Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated-localization microscopy (PALM) to study complexes downstream of the T cell antigen receptor (TCR) in single molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adapter LAT in activated cells thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adapter SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Calcineurin is a phosphatase whose primary targets in T cells are NFAT transcription factors, and inhibition of calcineurin activity by treatment with cyclosporin A (CsA) or FK506 is a cornerstone of immunosuppressive therapies. Here we found that calcineurin was recruited to the T cell antigen receptor (TCR) signaling complex, where it reversed inhibitory phosphorylation of the tyrosine kinase Lck on Ser59 (Lck). Loss of calcineurin activity impaired phosphorylation of Tyr493 of the tyrosine kinase ZAP-70 (ZAP-70), as well as some downstream pathways in a manner consistent with signaling in cells expressing Lck (Lck that cannot be phosphorylated) or Lck (a phosphomimetic mutant). Notably, CsA inhibited integrin-LFA-1-dependent and NFAT-independent adhesion of T cells to the intercellular adhesion molecule ICAM-1, with little effect on cells expressing mutant Lck. These results provide new understanding of how widely used immunosuppressive drugs interfere with essential processes in the immune response.

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 ...