Growth factor receptor-bound protein 2 SH2/SH3 domain binding to CD28 and its role in co-signaling.
The co-stimulatory antigen CD28 has been shown to bind to several intracellular proteins including phosphatidylinositol 3-kinase, growth factor receptor-bound protein 2 (Grb2), and ITK. Paradoxically, Grb2 and phosphatidylinositol 3-kinase binding has been mapped to a similar pYMNM motif within the CD28 cytoplasmic tail. Given the importance of CD28 co-signaling to T cell function, questions exist regarding the mechanism by which Grb2 binds to CD28, and whether the interaction plays a role in co-stimulation. To biochemically characterize Grb2/CD28 binding, we initially utilized glutathione S-transferase-Grb2 fusion proteins carrying inactivating mutations within the SH2 and SH3 domains of Grb2, and assessed their ability to bind to CD28. In vitro binding experiments indicated that the Grb2 SH2 domain is critical for the association, while the SH3 domain plays an additional role in facilitating optimal binding. Enhanced binding via the SH3 domains was not observed when the C-terminal PXXP motif within CD28 was disrupted, thereby indicating that both SH2 and SH3 domains contribute to CD28 binding. Mutations that alter Grb2 binding were found to block the CD28-dependent interleukin-2 production. Further, tyrosine phosphorylation of Vav and the costimulation-dependent activation of Jun N-terminal kinase was blocked in cells defective in CD28/Grb2 binding. These results provide evidence for an alternate CD28-mediated signaling process involving Grb2 binding to the co-receptor.
Kim
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Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
Tharayil
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Rudd
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D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D018106:CD28 Antigens / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D051380:GRB2 Adaptor Protein; D051379:Mice; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D011506:Proteins; D011994:Recombinant Proteins