Abstract
The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.
Highlights
The biological activities of the tumor necrosis factor-␣ (TNF) pro-inflammatory cytokine are resolved by two distinct cell surface receptors, TNFR13 and TNFR2, which elicit a diversity of cellular responses, such as inflammation, cell prolifera
Ligand binding and trimerization of TNFR1 enables the recruitment of TNFR1associated death domain protein (TRADD) [12, 13], which functions as a scaffold enabling the recruitment of receptorinteracting protein kinase 1 (RIPK1) (14 –16), TNF receptorassociated factor 2 (TRAF2) or TRAF5 [12], and the cellular inhibitor of apoptosis proteins cIAP1 and cIAP2 [11], which collectively form a signaling composite called complex I [17,18,19]
Western blotting analysis revealed that constitutive generation of both TNFR1 C-terminal fragment (CTF) and TNFR1 intracellular domain (ICD) was observed in wild type murine embryonic fibroblasts (MEFs), and phorbol 12-myristate 13-acetate (PMA) stimulation increased generation of the ICD fragment (Fig. 3, d and e)
Summary
Reagents and Antibodies—␥-Secretase inhibitors DAPT and L-685,458, protein synthesis inhibitor cycloheximide, TACE/ ADAM17-specific metalloprotease inhibitor TAPI-1, proteasomal inhibitor epoxomicin, and phorbol 12-myristate 13-acetate (PMA) were purchased from Calbiochem. Cells were kept on ice. Cells were incubated for 1 h with primary antibody (TNFR1 H5, Santa Cruz Biotechnology) in wash buffer, followed by three washes. Cells were incubated for 30 min with secondary antibody (Alexa Fluor anti-mouse 488) diluted in wash buffer. Cells were harvested in ice-cold PBS and lysed with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) freshly supplemented with 1 mM sodium orthovanadate and protease inhibitor mixture (CompleteTM, Molecular Biochemicals) on ice. Lysates were centrifuged (13,200 rpm, 20 min, 4 °C), the supernatants were collected, and protein yield was quantified using a bicinchoninic acid (BCA) assay (Pierce). A p value of 0.05 was considered significant (*, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
