title: Tyrosine phosphorylation of the CD3-epsilon subunit of the T cell antigen receptor mediates enhanced association with phosphatidylinositol 3-kinase in Jurkat T cells.
abstract: T cell receptor signaling results both in T cell proliferation and apoptosis. A key enzyme at the intersection of these downstream pathways is phosphatidylinositol 3'-kinase (PI 3-kinase). In a previous report, we showed that the p85alpha subunit of the PI 3-kinase preferentially associated with the CD3-zeta membrane-proximal immunoreceptor tyrosine-based activation motif of the zeta chain (zetaA-ITAM) (Exley, M., Varticovski, L., Peter, M., Sancho, J., and Terhorst, C. (1994) J. Biol. Chem. 269, 15140-15146). Here, we demonstrate that tyrosine phosphorylation of CD3-epsilon can recruit the PI 3-kinase enzyme in a T cell activation-dependent manner. In vivo studies with Jurkat cells stably transfected with a CD8-CD3-epsilon chimera (termed CD8-epsilon) shows that ligation of endogenous CD3-epsilon or CD8-epsilon by specific antibodies induces tyrosine phosphorylation of CD3-epsilon or CD8-epsilon, respectively. Increased tyrosine phosphorylation correlates with increased binding of p85alpha PI 3-kinase and recruitment of PI 3-kinase enzymatic activity to CD3-epsilon or CD8-epsilon proteins. Mutagenesis studies in COS-7 cells, transiently transfected with CD8-epsilon, p85alpha, and Fyn cDNAs in various combinations, show that both Tyr170 and Tyr181 within the CD3-epsilon-ITAM are required for efficient binding of p85alpha PI 3-kinase. Thus, replacement of Tyr170 by Phe (Y170F), or Tyr181 by Phe (Y181F) significantly reduces binding of p85alpha PI 3-kinase, whereas it does not affect binding of Fyn. Further in vitro experiments suggest that a direct binding of the tandem SH2 domains of p85alpha PI 3-kinase to the two phosphorylated tyrosines in a single CD3-epsilon-ITAM may occur. The data also support a model in which a single CD3 subunit can recruit distinct effector molecules by means of TCR-mediated differential ITAM phosphorylation.
authors.affiliation: Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Ventanilla 11, 18001 Granada, Spain.
authors.forename: I
authors.initials: I
authors.lastname: de Aós
authors.forename: M H
authors.initials: MH
authors.lastname: Metzger
authors.forename: M
authors.initials: M
authors.lastname: Exley
authors.forename: C E
authors.initials: CE
authors.lastname: Dahl
authors.forename: S
authors.initials: S
authors.lastname: Misra
authors.forename: D
authors.initials: D
authors.lastname: Zheng
authors.forename: L
authors.initials: L
authors.lastname: Varticovski
authors.forename: C
authors.initials: C
authors.lastname: Terhorst
authors.forename: J
authors.initials: J
authors.lastname: Sancho
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine; D017354:Point Mutation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013601:T-Lymphocytes
chemical_list: D000906:Antibodies; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C061836:antigen T cell receptor, epsilon chain; D019000:Phosphotyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor)