title: Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.
abstract: T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.
authors.affiliation: Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
authors.forename: Jennie
authors.initials: J
authors.lastname: Lugassy
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen 37077, Germany.
authors.forename: Jasmin
authors.initials: J
authors.lastname: Corso
authors.affiliation: Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
authors.forename: Dvora
authors.initials: D
authors.lastname: Beach
authors.affiliation: Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.
authors.forename: Thomas
authors.initials: T
authors.lastname: Petrik
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen 37077, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Oellerich
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Bioanalytical Mass Spectrometry Group, Göttingen 37077, Germany; Bioanalytics, Institute for Clinical Chemistry, University Medical Center, Göttingen 37075, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel. Electronic address: debya@tx.technion.ac.il.
authors.forename: Deborah
authors.initials: D
authors.lastname: Yablonski
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D015703:Antigens, CD / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000378:metabolism; D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D019169:Jurkat Cells; D037181:Lectins, C-Type / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D051966:Phospholipase C gamma / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D000099192:CD69 Antigens
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D004720:Endonucleases; D037181:Lectins, C-Type; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D010769:Phosphothreonine; D011948:Receptors, Antigen, T-Cell; D015534:Trans-Activators; D000099192:CD69 Antigens; C409587:GRAP2 protein, human; C092947:SLP-76 signal Transducing adaptor proteins
keywords: Adaptor protein; Gads; T cell receptor (TCR); Transcription activator-like effector nuclease (TALEN)