Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.

Cellular IAP12 and IAP2 (c-IAP1 and c-IAP2) were identified in a search for proteins associated with TNF receptors (TNFRs) (1). Through binding to TNFR-associated factor 2 (TRAF2), c-IAP1 and c-IAP2 are recruited to TNFR signaling complexes, where they regulate the activation of caspase-8 (1, 2). c-IAP1 and c-IAP2 were also proposed to modulate activation of the canonical NF-B pathway, although most of these studies relied on overexpression (3, 4). In contrast, however, targeted deletion of c-IAP1 or c-IAP2 genes in mice did not reveal any abnormalities in TNF␣-induced NF-B (nuclear factor B) activation (5, 6). The absence of any appreciable phenotype in single c-IAP knock-out mice has been attributed to the putative redundancy of c-IAP1 and c-IAP2 due to their high level of sequence and functional similarities (7). Thus, combined deficiency of cellular IAPs might expose their role in this signaling pathway. In support of this possibility, a null mutation in the sole cellular IAP in zebrafish results in severe defects in NF-B activation (8). c-IAP1 and c-IAP2 are also RING domain-containing ubiquitin ligases capable of promoting ubiquitination of several of their binding partners, including TRAF2 and SMAC (second mitochondrial activator of caspases) (4, 5, 9 -12).TNFR1 mediates activation of several signaling pathways, among them the canonical NF-B pathway (13). Binding of TNF␣ to TNFR1 induces recruitment of the adaptor protein TNFR-associated death domain (TRADD) to the death domain of the receptor (14). Through its death domain and amino-terminal region, TRADD recruits RIP1 (receptor-interacting protein), TRAF2, and through its interaction with TRAF2, c-IAP1 and c-IAP2 (13). Following binding to TRADD, TRAF2 was thought to mediate non-degradative Lys-63-linked polyubiquitination of RIP1 via its RING E3 ligase domain (15,16). This RIP1 modification induces assembly of two RIP1-associated kinase complexes, TAK1-TABs (transforming growth factor ␤-activated kinase 1-TAK1-binding proteins) and IB kinase (IKK) (17)(18)(19). Binding of these two complexes to Lys-63-linked polyubiquitin chains on RIP1 leads to phosphorylation of IKK␤ and subsequent phosphorylation and proteasomal degradation of IB (20). Loss of IB allows translocation of p50/RelA dimer to the nucleus and induction of gene expression (20).In the present study, we investigate the role of c-IAP1 and c-IAP2 in TNF␣-induced NF-B activation. We discover that c-IAP proteins are important mediators of canonical NF-B signaling and demonstrate that the absence of c-IAPs severely attenuates TNF␣-induced NF-B activation. Finally, we show that c-IAPs are ubiquitin ligases capable of promoting polymerization of Lys-63-linked polyubiquitin chains on the critical adapter in the canonical NF-B signaling pathway, RIP1. was from Genentech, Inc. The primary antibodies against mouse c-IAP1 were kindly provided by Drs. John Silke and David Vaux; anti-human c-IAP1 antibodies were purchased from R&D (affinity-purified goat antibody) or Protein Tech Group Inc.; pan c-IAP1/2 ...

Telomere maintenance is essential for the continuous growth of tumor cells. In most human tumors telomeres are maintained by telomerase, a specialized reverse transcriptase. Tankyrase 1, a human telomeric poly(ADP-ribose) polymerase (PARP), positively regulates telomere length through its interaction with TRF1, a telomeric DNA-binding protein. Tankyrase 1 ADP-ribosylates TRF1, inhibiting its binding to telomeric DNA. Overexpression of tankyrase 1 in the nucleus promotes telomere elongation, suggesting that tankyrase 1 regulates access of telomerase to the telomeric complex. The recent identification of a closely related homolog of tankyrase 1, tankyrase 2, opens the possibility for a second PARP at telomeres. We therefore sought to establish the role of tankyrase 1 at telomeres and to determine if tankyrase 2 might have a telomeric function. We show that endogenous tankyrase 1 is a component of the human telomeric complex. We demonstrate that telomere elongation by tankyrase 1 requires the catalytic activity of the PARP domain and does not occur in telomerase-negative primary human cells. To investigate a potential role for tankyrase 2 at telomeres, recombinant tankyrase 2 was subjected to an in vitro PARP assay. Tankyrase 2 poly(ADP-ribosyl)ated itself and TRF1. Overexpression of tankyrase 2 in the nucleus released endogenous TRF1 from telomeres. These findings establish tankyrase 2 as a bona fide PARP, with itself and TRF1 as acceptors of ADP-ribosylation, and suggest the possibility of a role for tankyrase 2 at telomeres.Telomere integrity is essential for chromosome stability, and the maintenance of telomeric DNA is required for long-term proliferation of eukaryotic cells. Telomeres are maintained by telomerase, a reverse transcriptase that adds telomeric repeats to chromosome ends (14; reviewed in reference 31). In most normal human somatic tissue telomerase is repressed, and as a result, telomeres shorten (17, 18). Critically short telomeres lose their ability to protect chromosome ends, resulting in chromosomal degradation and fusion. In contrast to normal somatic human cells, immortalized cells (including cancer cells) and germ cells express telomerase (21, 34) and maintain their telomeres. In these cells telomere maintenance is regulated by a homeostatic mechanism (reviewed in reference 29). Thus, in the mammalian germ line telomeres show a speciesspecific telomere length setting which is constant over the generations (23). Regulation is also apparent in many human tumor cell lines, where despite the presence of high levels of telomerase telomeres do not grow, but rather, they are stably maintained within a given size range (9, 10).Mammalian telomeres consist of long tandem arrays of TTAGGG repeats bound by the DNA-binding proteins, TRF1 and TRF2 (4, 5, 7; reviewed in reference 8). The TRFs are related in their primary structure; both contain carboxy-terminal Myb-type DNA-binding motifs and internal, conserved domains required for homodimerization (2, 5). The proteins do not form heterodimers (5). A dis...

Cohesins keep sister chromatids associated from the time of their replication in S phase until the onset of anaphase. In vertebrate cells, two distinct pathways dissociate cohesins, one acts on chromosome arms and the other on centromeres. Here, we describe a third pathway that acts on telomeres. Knockdown of tankyrase 1, a telomeric poly(ADP-ribose) polymerase caused mitotic arrest. Chromosomes aligned normally on the metaphase plate but were unable to segregate. Sister chromatids separated at centromeres and arms but remained associated at telomeres, apparently through proteinaceous bridges. Thus, telomeres may require a unique tankyrase 1-dependent mechanism for sister chromatid resolution before anaphase.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-jB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFa-stimulated NF-jB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-jB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.