Grb2 and Gads Exhibit Different Interactions with CD28 and Play Distinct Roles in CD28-Mediated Costimulation

Abstract

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-κB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-κB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-κB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-κB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.

Intracellular Signals of T Cell Costimulation

“…CD28 has several Src homology (SH)-2 binding domains (e.g., YMNM) which are able to bind p85, the regulatory subunit of PI3K, and is a potent activator of PI3K (19). CD28 has a cytoplasmic domain of approximately 40 amino acid residues that is highly conserved across species and that contains four tyrosine residues (Y170, 185, 188, 197), potentially mediating protein-protein interactions.…”

“…In the presence of costimulation, T cells would secrete large amounts of IL-2, IL-4, interferon (IFN)-γ, etc., and undergo differentiation into Th1/Th2/Th17 (34-37). CD28 engagement induces massive IL-2 secretion, which is dependent upon the activation of mitogen-activated protein (MAP) kinase (19,38). Using yeast-hybrid technique, a novel CD28 cytoplasmic tail (CD28 CYT) interacting protein, the MAP kinase phosphatase-6 (MKP6) was isolated, which showed to inactivate MAP kinases, so it is associated with cytokine secretion (39).…”

“…Similarly to PKC isotypes, CD28 binding Grb2-related adaptor protein 2 (Gads) is also essential for CD28-mediated NF-κB activation, and its binding requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. The mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduced their association with Gads and induced strong activity of the NF-AT/AP-1 reporter comparably, but weak activity of the NF-κB reporter, which was consistent with the weak activation of the IL-2 promoter (19). Consistent with this result, a mutation in the C-terminal proline-rich region of the cytoplasmic tail of CD28 showed that this motif is essential for CD28-dependent regulation of IL-2 secretion and T cell proliferation.…”

Grb2 Is Important for T Cell Development, Th Cell Differentiation, and Induction of Experimental Autoimmune Encephalomyelitis

“…Therefore, it is more likely that CD4 + cells are the main drivers of the EAE phenotype observed in Grb2 fl/fl CD4cre tg mice. Mechanistically, it was demonstrated that Grb2 is necessary for the induction of IL-2 secretion via CD28 (40), and it is also necessary to induce a mitogenic response via IL-2R (31,41). This might explain why proliferation of Grb2-deficient CD4 + T cells is diminished after stimulation with IL-2.…”

CD28 ligation in the absence of TCR promotes RelA/NF‐κB recruitment and trans‐activation of the HIV‐1 LTR

“…Y191 is contained in the YMNM motif involved in the recruitment of both Grb-2 [13] and the p85 subunit of the PI3K [14]. Y206 and Y209 are contained in the C-terminal prolinerich motif (YQPYAPP) that has been recently involved in the regulation of several CD28 functions [15][16][17][18]. The two tyrosine residues Y206/209, within the C-terminal proline-rich motif of CD28, have been described to exert a key role in CD28-regulated HIV-1 transcription, whereas disruption of the PI3K binding motif, by mutating Y191, increased HIV-1 transcription [1,19].…”