title: Nck Binds to the T Cell Antigen Receptor Using Its SH3.1 and SH2 Domains in a Cooperative Manner, Promoting TCR Functioning.
abstract: Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
authors.forename: Pussadee
authors.initials: P
authors.lastname: Paensuwan
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany;
authors.forename: Frederike A
authors.initials: FA
authors.lastname: Hartl
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany; Spemann Graduate School of Biology and Medicine, Albert Ludwigs University Freiburg, Freiburg 79104, Germany;
authors.forename: O Sascha
authors.identifier: http://orcid.org/0000-0001-5304-729X
authors.initials: OS
authors.lastname: Yousefi
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
authors.forename: Jatuporn
authors.initials: J
authors.lastname: Ngoenkam
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
authors.forename: Piyamaporn
authors.initials: P
authors.lastname: Wipa
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany; International Max Planck Research School for Molecular and Cellular Biology, Freiburg 79108, Germany;
authors.forename: Esmeralda
authors.initials: E
authors.lastname: Beck-Garcia
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany;
authors.forename: Elaine P
authors.initials: EP
authors.lastname: Dopfer
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
authors.forename: Boonruang
authors.initials: B
authors.lastname: Khamsri
authors.affiliation: Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand;
authors.forename: Donruedee
authors.initials: D
authors.lastname: Sanguansermsri
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany;
authors.forename: Susana
authors.initials: S
authors.lastname: Minguet
authors.affiliation: Department of Molecular Immunology, Faculty of Biology, BIOSS Centre for Biological Signaling Studies and Centre of Chronic Immunodeficiency, University of Freiburg, Freiburg 79108, Germany; sutatipp@nu.ac.th wolfgang.schamel@biologie.uni-freiburg.de.
authors.forename: Wolfgang W
authors.initials: WW
authors.lastname: Schamel
authors.affiliation: Centre of Excellence in Medical Biotechnology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand; Research Center for Academic Excellence in Petroleum, Petrochemical and Advanced Materials, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand; and Department of Medicine, Faculty of Medicine, Naresuan University, Phitsanulok 65000, Thailand sutatipp@nu.ac.th wolfgang.schamel@biologie.uni-freiburg.de.
authors.forename: Sutatip
authors.initials: S
authors.lastname: Pongcharoen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D017252:CD3 Complex / Q000378:metabolism; D045744:Cell Line, Tumor; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000276:immunology*; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D055232:Proline-Rich Protein Domains; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; C115335:CD3E protein, human; C078332:Nck protein; D015513:Oncogene Proteins; D011948:Receptors, Antigen, T-Cell