Ligation of CD28 Stimulates the Formation of a Multimeric Signaling Complex Involving Grb-2-Associated Binder 2 (Gab2), Src Homology Phosphatase-2, and Phosphatidylinositol 3-Kinase: Evidence That Negative Regulation of CD28 Signaling Requires the Gab2 Pleckstrin Homology Domain

Abstract

Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-κB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.

A novel association between filamin A and NF-κB inducing kinase couples CD28 to inhibitor of NF-κB kinase α and NF-κB activation

“…We found that mutations in the YMNM motif up-regulates both IKK␣ and NF-B activation when CD28 is stimulated alone. These data may be consistent with the observations of Parry et al showing that CD28 induces the association of PI3K with the negative regulator Gab2, when engaged by B7.1 alone [56]. Other cytoplasmic motifs of CD28 outside of the YMNM, such as the N-terminal and C-terminal proline-rich motifs [37,57], are also required for TCR/CD28-mediated NF-B activation.…”

Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells

“…The YMNM motif in the intracellular region of CD28 is phosphorylated, thereby binding the p85 subunit of PI3K, which initiates activation of the phosphatidylinositol molecules 3,4 biphospate (PI(3,4)P 2 ) and 3,4,5-triphospate (PIP 3 ). 22 Co-stimulation via CD28 reduces the levels of the cyclin-dependent kinase p27 Kip1 which may explain increased proliferation in co-stimulated T cells. 23 T cells that lack CD28 activation become anergized.…”

NF-κB, AP-1, Zinc-deficiency and aging

“…Critical role for Zn in both NF-jB and AP-1 activations following CD3/CD28 costimulation of PBLs isolated from young and elderly healthy individuals Since CD3/CD28 costimulation has been reported to result in both NF-jB and AP-1 activation in primary T cells (Parry et al 2006) leading to IL-2 production, we investigated the role of Zn in both pathways (Fig. 3).…”