Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10815154 | Cellular Signalling | 2015 | 10 Pages |
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.
Keywords
InDelGrb2-related adaptor downstream of ShcSH2 domain-containing leukocyte protein of 76 kDaSLP-76T cell receptor (TCR)ZFNPLC-γ1GRAPGRB2TCRTITKIRESZAP-70phorbol 12-myristate 13-acetateSOSNFATIP3GFPInositol trisphosphateNHEJPMAMAPKstable isotope labeling with amino acids in cell cultureDAGTranscription activator-like effector nucleaseTALENLATGADSinternal ribosome entry siteSILACNuclear Factor of Activated T Cellsnon-homologous end joiningphospholipase C-γ1Linker for activation of T cellszinc finger nucleaseSrc homologyadaptor proteingreen fluorescent proteinmitogen-activated protein kinaseson of sevenlessCell receptor
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Authors
Jennie Lugassy, Jasmin Corso, Dvora Beach, Thomas Petrik, Thomas Oellerich, Henning Urlaub, Deborah Yablonski,