduction Pathways

Stephen J Lynch,Jiri Zavadil,Angel Pellicer,Anna Tramontano

doi:10.1371/journal.pone.0063193

Stephen J Lynch, Jiri Zavadil + Show 2 more

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https://doi.org/10.1371/journal.pone.0063193

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Journal: PLoS ONE	Publication Date: Jun 3, 2013
Citations: 32	License type: CC BY 4.0

Affiliation: New York University Langone Medical Center

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.

Highlights

The three primary Ras isoforms (N-ras, H-ras and K-ras) share a high degree of structural and functional similarity, which initially led some researchers to propose that the Ras proteins are functionally redundant
Thymocytes were isolated from 6 week old N-ras knockout mice (KO), H-ras KO and wild type (WT) mice, and differential display expression analyses were performed with cDNA derived from total RNA isolated from these mice
To determine if N-ras and H-ras regulate different sets of genes in thymocytes, a comparison was made between the set of genes that were differentially regulated by N-ras in the [WT] vs. [N-ras KO] comparison and the set of genes that were differentially regulated by H-ras in the [WT] vs. [H-ras KO] comparison

Summary

Introduction

The three primary Ras isoforms (N-ras, H-ras and K-ras) share a high degree of structural and functional similarity, which initially led some researchers to propose that the Ras proteins are functionally redundant. Ras-mediated signal transduction in T-cells is initiated through ligation of the T-cell receptor (TCR) by antigen:MHC complexes present on the surface of the antigen presenting cell (APC) at a specialized cellular structure known as the immunological synapse [10]. Full TCR-mediated signaling leads to recruitment of Grb and Sos, that have been shown to be directly responsible for the activation of Ras at the plasma membrane (PM) in T-cells. A number of signal transduction pathways are downstream of activated Ras in T-cells [11,12,13]

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