Abstract
Survivin is a multifunctional member of the IAP (inhibitor of apoptosis) family, but its molecular interactions in protection from cell death and regulation of cell division have not been completely elucidated. In a proteomics screening to identify novel survivin-binding partners, we found that the aryl hydrocarbon receptor-interacting protein (AIP) directly associates with survivin in vitro and in co-immunoprecipitation experiments in vivo. This interaction is mediated by the carboxyl-terminal end of AIP, which contains three tetratricopeptide motifs, and involves the carboxyl terminus coiled coil in survivin with critical roles of Asp(142) in AIP recognition. A survivin mutant lacking only Asp(142) fails to bind AIP and exhibits accelerated degradation in vivo in a reaction reversed by a proteasome inhibitor. Acute knock-down of AIP by short interference RNA or competition of the survivin-AIP complex by peptidyl mimicry destabilizes survivin levels in cells, with enhanced apoptosis but no changes in cell cycle progression. Therefore, AIP regulates survivin stability, thus elevating a cellular anti-apoptotic threshold. The survivin-AIP complex may influence the cellular xenobiotic response to environmental toxin(s) and contribute to subcellular chaperone trafficking during cell death regulation.
Highlights
Idly in response to noxious environmental stimuli, including hypoxia, irradiation, exposure to chemotherapeutic drugs, and heat shock (5, 6)
We found that the aryl hydrocarbon receptor-interacting protein (AIP)[3] (11), a critical modulator of the cellular xenobiotic response to environmental toxin(s) and a component of the Hsp[90] chaperone system (12), directly associates with survivin, preserving protein stability and a cytosolic anti-apoptotic threshold
Survivin associates with AIP directly in vitro and in vivo through a recognition largely mediated by the last carboxyl terminus residue in survivin, Asp[142]
Summary
Cell Culture and Reagents—Cervical carcinoma HeLa, breast adenocarcinoma MCF-7, and lymphoblastoid Raji cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in culture as recommended by the supplier. The lysate was mixed with control GST beads or GST beads coupled to recombinant survivin and washed with 20 bead volume of PBS, and bound proteins were eluted in 20 mM Tris, pH 7.4, 2 mM EDTA, 0.1% CHAPS, and 1 M NaCl. The eluates were concentrated by precipitation with ProteoExtract protein precipitation kit (Calbiochem), and the pellets were analyzed by twodimensional gel electrophoresis, followed by silver staining (Genomine, Pohang, Kyungbuk, South Korea). 35S-labeled survivin or 35S-labeled AIP was generated using a TNT quick coupled transcription/translation system (Promega) in the presence of [35S]methionine (Amersham Biosciences), mixed with GST, GST-AIP, or GST-survivin, and bound proteins were detected by autoradiography In another series of experiments, Raji cell extracts were incubated with control IgG or an antibody to survivin, and the immune complexes were precipitated by the addition of protein A-Sepharose beads (Amersham Biosciences) in buffer containing 50 mM Tris, pH 7.4, 120 mM NaCl, 0.5% Triton X-100. Phenylindole for 1 min, rinsed, mounted in 90% glycerol, and sealed with nail polish, as described (13)
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