ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters

Abstract

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion, and enables the upregulation of CD69. Using point mutants and a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, un-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, calcium-competent microclusters into stable adhesive junctions with enhanced signaling potential.