title: TCR and CD28 activate the transcription factor NF-κB in T-cells via distinct adaptor signaling complexes.
abstract: The transcription factor NF-κB is needed for the induction of inflammatory responses in T-cells. Whether its activation by the antigen-receptor and CD28 is mediated by the same or different intracellular signaling pathways has been unclear. Here, using T-cells from various knock-out (Cd28(-/-), adap(-/-)) and knock-in (i.e. Cd28 Y-170F) mice in conjunction with transfected Jurkat T-cells, we show that the TCR and CD28 use distinct pathways to activate NF-κB in T-cells. Anti-CD28 ligation alone activated NF-κB in primary and Jurkat T-cells as measured by NF-κB reporter and EMSA assays. Anti-CD28 also activated NF-κB normally in primary T-cells from adap(-/-) mice, while anti-CD3 stimulation required the adaptor ADAP. Over-expression of ADAP or its binding partner SKAP1 failed to enhance anti-CD28 activation of NF-κB, while ADAP greatly increased anti-CD3 induced NF-κB activity. By contrast, CD28 activation of NF-κB depended on GRB-2 binding to CD28 as seen in CD28 deficient Jurkat T-cells reconstituted with the CD28 YMN-FM mutant, and in primary T-cells from CD28 Y170F mutant knock-in mice. CD28 associated with GRB-2, and GRB-2 siRNA impaired CD28 NF-κB activation. GRB-2 binding partner and guanine nucleotide exchange factor, VAV1, greatly enhanced anti-CD28 driven activation of NF-κB. Further, unlike in the case of anti-CD28, NF-κB activation by anti-CD3 and its cooperation with ADAP was strictly dependent on LAT expression. Overall, we provide evidence that CD28 and the TCR complex regulate NF-κB via different signaling modules of GRB-2/VAV1 and LAT/ADAP pathways respectively.
authors.affiliation: Cell Signalling Section, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom. Electronic address: yrt20@cam.ac.uk.
authors.forename: Youg Raj
authors.initials: YR
authors.lastname: Thaker
authors.affiliation: Cell Signalling Section, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom.
authors.forename: Helga
authors.initials: H
authors.lastname: Schneider
authors.affiliation: Cell Signalling Section, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, United Kingdom.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Rudd
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000276:immunology*; D000818:Animals; D018106:CD28 Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018106:CD28 Antigens; D016328:NF-kappa B; D011948:Receptors, Antigen, T-Cell
keywords: ADAP; Adaptors; GRB-2; NF-κB; VAV-1