title: Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phosphorylation of a common site in CD3epsilon.
abstract: Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3epsilon to Eps8L1 and Nck SH3 domains. CD3epsilon bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3epsilon to interact with either SH3 or SH2 domain-containing proteins.
authors.affiliation: Department of Virology, Haartman Institute, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland.
authors.forename: Tapio
authors.initials: T
authors.lastname: Kesti
authors.forename: Anja
authors.initials: A
authors.lastname: Ruppelt
authors.forename: Jing-Huan
authors.initials: JH
authors.lastname: Wang
authors.forename: Michael
authors.initials: M
authors.lastname: Liss
authors.forename: Ralf
authors.initials: R
authors.lastname: Wagner
authors.forename: Kjetil
authors.initials: K
authors.lastname: Taskén
authors.forename: Kalle
authors.initials: K
authors.lastname: Saksela
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000502:physiology; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D019151:Peptide Library; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000276:immunology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D017252:CD3 Complex; C489989:EPS8 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C485544:NCK2 protein, human; C078332:Nck protein; D015513:Oncogene Proteins; D019151:Peptide Library; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine