TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1–TRAF2 complex to sensitize tumor cells to TNFα

Synthetic inhibitor of apoptosis (IAP) antagonists induce degradation of IAP proteins such as cellular IAP1 (cIAP1), activate nuclear factor κB (NF-κB) signaling, and sensitize cells to tumor necrosis factor α (TNFα). The physiological relevance of these discoveries to cIAP1 function remains undetermined. We show that upon ligand binding, the TNF superfamily receptor FN14 recruits a cIAP1–Tnf receptor-associated factor 2 (TRAF2) complex. Unlike IAP antagonists that cause rapid proteasomal degradation of cIAP1, signaling by FN14 promotes the lysosomal degradation of cIAP1–TRAF2 in a cIAP1-dependent manner. TNF-like weak inducer of apoptosis (TWEAK)/FN14 signaling nevertheless promotes the same noncanonical NF-κB signaling elicited by IAP antagonists and, in sensitive cells, the same autocrine TNFα-induced death occurs. TWEAK-induced loss of the cIAP1–TRAF2 complex sensitizes immortalized and minimally passaged tumor cells to TNFα-induced death, whereas primary cells remain resistant. Conversely, cIAP1–TRAF2 complex overexpression limits FN14 signaling and protects tumor cells from TWEAK-induced TNFα sensitization. Lysosomal degradation of cIAP1–TRAF2 by TWEAK/FN14 therefore critically alters the balance of life/death signals emanating from TNF-R1 in immortalized cells

TAK1 Is Required for Survival of Mouse Fibroblasts Treated with TRAIL, and Does So by NF-κB Dependent Induction of cFLIPL

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a “death ligand”—a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-κB and JNK signalling pathways. To determine the role of TGF-β-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1−/− MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-κB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1−/− MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1–NF-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance

Identification of an Xiap-Like Pseudogene on Mouse Chromosome 7

The most thoroughly characterized mammalian IAP is XIAP/BIRC4, which can inhibit caspases 9, 3 and 7, but may also regulate apoptosis through interactions with other proteins such as Smac/DIABLO, HtrA2/Omi, XAF1, TAK1, cIAP1, and cIAP2. High throughput sequencing of the mouse genome revealed the existence of a gene resembling Xiap/Birc4 on mouse chromosome 7. To confirm the existence of this gene, and to determine its functional significance, we performed Southern and Northern blot analysis. This showed the presence of the Xiap-like gene in both wild-type and Xiap gene knock-out mice, but the corresponding mRNA was not detected in any tissues examined by Northern blot. Analysis of the gene sequence in all three possible reading frames predicts that expression of this gene would not give rise to a full-length protein, but only non-functional truncated polypeptides. Because its nucleotide sequence is 92% identical to Xiap, but it has no introns corresponding to those of Xiap, we conclude that Xiap-ps1 is a pseudogene generated by retro-transposition of a spliced Xiap message to chromosome 7

Schematic diagram of the <i>Xiap</i> genes.

<p>(a) The murine <i>Xiap</i> gene spans 42 kb on the X-chromosome and consists of 7 exons. <i>Xiap</i> K/O locus has exon 2 removed via homologous recombination. The <i>Xiap-</i>like gene is found on chromosome 7 and lacks any intronic sequences, giving rise to one exon that is 92% identical to spliced WT <i>Xiap</i>. A DNA probe designed to detect this pseudogene was produced from a 510 bp region of exon 2 in WT <i>Xiap</i> that is unable to hybridise to the <i>Xiap</i> K/O locus.(b) The splicing of the 7 exons of <i>Xiap</i> gives rise to mRNA encoding the XIAP protein. Two codons from the beginning and the end of each exon were aligned to <i>Xiap-ps1</i>. The nucleotide sequences of <i>Xiap</i> and <i>Xiap-ps1</i> are identical around the exon boundaries with the exceptions of two C>T transitions, one silent at the beginning of exon 4 and another coding at the end of exon 5.</p

Confirmation of RNA expression by Northern analysis.

<p>Total RNA was harvested from C57BL/6 and <i>Xiap</i> K/O tissues, separated by electrophoresis, blotted and probed with a 510 bp <i>Xiap</i> fragment as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008078#pone-0008078-g001" target="_blank">Figure 1</a>. The only band detected (∼6.6 kb) is the reported size for <i>Xiap</i> mRNA, and was only found in wild-type tissues and not in the <i>Xiap</i> knockout tissues. A β-actin probe was used to re-probe the same blots to show relative loading of lanes.</p

Alignment of translated pseudogene to WT XIAP protein.

<p>Three small methionine-initiated peptides are predicted from translation of the pseudogene sequence in all three frames. Functional domains are highlighted by background shading. XIAP-ps1 peptides encompassing BIR 1 and the RING domain are translations from the first frame, whereas the peptide containing BIR3 is a translation in the 3^rd frame. Alignment of these peptides to WT XIAP shows significant similarity with regions of XIAP, as shown in pale grey lettering, but no full length protein could be produced.</p

Detection of an <i>Xiap</i> pseudogene by Southern analysis.

<p>Genomic DNA was digested to completion with <i>Bam</i>HI (B), <i>Eco</i>RI (E), <i>Hin</i>dIII (H), or <i>Xba</i>I (X), and probed with a 510 bp fragment from exon 2 of <i>Xiap</i>. Bands of the expected size for the <i>Xiap</i> gene were revealed in the WT DNA. In addition, both WT and <i>Xiap</i> K/O DNA showed bands of the sizes predicted from the sequence of the <i>Xiap</i>-like gene on chromosome 7.</p

Mature DIABLO/Smac Is Produced by the IMP Protease Complex on the Mitochondrial Inner Membrane

DIABLO/Smac is a mitochondrial protein that can promote apoptosis by promoting the release and activation of caspases. To do so, DIABLO/Smac must first be processed by a mitochondrial protease and then released into the cytosol, and we show this in an intact cellular system. We propose that the precursor form of DIABLO/Smac enters the mitochondria through a stop-transfer pathway and is processed to its active form by the inner membrane peptidase (IMP) complex. Catalytic subunits of the mammalian IMP complex were identified based on sequence conservation and functional complementation, and the novel sequence motif RX(5)P in Imp1 and NX(5)S in Imp2 distinguish the two catalytic subunits. DIABLO/Smac is one of only a few specific proteins identified as substrates for the IMP complex in the mitochondrial intermembrane space