Regulation of Linear Ubiquitin Chain Assembly Complex by Caspase-Mediated Cleavage of RNF31

Abstract

Cell death and survival signaling pathways have opposed but fundamental functions for various cellular processes and maintain cell homeostasis through cross talk. Here we report a novel mechanism of interaction between these two pathways through the cleavage of RNF31 by caspases. RNF31, a component of the linear ubiquitin chain assembly complex (LUBAC), regulates cell survival by inducing linear ubiquitination of NF-B signaling components. We found that RNF31 is cleaved under apoptosis conditions through various stimulations. The effector caspases caspase 3 and caspase 6 are responsible for this event, and aspartates 348, 387, and 390 were identified as target sites for this cleavage. Cleavage of RNF31 suppressed its ability to activate NF-B signaling; thus, mutation of cleavage sites inhibited the induction of apoptosis by treatment with tumor necrosis factor alpha (TNF-␣). Our findings elucidate a novel regulatory loop between cell death and the survival signal and may provide guidance for the development of therapeutic strategies for cancers through the sensitization of tumor cells to death-inducing drugs. The nuclear factor B (NF-B) signaling pathway plays a critical role in various cellular processes, including proliferation, differentiation, survival, and death. In the resting state, inhibitor of B-␣ (IB-␣) sequesters the NF-B complex in the cytoplasm by interacting with it. Through the activation of the IB kinase (IKK) complex (composed of IKK␣, -␤, and -␥), followed by the phosphorylation and consequent degradation of IB-␣, free NF-B complex acquires the ability to enter the nucleus and induce target gene expression (1). Previous studies have revealed that K63-linked polyubiquitination of IKK␥ (also called NEMO) is critical for NF-B activation (2). Recently, linear ubiquitination was identified as a novel type of ubiquitination that forms the ubiquitin linkage between the N-terminal Met of one ubiquitin and the C-terminal Gly of another (3-5). To date, the linear ubiquitin chain assembly complex (LUBAC), which is composed of one main E3 ligase, ring finger protein 31 (RNF31; also known as HOIP), and two associated proteins, HOIL-1 and Sharpin, is the only E3 ligase complex for linear ubiquitination. Upstream activation leads to the linear ubiquitination of NEMO. Then these modified molecules function as a bridge between the receptor complex and the downstream IKK complex to activate NF-B signaling (6). Genetic studies have shown that defects in HOIL-1 or Sharpin result in reduced phosphorylation and degradation of IB-␣, impaired and delayed nuclear translocation of the NF-B subunit p65, diminished overall gene induction, and increased tumor necrosis factor (TNF)-induced cell death (3-5).The activation of NF-B signaling not only directly prompts cell growth and proliferation but also suppresses cell death by upregulating antiapoptotic molecules that inhibit the function of caspases. Caspases are regulatory proteases that are essential for apoptosis activation. Briefly, diverse factors, including TNF-␣, TN...

The linear ubiquitin chain assembly complex regulates TRAIL‐induced gene activation and cell death

“…Conversely, the shorter N‐terminal fragment was still visible in cells mutated for D387A (Fig E). Another study recently described that HOIP is cleaved at D390, in addition to D348 and D387 upon TNF stimulation (Joo et al , ). In accordance, HOIP D348A/D387A/D390A (HOIP AAA ) is completely resistant to TRAIL‐induced cleavage (Fig E).…”

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) regulates deubiquitinase USP5 in tumor cells

“…Labeling of DUBs using HA-Ub-Vs showed that at least 4-5 DUBs were consistently suppressed or inhibited by TRAIL in sensitive cells. This labeling can only recognize USP (ubiquitin specific protease) family members, and thus TRAIL may also affect other ubiquitin and DUB family proteins including E1, E2 and E3 ligases; in fact, cleavage of the E3 ligase RNF31 was shown to be TRAIL dependent [30]. The few DUBs inhibited by TRAIL may not be direct targets and should be further investigated.…”

Linear Ubiquitin Code: Its Writer, Erasers, Decoders, Inhibitors, and Implications in Disorders

“…However, under conditions where the expression of NF-κB-target genes is suppressed, such as by the protein synthesis inhibitor cycloheximide, TNF-α stimulation extensively induces apoptosis through the generation of TNFR complex IIa, which is composed of RIP1, FADD, and caspase 8 [55] (Figure 2). Subsequently, caspase 8 activates caspase 3 to induce extrinsic apoptosis, and the activated caspases cleave the N-terminal portion of HOIP [39,56]. A genetic deficiency of LUBAC subunits causes reduced expression of NF-κB genes, and thus efficiently induces TNF-α-mediated apoptosis in mice [11][12][13]38,57].…”