Itk Promotes the Integration of TCR and CD28 Costimulation through Its Direct Substrates SLP-76 and Gads.

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

Dimerization of the adaptor Gads facilitates antigen receptor signaling by promoting the cooperative binding of Gads to the adaptor LAT.

The accurate assembly of signalosomes centered on the adaptor protein LAT (linker of activated T cells) is required for antigen receptor signaling in T cells and mast cells. During signalosome assembly, members of the growth factor receptor-bound protein 2 (Grb2) family of cytosolic adaptor proteins bind cooperatively to LAT through interactions with its phosphorylated tyrosine (pTyr) residues. We demonstrated the Src homology 2 (SH2) domain-mediated dimerization of the Grb2 family member, Grb2-related adaptor downstream of Shc (Gads). Gads dimerization was mediated by an SH2 domain interface, which is distinct from the pTyr binding pocket and which promoted cooperative, preferential binding of paired Gads to LAT. This SH2 domain-intrinsic mechanism of cooperativity, which we quantified by mathematical modeling, enabled Gads to discriminate between dually and singly phosphorylated LAT molecules. Mutational inactivation of the dimerization interface reduced cooperativity and abrogated Gads signaling in T cells and mast cells. The dimerization-dependent, cooperative binding of Gads to LAT may increase antigen receptor sensitivity by reducing signalosome formation at incompletely phosphorylated LAT molecules, thereby prioritizing the formation of complete signalosomes.