title: Nck recruitment to the TCR required for ZAP70 activation during thymic development.
abstract: The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4(+)CD8(+) thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid 28049, Spain.
authors.forename: Aldo
authors.initials: A
authors.lastname: Borroto
authors.forename: Irene
authors.initials: I
authors.lastname: Arellano
authors.forename: Elaine P
authors.initials: EP
authors.lastname: Dopfer
authors.forename: Marek
authors.initials: M
authors.lastname: Prouza
authors.forename: Miloslav
authors.initials: M
authors.lastname: Suchànek
authors.forename: Manuel
authors.initials: M
authors.lastname: Fuentes
authors.forename: Alberto
authors.initials: A
authors.lastname: Orfao
authors.forename: Wolfgang W
authors.initials: WW
authors.lastname: Schamel
authors.forename: Balbino
authors.initials: B
authors.lastname: Alarcón
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017951:Antigen Presentation; D017252:CD3 Complex / Q000235:genetics* / Q000276:immunology; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005260:Female; D055879:Gene Knock-In Techniques; D057927:Hydrophobic and Hydrophilic Interactions; D038041:Lymphopoiesis / Q000502:physiology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000378:metabolism*; D055232:Proline-Rich Protein Domains / Q000235:genetics; D021381:Protein Transport; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000378:metabolism*; D060168:Thymocytes / Q000166:cytology / Q000378:metabolism*; D013950:Thymus Gland / Q000254:growth & development / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; D015704:CD4 Antigens; D016827:CD8 Antigens; C485468:Cd3e protein, mouse; C078332:Nck protein; D015513:Oncogene Proteins; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011993:Recombinant Fusion Proteins; D051746:ZAP-70 Protein-Tyrosine Kinase; C497262:Zap70 protein, mouse