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.
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.
Parry
Richard V
RV
Lymphocyte Activation Group, Department of Pharmacy and Pharmacology, University of Bath, Bath, UK.
Whittaker
Gillian C
GC
Sims
Martin
M
Edmead
Christine E
CE
Welham
Melanie J
MJ
Ward
Stephen G
SG
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins / Q000276:immunology / Q000378:metabolism; D018106:CD28 Antigens / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology* / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000276:immunology* / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D054648:Protein Phosphatase 2; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000276:immunology* / Q000378:metabolism; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases; D013601:T-Lymphocytes / Q000276:immunology; D018808:Transcription Factor AP-1 / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D018909:src Homology Domains / Q000276:immunology*
D048868:Adaptor Proteins, Signal Transducing; D001798:Blood Proteins; D018106:CD28 Antigens; C490895:GAB2 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D010750:Phosphoproteins; D018808:Transcription Factor AP-1; C039077:platelet protein P47; D019869:Phosphatidylinositol 3-Kinases; D054648:Protein Phosphatase 2; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D054593:SH2 Domain-Containing Protein Tyrosine Phosphatases