title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome.
abstract: T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Hyun Mu
authors.initials: HM
authors.lastname: Shin
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Mulualem E
authors.initials: ME
authors.lastname: Tilahun
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Ok Hyun
authors.initials: OH
authors.lastname: Cho
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Karthik
authors.initials: K
authors.lastname: Chandiran
authors.affiliation: Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Christina Arieta
authors.initials: CA
authors.lastname: Kuksin
authors.affiliation: Program in Molecular Biology, Loyola University Medical Center , Maywood, IL , USA.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Keerthivasan
authors.affiliation: Chemical Synthesis Core Facility, Mayo Clinic , Jacksonville, FL , USA.
authors.forename: Abdul H
authors.initials: AH
authors.lastname: Fauq
authors.affiliation: Center for Translational Research in Neurodegenerative Disease, University of Florida , Gainesville, FL , USA ; Department of Neuroscience, College of Medicine, University of Florida , Gainesville, FL , USA.
authors.forename: Todd E
authors.initials: TE
authors.lastname: Golde
authors.affiliation: Department of Medicine and Pharmacology, University of Mississippi Medical Center, University of Mississippi Cancer Institute , Jackson, MS , USA.
authors.forename: Lucio
authors.initials: L
authors.lastname: Miele
authors.affiliation: Department of Biochemistry, Center of Immunity and Infection, University of Lausanne , Epalinges , Switzerland.
authors.forename: Margot
authors.initials: M
authors.lastname: Thome
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts/Amherst , Amherst, MA , USA ; Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Osborne
authors.affiliation: Program in Molecular and Cellular Biology, University of Massachusetts/Amherst , Amherst, MA , USA ; Department of Veterinary and Animal Sciences, University of Massachusetts/Amherst , Amherst, MA , USA.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Minter
keywords: CARMA1; NF-κB; NOTCH1; PKCθ; T cell subject category: immunology; cytosolic; non-canonical; signal transduction