title: 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.
abstract: Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)(1). Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3(N) and microscale thermophoresis (MST) indicates an intermediate binding affinity (Kd = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.
authors.affiliation: From the ‡Freie Universität Berlin, Institut für Chemie und Biochemie, Protein Biochemistry group, Thielallee 63, 14195 Berlin, Germany; §Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany;
authors.forename: Benno
authors.initials: B
authors.lastname: Kuropka
authors.affiliation: ¶Otto-von-Guericke-University, Institute of Molecular and Clinical Immunology, Leipziger Strasse 44, 39120 Magdeburg, Germany;
authors.forename: Amelie
authors.initials: A
authors.lastname: Witte
authors.affiliation: From the ‡Freie Universität Berlin, Institut für Chemie und Biochemie, Protein Biochemistry group, Thielallee 63, 14195 Berlin, Germany;
authors.forename: Jana
authors.initials: J
authors.lastname: Sticht
authors.affiliation: ¶Otto-von-Guericke-University, Institute of Molecular and Clinical Immunology, Leipziger Strasse 44, 39120 Magdeburg, Germany;
authors.forename: Natalie
authors.initials: N
authors.lastname: Waldt
authors.affiliation: §Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany; ‖RiNA GmbH, Volmerstrasse 9, 12489 Berlin, Germany;
authors.forename: Paul
authors.initials: P
authors.lastname: Majkut
authors.affiliation: §Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany;
authors.forename: Christian P R
authors.initials: CP
authors.lastname: Hackenberger
authors.affiliation: ¶Otto-von-Guericke-University, Institute of Molecular and Clinical Immunology, Leipziger Strasse 44, 39120 Magdeburg, Germany; **Helmholtz Center for Infection Research (HZI), Department of Immune Control, Inhoffenstrasse 7, 38124 Braunschweig, Germany.
authors.forename: Burkhart
authors.initials: B
authors.lastname: Schraven
authors.affiliation: §Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, 13125 Berlin, Germany; christian.freund@fu-berlin.de ekrause@fmp-berlin.de stefanie.kliche@med.ovgu.de.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.affiliation: ¶Otto-von-Guericke-University, Institute of Molecular and Clinical Immunology, Leipziger Strasse 44, 39120 Magdeburg, Germany; christian.freund@fu-berlin.de ekrause@fmp-berlin.de stefanie.kliche@med.ovgu.de.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Kliche
authors.affiliation: From the ‡Freie Universität Berlin, Institut für Chemie und Biochemie, Protein Biochemistry group, Thielallee 63, 14195 Berlin, Germany; christian.freund@fu-berlin.de ekrause@fmp-berlin.de stefanie.kliche@med.ovgu.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Freund
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000276:immunology; D001665:Binding Sites; D002448:Cell Adhesion / Q000187:drug effects; D015550:Cell Degranulation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D054377:Chemokine CXCL12 / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008958:Models, Molecular; D010766:Phosphorylation / Q000187:drug effects; D061251:Primary Cell Culture; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics* / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; C106957:FYB1 protein, human; D014443:Tyrosine; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human