PROXIMITY AND ORIENTATION UNDERLIE SIGNALING BY THE NONRECEPTOR TYROSINE KINASE ZAP70

Signaling by the antigen receptor of T lymphocytes initiates different developmental transitions, each of which require the tyrosine kinase ZAP70, Previous studies with agonist and antagonist peptides have indi cated that ZAP70 might respond differently to different structures of the TCR-CD3 complex induced by bound peptides, The roles of membrane p roximity and orientation in activation of ZAP70 signaling were explore d using synthetic ligands and their binding proteins designed to produ ce different architectures of membrane-bound complexes composed of ZAP 70 fusion proteins, Transient membrane recruitment of physiological le vels of ZAP70 with the membrane-permeable synthetic ligand FK1012A lea ds to rapid phosphorylation of ZAP70 and activation of the ras/MAPK an d Ca2+/calcineurin signaling pathways. ZAP70 SH2 domains are not requi red for signaling when the kinase is artifically recruited to the memb rane, indicating that the SH2 domains function solely in recruitment a nd not in kinase activation, Using additional synthetic ligands and th eir binding proteins that recruit ZAP70 equally well but orient it at the cell membrane in different ways, we define a requirement for a spe cific presentation of ZAP70 to its downstream targets, These results p rovide a mechanism by which ZAP70, bound to the phosphorylated recepto r, could discriminate between conformational changes induced by the bi nding of different MHC-peptide complexes to the antigen receptor and i ntroduce an approach to exploring the role of spatial orientation of s ignaling complexes in living cells.