title: Characterization of the roles of SH2 domain-containing proteins in T-lymphocyte activation by using dominant negative SH2 domains.
abstract: Activation of the T-cell antigen receptor initiates a complex signaling cascade leading to changes in cytokine gene transcription. Several proteins containing Src homology 2 (SH2) domains, capable of interacting with phosphotyrosine-containing sequences within other proteins, are involved in the activation of signaling intermediates such as p2l(ras) and phospholipase Cgamma1. In this study, we used dominant negative SH2 domains to determine the importance of SH2 domain-containing proteins for T-cell activation. We show that tandem SH2 domains of either Zap70 or Syk tyrosine kinase are potent inhibitors of signaling initiated by the T-cell receptor zeta chain in vivo while individual SH2 domains are ineffective. Thus, while only the C-terminal SH2 domains appear to have significant affinity for immunoreceptor tyrosine-based activation motifs in vitro, the N-terminal SH2 domains are necessary in vivo. We find the spacing between the tandem SH2 domains of Zap70 to be critical for in vivo interactions. The SH2 domain of the adapter protein Grb2 is an effective inhibitor in our dominant negative assay, although it has little affinity for immunoreceptor tyrosine-based activation motifs. A single point mutation that abolishes phosphotyrosine binding renders the Grb2 SH2 domain incapable of this inhibition. In contrast, the SH2 domain of Shc does not inhibit this signaling cascade. We conclude that Grb2, but not Shc, is involved in T-cell receptor signaling.
authors.affiliation: Affymax Research Institute, Santa Clara, California, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Northrop
authors.forename: M J
authors.initials: MJ
authors.lastname: Pustelnik
authors.forename: A T
authors.initials: AT
authors.lastname: Lu
authors.forename: J R
authors.initials: JR
authors.lastname: Grove
mesh_terms: D000595:Amino Acid Sequence; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis; D002461:Cell Line, Transformed; D016207:Cytokines / Q000096:biosynthesis*; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D013539:Simian virus 40 / Q000235:genetics; D000072377:Syk Kinase; D013601:T-Lymphocytes / Q000276:immunology*; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
chemical_list: D000952:Antigens, Polyomavirus Transforming; D016207:Cytokines; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human