Regulation of Interleukin-2 Transcription by Inducible Stable Expression of Dominant Negative and Dominant Active Mitogen-activated Protein Kinase Kinase Kinase in Jurkat T Cells: EVIDENCE FOR THE IMPORTANCE OF Ras IN A PATHWAY THAT IS CONTROLLED BY DUAL RECEPTOR STIMULATION

Mary Faris,Niels Kokot,Leo Lee,Andre E Nel

doi:10.1074/jbc.271.44.27366

Abstract

Engagement of the T cell receptor induces the activation of several mitogen-activated protein kinase modules, including the extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) cascades. Whereas extracellular signal-regulated kinase is activated by T cell receptor/CD3 ligation alone, activation of JNK requires co-stimulation by the CD28 receptor. Activation of MEKK-1, which acts as a mitogen-activated protein kinase kinase kinase in the JNK pathway, was also induced by CD3 plus CD28 (CD3/CD28) ligation in Jurkat cells. To study the significance of the JNK cascade in T lymphocytes, we established stable Jurkat cell lines that inducibly express dominant active (DA) or dominant negative (DN) MEKK-1. Whereas expression of DA-MEKK-1 resulted in the constitutive activation of JNK along with the transcriptional activation of the minimal interleukin-2 (IL-2) promoter, DN-MEKK-1 inhibited JNK responsiveness during CD3/CD28 co-stimulation. In addition to inhibiting CD3/CD28-induced IL-2 mRNA expression, DN-MEKK-1 abrogated the transcriptional activation of the IL-2 promoter and the distal nuclear factor of activated T cells (NFAT)-activating protein 1 (AP-1) response element in that promoter. A c-Jun mutant lacking activation sites for JNK also interfered with the activation of the distal NFAT/AP-1 complex, suggesting that the JNK pathway functions by controlling AP-1 response elements in the IL-2 promoter. Using inducible stable expression of DA- and DN-Ras in Jurkat cells, we found that Ras regulates JNK activation in these cells. Our results suggest that the dual ligation of CD3 and CD28 in T cells triggers a cascade of events that involve Ras, the JNK cascade, and one or more AP-1 response elements in the IL-2 promoter.

Highlights

The role of Jun N-terminal kinase (JNK) in T cell activation is not well understood
Our results suggest that the dual ligation of CD3 and CD28 in T cells triggers a cascade of events that involve Ras, the JNK cascade, and one or more activating protein 1 (AP-1) response elements in the IL-2 promoter
In light of the above, we were interested in determining whether the simultaneous ligation of T cell antigen receptor (TCR)/CD3 and CD28 activates the JNK pathway in T lymphocytes in an MEKK-1-dependent fashion and whether this pathway contributes to activation of the IL-2 promoter

Summary

Introduction

The role of JNK in T cell activation is not well understood. Several lines of evidence suggest, that JNKs may be involved in inducing the transcriptional activation of AP-1 response elements in TCR-responsive genes [10]. Both c-Fos and c-Jun contribute to the expression of the IL-2 gene, which, as for JNK activation, is dependent on co-ligation of CD3 and CD28 [10, 16, 32, 33] This suggests that JNK may play a role in transcriptional activation of the IL-2 promoter. In order to accomplish our goals, we established stable transfected Jurkat cell lines with inducible expression of dominant active (DA)-MEKK-1, dominant negative (DN)-MEKK-1, DA-Ras, or DN-Ras under a tetracycline-controlled transactivator (tTA) protein We used these cell lines to study the effect of MEKK-1 on IL-2 mRNA expression, including the effect of this kinase on transcriptional activation of the IL-2 promoter and the distal NFAT response element. DN-Ras interfered with activation of the JNK cascade during CD3/CD28 co-ligation, whereas DA-Ras enhanced JNK activation by the same stimulatory combination, including treatment with antiCD3 mAb alone

Methods

Results

Conclusion