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

Abstract

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.

SILAC Phosphoproteomics Reveals Unique Signaling Circuits in CAR-T Cells and the Inhibition of B Cell-Activating Phosphorylation in Target Cells

“…The appearance of ADAP Y571 was particularly interesting as it promotes both ADAP/STAT3 interaction and subsequent STAT3 phosphorylation, the latter of which was also observed in the form of STAT3 T716S727 by TiO 2 enrichment (Figure A). Crucially, sSH2 enrichment indicated increased phosphorylation of GADS Y45 (Figure A), an Itk substrate site that is phosphorylated exclusively within the LAT-SLP76 complex . The primary function of SLP76 complex is to activate critical signaling pathways downstream of the TCR, including p38/Jnk, MAPK/Erk1/2 and subsequent pathways regulating the actin cytoskeleton. , The guanine exchange factor, Vav1, is a component of the SLP76 complex known to activate Rho/Rac GTPases after TCR stimulation. , sSH2 pTyr enrichment revealed that Vav1 Y791 , a pTyr site known to increase in response to α-CD3 and α-CD28 treatment, significantly increased upon CAR activation.…”

“…Crucially, sSH2 enrichment indicated increased phosphorylation of GADS Y45 (Figure 5A), an Itk substrate site that is phosphorylated exclusively within the LAT-SLP76 complex. 76 The primary function of SLP76 complex is to activate critical signaling pathways downstream of the TCR, including p38/Jnk, MAPK/Erk1/2 and subsequent pathways regulating the actin cytoskeleton. 15,77 The guanine exchange factor, Vav1, is a component of the SLP76 complex known to activate Rho/Rac GTPases after TCR stimulation.…”

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation

“…It will also be important to map the CD28-downstream signaling pathway to understand both the increase and subsequent decrease in TCR translation that occur during the translation burst. We found that AKT influences the rapid rise in translation ( Figure 4E–F ), but other kinases such as Tec-kinases ( Andreotti et al, 2018 ; Gallagher et al, 2021 ; Hallumi et al, 2021 ) could also impact the translation burst.…”

Mapping the SLP76 interactome in T cells lacking each of the GRB2-family adaptors reveals molecular plasticity of the TCR signaling pathway

“…Previous studies also indicated that proper T cell activation requires cooperative interactions between several members of the LAT-SLP76 signalosomes leading to the formation of large multiprotein complexes (9)(10)(11)(12). For example, multipoint binding facilitates recruitment to the LAT-SLP76 complex of the ITK and PLCg1 effectors which are involved in calcium release (13)(14)(15)(16). TCR signal diversification is also enhanced by the phosphorylation of transmembrane molecules which provide additional docking sites for adaptors localized near the priming module.…”