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

• Published in: Journal of Immunology Vol. 176; no. 1; pp. 594 - 602
• Main Authors: Parry, Richard V, Whittaker, Gillian C, Sims, Martin, Edmead, Christine E, Welham, Melanie J, Ward, Stephen G
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 01.01.2006
• Subjects: Adaptor Proteins, Signal Transducing; Blood Proteins - immunology; Blood Proteins - metabolism; CD28 Antigens - immunology; CD28 Antigens - metabolism; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins - immunology; Intracellular Signaling Peptides and Proteins - metabolism; Jurkat Cells; Lymphocyte Activation - immunology; NF-kappa B - immunology; NF-kappa B - metabolism; Phosphatidylinositol 3-Kinases - immunology; Phosphatidylinositol 3-Kinases - metabolism; Phosphoproteins - immunology; Phosphoproteins - metabolism; Phosphorylation; Protein Phosphatase 2; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatases - immunology; Protein Tyrosine Phosphatases - metabolism; SH2 Domain-Containing Protein Tyrosine Phosphatases; src Homology Domains - immunology; T-Lymphocytes - immunology; Transcription Factor AP-1 - immunology; Transcription Factor AP-1 - metabolism; Transfection
• ISSN: 0022-1767; 1550-6606; 1365-2567
• DOI: 10.4049/jimmunol.176.1.594

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-kappaB 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.