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.
Highlights
High affinity interactions between MHC:peptide complexes that match their clonotypic TCR lead to stable contact formation of antigen-presenting cells and T cells
Novel phosphorylation sites In this report, we comprehensively describe tyrosine phosphorylation sites within the T cell protein adapter protein (ADAP) and demonstrate their role for T cell adhesion and migration
Individual phosphorylation sites and their functional role had been described in Jurkat T cells
Summary
High affinity interactions between MHC:peptide complexes that match their clonotypic TCR lead to stable contact formation of antigen-presenting cells and T cells. Tyrosine-phosphorylation of receptors and receptor-proximal signaling molecules lead to the recruitment of SH2 domain containing proteins that in turn transmit information to modulators of integrin activity. Three critical tyrosine motifs of ADAP are thought to coordinate the changes in protein assembly that accompany inside-out signaling. One of these sites (Y625) is assumed to bind to the Src family kinase Fyn, a kinase that can phosphorylate ADAP in vivo [10]. The constitutive interaction of Gads with SLP-76 recruits this complex to phosphorylated LAT, thereby linking the TCR to ADAP and integrin adhesion [12]
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