CLIPR-59 regulates TNF-α-induced apoptosis by controlling ubiquitination of RIP1

D Fujikura,T Miyazaki,F Perez,S Chiba,J C Reed,T Uede,M Ito,T Harada

doi:10.1038/cddis.2012.3

Abstract

Tumor necrosis factor-α (TNF-α) has important roles in several immunological events by regulating apoptosis and transcriptional activation of cytokine genes. Intracellular signaling mediated by TNF-receptor-type 1 (TNFR1) is constituted by two sequential protein complexes: Complex-I containing the receptor and Complex-II-containing Caspase-8. Protein modifications, particularly ubiquitination, are associated with the regulation of the formation of these complexes. However, the underlying mechanisms remain poorly defined. Here, we identified CLIP-170-related 59 kDa protein (CLIPR-59) as a novel adaptor protein for TNFR1. Experimental reduction of CLIPR-59 levels prevented induction of apoptosis and activation of caspases in the context of TNF-α signaling. CLIPR-59 binds TNFR1 but dissociates in response to TNF-α stimulation. However, CLIPR-59 is also involved in and needed for the formation of Complex-II. Moreover, CLIPR-59 regulates TNF-α-induced ubiquitination of receptor-interacting protein 1 (RIP1) by its association with CYLD, a de-ubiquitinating enzyme. These findings suggest that CLIPR-59 modulates ubiquitination of RIP1, resulting in the formation of Complex-II and thus promoting Caspase-8 activation to induce apoptosis by TNF-α.

Highlights

It was demonstrated that the intracellular signaling mediated by TNF-receptortype 1 (TNFR1) is constituted by formation of two sequential protein complexes.[8,9] Aggregation of TNFR1 mediated by its specific ligand induces the recruitment of adaptor proteins tumor necrosis factor (TNF) receptor-associated death domain protein (TRADD) and receptor-interacting protein 1 (RIP1) to the cytoplasmic domain of TNFR1 (Complex-I)
In HeLa cells, co-IP assays using anti-TNFR1-specific antibody (Ab) showed that CLIPR-59 was associated with TNFR1 before ligand stimulation and this association was significantly reduced at 120 min after stimulation (Figure 1f)
It was demonstrated that CYLD is essential for the induction of apoptosis and Complex-II formation by TNF-a in the presence of a Smac mimetic compounds that induces auto-degradation of endogenous anti-apoptotic proteins, c-inhibitor of apoptosis (IAP)1/2.26 We examined the effect of CLIPR-59 and CYLD knockdown on the induction of apoptosis by treatment of tumor cell lines with TNF-a plus Smac mimetics or plus c-IAPs knockdown

Summary

Introduction

It was demonstrated that the intracellular signaling mediated by TNFR1 is constituted by formation of two sequential protein complexes.[8,9] Aggregation of TNFR1 mediated by its specific ligand induces the recruitment of adaptor proteins TNF receptor-associated death domain protein (TRADD) and receptor-interacting protein 1 (RIP1) to the cytoplasmic domain of TNFR1 (Complex-I) These proteins dissociate from Complex-I, facilitating the formation of a second protein complex (Complex-II)containing Fas-associated protein with death domain (FADD) and Caspase-8, which is an initiator of the proteolytic cascade resulting in apoptosis.[4,10,11] In addition, it is reported that the transition from Complex-I to Complex-II is regulated by protein modifications, ubiquitination of RIP1.12–14. These results suggest that de-ubiquitination of RIP1 regulates the formation of Complex-II in apoptotic signaling by TNF-a

Methods

Results

Conclusion