Adhesion and Degranulation Promoting Adapter Protein (ADAP) Is a Central Hub for Phosphotyrosine-Mediated Interactions in T Cells

Abstract

TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize in vitro phosphorylation and mass spectrometry to map novel phosphotyrosine sites in the C-terminal part of human ADAP (486–783). Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCγ, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

“…For example, because the surface residues of protein domains such as SH2 are less well conserved for regions outside the binding pocket, the ADAP hSH3 N domain surface geometry and charge distribution in the vicinity of Y571 might well be incompatible with the defined docking of certain SH2 domain containing proteins. In line with this argument and considering that we use a large excess of bait protein in our domain-based pull-down we do not enrich any of the other SH2 domain containing proteins when compared with previous phosphopeptide enrichments (18,22). Phosphorylated Y571 is predominantly recognized by the N-terminal SH2 (N-SH2) domain of ZAP70 and the residues displaying the largest signal reduction in our NMR studies (R17, L40) are identical to those that are line-broadened when ZAP70-tSH2 binds to the singly phosphorylated ITAM peptides pYNEL or pYDVL (37).…”

“…S5) (49). An indirect association of ADAP-Y571F with ZAP70 after TCR triggering is also supported by the finding that the other major tyrosine phosphorylation sites of ADAP are not recognized by ZAP70 in pull-down experiments (18,22,56). Also, when the TCR is fully phosphorylated in its cytoplasmic domains upon TCR stimulation, it will be the prime target for the tandem SH2 domains of ZAP70, which is, in this way, recruited to the membrane for further activation by Lck.…”

“…Therefore we aimed to confirm the interaction in primary cells and used lysate from purified human T cells in combination with 18 O/ 16 O labeling to confirm the interaction. Indeed, we again characterized ZAP70 as one of the most highly enriched proteins, in addition to the tyrosine kinase MATK and the lipid phosphatases SHIP-1 and SHIP-2 (Figs.…”

“…For the control experiment no ATP was added. Tyrosine phosphorylation was detected by Western blotting with an anti-phosphotyrosine antibody as described elsewhere (18). Phosphorylation degrees were detected by mass spectrometry after SDS-PAGE and tryptic in-gel digestion as described further below.…”

“…Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig. 1A) (17)(18)(19)(20)(21). Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig.…”