Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells

Abstract

The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Engagement of a T cell antigen receptor (TCR) by a cognate peptide-major histocompatibility complex (MHC) on an antigen-presenting cell triggers complex molecular cascades that control signaling pathways crucial for gene transcription, cytokine production, cell adhesion, mobility, proliferation, and differentiation (1). Appropriate T cell responses depend on the formation of multiprotein complexes at the plasma membrane that regulate efficient signal transduction via multiple pathways. TCR stimulation facilitates phosphorylation of the TCR chains by the Src family kinase Lck, which allows for the recruitment and activation of the protein tyrosine kinase Zap-70. Phosphorylation of the essential adapter proteins LAT and SLP-76 by Zap-70 creates docking sites for SH2 domain-containing adapter and effector proteins. The adapter, Grb2, binds LAT and recruits the guanine exchange factor, Sos, while the Grb2-related adapter, Gads, recruits the adapter SLP-76 (1). LAT and SLP-76 recruit other signaling proteins, thereby inducing the assembly of multiprotein complexes. Molecular associations within the multiprotein complexes are dynamic and highly cooperative, which might allow tight regulation of signal transduction pathways in T cells (2-5, 9, 32).In confocal imaging studies of T cell lines and peripheral blood lymphocytes stimulated on anti-CD3-coated glass coverslips, within seconds of TCR engagement LAT and SLP-76 are visualized in microclusters, which contain many protein complexes (6, 7). Numerous reports indicate that effective TCR signaling depends on the assembly and persistence of the microclusters that contain . Microclusters containing LAT are abolished by tyrosine-to-phenylalanine (Y-F) changes of critical Gads and Grb2 binding sites on LAT (8,9,14). Microclusters containing SLP-76 are abolished by a mutation to the Gads binding motif of SLP-76, which prevents recruitment to LAT. In both cases, losses of microclusters are associated with defective signaling. A mutation of the C-terminal SH2 domain of SLP-76 also resulted in a similar loss of SLP-76 microclusters along with severely reduced CD69 upregulation and NFAT activity (8). This result was completely unanticipated, because the mutation is not expected to prevent recruitment to LAT. Instead, the result indicated that the SH2 domain plays a central and essential role in the stabilization of SLP-76 microclusters and signal transduction in T cells.The adapter protein, ADAP, binds the SLP-76 SH2 domain, and the interaction appears to affect TCR signaling (15, 16). SLP-76-deficient J14 cells transfected with SLP-76 containing an SH2 mutation (SLP-76-SH2*) showed reduced ...

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

“…Most phosphotyrosines are contained in short motifs of intrinsically unstructured regions of the corresponding signaling proteins. Specificity for these short motifs is usually limited, leading to promiscuous binding that allows for the dynamic exchange of signaling molecules (53). In contrast, phosphorylation within or in the vicinity of a defined three- dimensional protein fold harbors the potential that specificity is tuned by a conformational epitope rather than a linear motif.…”

SHP-1 Acts as a Key Regulator of Alloresponses by Modulating LFA-1-Mediated Adhesion in Primary Murine T Cells

“…Published data favor either YDDV tyrosine motif of ADAP (Y595, Y651 [isoform ADAP-120] or Y595, Y697 [isoform ADAP-130]) as a major binding site for the SLP-76 SH2 domain and suggest a requirement of both sites for optimal SLP-76 binding (18)(19)(20)(21)(22)(23). Moreover, the YDGI tyrosine motif (Y625) plays an additional role in binding to SLP-76.…”

“…Several phosphotyrosine residues of ADAP have been reported to interact with the SLP-76 SH2 domain (18)(19)(20)(21)(22)(23). We therefore next examined the effect of SHP-1 on the phosphorylation of ADAP by using an anti-ADAP antibody to precipitate the adaptor protein followed by blotting with a monoclonal antibody to phosphotyrosine.…”

Development of nanoscale structure in LAT-based signaling complexes

“…Grb2 binds to the same phosphorylation sites used by Gads, so SLP-76 might be excluded from areas where oligomerized LAT is bound to Grb2. In addition, crosslinking of SLP-76 by ADAP (Coussens et al, 2013) . Actin polymerization might then pull these SLP-76 molecules to the edges of LAT clusters.…”

“…Removing two of these sites prevents crosslinking and leads to decreased Ca 2+ flux. Thus, it appears that some level of oligomerization of LAT and SLP-76 is required to produce proper T cell activation (Coussens et al, 2013).…”