title: Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70.
abstract: 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 indicated that ZAP70 might respond differently to different structures of the TCR-CD3 complex induced by bound peptides. The roles of membrane proximity and orientation in activation of ZAP70 signaling were explored using synthetic ligands and their binding proteins designed to produce different architectures of membrane-bound complexes composed of ZAP70 fusion proteins. Transient membrane recruitment of physiological levels of ZAP70 with the membrane-permeable synthetic ligand FK1012A leads to rapid phosphorylation of ZAP70 and activation of the ras/MAPK and Ca2+/calcineurin signaling pathways. ZAP70 SH2 domains are not required for signaling when the kinase is artifically recruited to the membrane, indicating that the SH2 domains function solely in recruitment and not in kinase activation. Using additional synthetic ligands and their binding proteins that recruit ZAP70 equally well but orient it at the cell membrane in different ways, we define a requirement for a specific presentation of ZAP70 to its downstream targets. These results provide a mechanism by which ZAP70, bound to the phosphorylated receptor, could discriminate between conformational changes induced by the binding of different MHC-peptide complexes to the antigen receptor and introduce an approach to exploring the role of spatial orientation of signaling complexes in living cells.
authors.affiliation: Department of Developmental Biology, Howard Hughes Institute at Stanford University, 300 Pasteur Drive, Beckman Center Room B211, Stanford University Medical School, Stanford, CA 94305, USA.
authors.forename: I A
authors.initials: IA
authors.lastname: Graef
authors.forename: L J
authors.initials: LJ
authors.lastname: Holsinger
authors.forename: S
authors.initials: S
authors.lastname: Diver
authors.forename: S L
authors.initials: SL
authors.lastname: Schreiber
authors.forename: G R
authors.initials: GR
authors.lastname: Crabtree
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology; D017930:Genes, Reporter; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D016559:Tacrolimus / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins; D014162:Transfection; D051746:ZAP-70 Protein-Tyrosine Kinase
chemical_list: D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus