Novel Role of Phosphatidylinositol 3-Kinase in CD28-mediated Costimulation

Yohsuke Harada,Eri Tanabe,Ryosuke Watanabe,Bonnie D Weiss,Akira Matsumoto,Hiroyoshi Ariga,Osamu Koiwai,Yasuhisa Fukui,Masato Kubo,Carl H June,Ryo Abe

doi:10.1074/jbc.m005051200

Abstract

Ligation of the CD28 surface receptor provides a major costimulatory signal for full scale T cell activation. Despite extensive studies, the intracellular signaling pathways delivered by CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants were expressed in Jurkat cells. We then measured IL-2 promoter activation after CD28 ligation. Our results showed that the Y189F mutant, which disrupts binding by PI3K, and the YMNM deletion mutant both demonstrated reduced but significant activity for IL-2 promoter activation. In contrast, the N191A mutant, which retains PI3K binding ability, resulted in a complete abrogation of activity, suggesting that PI3K mediates a negative effect upon transcriptional activation of the IL-2 gene. Consistent with this idea, we found that the addition of a PI3K pharmacological inhibitor augmented IL-2 promoter activity, whereas coexpression of a constitutively active form of PI3K reduced this activity. Taken together, these data indicate that PI3K, when associated with the YMNM motif, may act as a negative mediator in CD28-mediated IL-2 gene transcription.

Highlights

In addition to the signaling provided by recognition of antigen-major histocompatibility complex by the T cell receptor, other receptor-ligand interactions play critical roles for full activation of T cells by providing costimulatory signals to T cells
Mutational Analysis of CD28-mediated IL-2 Promoter Activation—In the CD28 cytoplasmic domain, four tyrosines that potentially bind to SH2 domains and two potential motifs (PXXP motif) that bind to SH3 domains exist (Fig. 1)
The YMNM motif, which exists in the CD28 cytoplasmic region, is known to associate with phosphatidylinositol 3-kinase (PI3K) and Grb-2 (4 – 8)

Summary

Introduction

In addition to the signaling provided by recognition of antigen-major histocompatibility complex by the T cell receptor, other receptor-ligand interactions play critical roles for full activation of T cells by providing costimulatory signals to T cells. Some investigators reported that wortmannin fails to block CD28-mediated costimulation of IL-2 production by Jurkat cells and murine CD4ϩ splenic T cells [14, 17,18,19,20] They showed that mutation of Tyr189 to Phe, which disrupts PI3K binding, had no effect on the ability of CD28 to deliver a costimulatory signal to Jurkat cells [17]. As a part of efforts to define the relationship between CD28-mediated intracellular signaling pathways and T cell functions, we constructed a panel of point and deletion mutants of CD28 including the critical PI3K binding motif, YMNM, and tested their costimulatory ability for activation of IL-2 gene transcription

Methods

Results

Conclusion