The FLIP-side of Fas signaling.
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Apoptosis↗
Biomedical subjects
Temesgen Samuel
Publications and source records attributed to Temesgen Samuel.
Distinct BIR domains of cIAP1 mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and second mitochondrial activator of caspases.
Inhibitor of apoptosis proteins (IAPs) regulate apoptosis primarily by inhibiting caspase-family proteases. However, many IAPs also possess E3 ligase (ubiquitin-protein isopeptide ligase) activities implicated in both caspase-dependent and -independent functions of these proteins. Here, we compared the structural features of cIAP1 responsible for its interactions with two known target proteins, TRAF2 and SMAC. The N-terminal (BIR1) and C-terminal (BIR3) BIR domains of cIAP1 were determined to be necessary and sufficient for binding TRAF2 and SMAC, respectively. Mutational analysis of the BIR1 and BIR3 domains identified critical residues required for TRAF2 and SMAC binding. Using these mutants, cIAP1-mediated ubiquitination of TRAF2 and SMAC in vitro was determined to be correspondingly dependent on intact binding sites on BIR1 and BIR3. Because TRAF2 regulates NF-kappaB activation, the effects of cIAP1 on TRAF2-mediated induction of NF-kappaB transcriptional activity were studied using reporter gene assays. Expression of a fragment of cIAP1 encompassing the three BIR domains (but not full-length cIAP1) greatly enhanced TRAF2-induced increases in NF-kappaB activity, providing a convenient assay for monitoring BIR-dependent effects of cIAP1 on TRAF2 in cells. BIR1 mutants of the BIR1-3 fragment of cIAP1 that failed to bind TRAF2 lost the ability to modulate NF-kappaB activity, demonstrating a requirement for BIR1-mediated interactions with TRAF2. Altogether, these findings demonstrate the modularity and diversification of BIR domains, showing that a single cIAP can direct its E3 ligase activity toward different substrates and can alter the cellular functions of different protein targets in accordance with differences in the specificity of individual BIR domains.
Amino Acid Sequence↗
cIAP1 Localizes to the nuclear compartment and modulates the cell cycle.
We explored the location and function of the human cIAP1 protein, a member of the inhibitor of apoptosis protein (IAP) family. Unlike family member X-linked IAP (XIAP), which was predominantly cytoplasmic, the cIAP1 protein localized almost exclusively to nuclei in cells, as determined by immunofluorescence microscopy and subcellular fractionation methods. Interestingly, apoptotic stimuli induced nuclear export of cIAP1, which was blocked by a chemical caspase inhibitor. In dividing cells, cIAP1 was released into the cytosol early in mitosis, then reaccumulated in nuclei in late anaphase and in telophase, with the exception of a pool of cIAP1 that associated with the midbody. Survivin, another IAP family member, and cIAP1 were both localized on midbody microtubules at telophase, and also interacted with each other during mitosis. Cells stably overexpressing cIAP1 accumulated in G(2)-M phase and grew slower than control-transfected cells. These cIAP1-overexpressing cells also exhibited cytokinesis defects over 10 times more often than control cells and displayed a mitotic checkpoint abnormality with production of polyploid cells when exposed to microtubule-targeting drugs nocodazole and paclitaxel (Taxol). Our findings demonstrate a role for overexpressed cIAP1 in genetic instability, possibly by interfering with mitotic functions of Survivin. These findings may have important implications for cancers in which cIAP1 overexpression occurs.
Apoptosis↗
Cellular, biochemical, and genetic analysis of mechanism of small molecule IAP inhibitors.
XIAP is member of the IAP family of anti-apoptotic proteins and is known for its ability to bind and suppress caspase family cell death proteases. A phenylurea series of chemical inhibitors of XIAP was recently generated by our laboratories (Schimmer, A. D., Welsh, K., Pinilla, C., Bonneau, M., Wang, Z., Pedersen, I. M., Scott, F. L., Glinsky, G. V., Scudiero, D. A., Sausville, E., Salvesen, G., Nefzi, A., Ostresh, J. M., Houghten, R. A., and Reed, J. C. (2004) Cancer Cell 5, 25-35). We examined the mechanisms of action of these chemical compounds using biochemical, molecular biological, and genetic methods. Active phenylurea-based compounds dissociated effector protease caspase-3 but not initiator protease caspase-9 from XIAP in vitro and restored caspase-3 but not caspase-9 enzymatic activity. When applied to tumor cell lines in culture, active phenylurea-based compounds induced apoptosis in a rapid, concentration-dependent manner, associated with activation of cellular caspases. Apoptosis induced by active phenylurea-based compounds was blocked by chemical inhibitors of caspases, with inhibitors of downstream effector caspases displaying more effective suppression than inhibitors of upstream initiator caspases. Phenylurea-based XIAP antagonists induced apoptosis (defined by annexin V staining) prior to mitochondrial membrane depolarization, in contrast to cytotoxic anticancer drugs. Consistent with these findings, apoptosis induced by phenylurea-based compounds was not altered by genetic alterations in the expression of Bcl-2 family proteins that control mitochondria-dependent cell death pathways, including over-expression of anti-apoptotic proteins Bcl-2 or Bcl-X(L) and genetic ablation of pro-apoptotic proteins Bax and Bak. Conversely, conditional over-expression of an active fragment of XIAP or genetic ablation of XIAP expression altered the apoptosis dose-response of the compounds. Altogether, these findings indicate that phenylurea-based XIAP antagonists block interaction of downstream effector caspases with XIAP, thus inducing apoptosis of tumor cell lines through a caspase-dependent, Bcl-2/Bax-independent mechanism.
Animals↗
An IAP-IAP complex inhibits apoptosis.
Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.
Animals↗
Retraction: The triterpenoid CDDO-Imidazolide induces apoptosis and enhances fludarabine-induced apoptosis of CLL B-cells.
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Retraction of Publication as Topic↗
Human p14(ARF)-mediated cell cycle arrest strictly depends on intact p53 signaling pathways.
The tumor suppressor ARF is transcribed from the INK4a/ARF locus in partly overlapping reading frames with the CDK inhibitor p16(Ink4a). ARF is able to antagonize the MDM2-mediated ubiquitination and degradation of p53, leading to either cell cycle arrest or apoptosis, depending on the cellular context. However, recent data point to additional p53-independent functions of mouse p19(ARF). Little is known about the dependency of human p14(ARF) function on p53 and its downstream genes. Therefore, we analysed the mechanism of p14(ARF)-induced cell cycle arrest in several human cell types. Wild-type HCT116 colon carcinoma cells (p53(+/+)p21(CIP1+/+) 14-3-3sigma(+/+)), but not p53(-/-) counterparts, underwent G(1) and G(2) cell cycle arrest following infection with a p14(ARF)-adenovirus. In p21(CIP1-/-) cells, p14(ARF) did not induce G(1) or G(2) arrest, while 14-3-3sigma(-/-) counterparts were mainly arrested in G(1), pointing to essential roles of p21(CIP1) in G(1) and G(2) arrest and cooperative roles of p21 and 14-3-3sigma in ARF-mediated G(2) arrest. Our data demonstrate a strict p53 and p21(CIP1) dependency of p14(ARF)-induced cell cycle arrest in human cells.
14-3-3 Proteins↗
Linking DNA damage to cell cycle checkpoints.
Cell cycle checkpoints constitute a network of signal transduction mechanisms to monitor DNA damage and replication and thereby regulate progression through the cell cycle. A series of events is triggered in cells upon DNA damage. Here we describe a framework for the understanding of the functions of the core components involved in the cell cycle response to DNA damage and the relevance to the origin of cancer.
Cell Cycle↗