High affinity interactions between MHC:peptide complexes that match their clonotypic TCR lead to stable contact formation of antigen-presenting cells and T cells. Formation and maintenance of the “immunological synapse” rely on integrins, adhesion molecules that are indirectly regulated by TCR or chemokine receptor stimulation 
. 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. ADAP is one of the scaffolding proteins that are central to integrin activation and it is heavily phosphorylated at multiple tyrosines upon TCR stimulation 
. Most of the known and putative tyrosine-phosphorylation sites in ADAP are located within the C-terminal half of the protein that also contains two helically extended SH3 (hSH3) domains 
. The C-terminal hSH3 domain preferentially interacts with negatively charged lipids, while the function of its N-terminal hSH3 domain, apart from displaying a weak lipid binding affinity, is still enigmatic 
. ADAP constitutively associates with SKAP55 via a proline-rich domain in its N-terminal region. summarizes the interaction motifs and domains of the protein as well as its known interaction partners. Three critical tyrosine motifs of ADAP are thought to coordinate the changes in protein assembly that accompany inside-out signaling. Interestingly, one of these sites (Y625) is assumed to bind to the Src family kinase Fyn, a kinase that can phosphorylate ADAP in vivo 
. In addition to the Fyn binding site, two YDDV motifs are thought to be recognized by the SH2 domain of the adapter protein SLP-76 
. SLP-76 in turn binds to the guanine-nucleotide exchange factor Vav1, the adapter protein Nck, and the Tec kinase Itk as well as to PLCγ1 and Gads. The constitutive interaction of Gads with SLP-76 recruits this complex to phosphorylated LAT, thereby linking the TCR to ADAP and integrin adhesion 
Sequence features of ADAP and three dimensional domain structures.
Simultaneous mutation of both YDDV motifs results in reduced cell conjugate formation and loss of LFA-1 polarization in stimulated Jurkat T cells 
. Despite the wealth of information on individual tyrosine phosphorylation motifs, two important questions remain unanswered: Can these motifs, upon phosphorylation, interact with other signaling molecules and secondly, are additional, functionally important tyrosine residues phosphorylated?
Here, we use in vitro phosphorylation to map tyrosine phosphorylation sites in ADAP (486–783) comprehensively. Mass spectrometric analysis reveals several sites of modification that comprise previously identified as well as novel sites. Two of these motifs are located in the folded hSH3 domains of ADAP at helix-sheet interfaces. Peptide pulldown experiments with a linear pYDDV-containing peptide show that several SH2 domain-containing proteins can bind to this motif in addition to SLP-76. Amongst these, actin cytoskeleton modulators Nck1 and Nck2 interact with the ADAP motif in a phosphorylation-dependent manner, thereby providing a direct link between integrin regulation and cytoskeletal rearrangements. Functionally, tyrosine to phenylalanine mutations of SLP-76/Nck interaction sites, of tyrosines in the hSH3 domains and in the C-terminus lead to an attenuation of Jurkat T cell adhesion and migration. This demonstrates that tyrosine phosphorylation of ADAP is more intricate than previously anticipated.