Direct Interaction between Survivin and Smac/DIABLO Is Essential for the Anti-apoptotic Activity of Survivin during Taxol-induced Apoptosis

Abstract

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that has been implicated in both apoptosis inhibition and cell cycle control. However, its inhibitory mechanism and subcellular localization remain controversial. In this report, we provided evidence for the first time that Survivin physically interacts with Smac/DIABLO both in vitro and in vivo. A point mutation (D71R) in the baculovirus IAP repeat motif and a C-terminal deletion mutant (Surv-BIR) of Survivin fail to bind to Smac/DIABLO and abrogate its ability to inhibit apoptosis. The N-terminal of mature Smac/DIABLO is absolutely required for Survivin⅐Smac complex formation. Subcellular distributions of Survivin and Smac/ DIABLO showed that they co-localized within the cytosol during interphase. In addition, Survivin was found to be incapable of binding to caspase. We also identified that the co-presence of Smac/DIABLO and XIAP was required for Survivin to inhibit caspase cleavage in a cell-free system. In conclusion, our results provide the first evidence that the interaction between Smac/DIA-BLO and Survivin is an essential step underling the inhibition of apoptosis induced by Taxol.

Livin promotes Smac/DIABLO degradation by ubiquitin–proteasome pathway

“…19,20 Protein stability was compared among wtLivin, LivinDRING, Livin-C124A and Livin-C252A with and without MG132 treatment. As shown in Figure 3, although the steady-state level of wt-Livin or Livin-C124A was considerably higher in the presence (lanes 2, 6) than in the absence of MG132 (lanes 1, 5) (densitometry data not shown), both RING deletion mutant LivinDRING and RING point mutant Livin-C252A were expressed at comparable levels before and after MG132 treatment (lanes 3,4,7,8), indicating that the RING domain, but not BIR domain, is responsible for Livin's ubiquitin-mediated proteasome degradation. We noticed that BIR mutant Livin-C124A always expressed at a lower level than wt-Livin, and moreover, MG132 treatment could not bring the level of BIR mutant back to a level similar to that of wtLivin.…”

“…Western blot analysis was performed according to the procedures described by Song et al 8 For immunoprecipitation, cells were first lysed in a Triton X-100-based lysis buffer (1% Triton X-100, 10% glycerol, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, protease inhibitor cocktail) for 1 h, and the nuclear and cellular debris was cleared by centrifugation. Cytosolic lysate was then incubated with first monoclonal antibody-bound Protein A/G-Sepharose.…”

“…3,4 Mature Smac/DIABLO was reported to interact with XIAP, cIAP-1, cIAP-2, Survivin or Livin to inhibit their antiapoptotic activity. [5][6][7][8][9] Although RING domains have been identified in various proteins with different functions such as DNA and protein interactions, 10 the most prominent role the RING domain in XIAP, cIAP-1 or cIAP-2 plays has been attributed to its E3 ubiquitin ligase activity: promoting the degradation of both targeted proteins and themselves through ubiquitination. Although XIAP and cIAP-1 have been shown to mediate their self-degradation during apoptosis of thymocytes, XIAP, cIAP-1 and cIAP-2 have also been attested as ubiquitin-protein ligase E3 for Smac/ DIABLO.…”

Antisense and nonantisense effects of antisense Bcl-2 on multiple roles of Bcl-2 as a chemosensitizer in cancer therapy

“…The apoptosome then catalytically activates effector caspase-3 by cleavage, resulting in wide-spread proteolysis of the substrates such as PARP and inhibitor of caspase-activated DNase leading to DNA fragmentation in apoptosis. 15 Smac/DIABLO, the second small molecule released from mitochondria, inhibits inhibitors of apoptosis proteins (IAPs) such as c-IAP1, c-IAP2 35 and survivin, 36 resulting in the activation of caspase-9. Smac/DIABLO also directly promotes the activation of caspase-3.…”

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

“…Although it is capable of binding to effector caspases under cell free conditions, under more physiological conditions it inhibits apoptosis by binding to second mitochondrial activator of caspases (Smac). 12 Survivin represents the fourth top gene expressed in cancers of the lung, colon, brain, breast and in melanoma, but is absent in most normal adult tissues. 11,13 Survivin overexpression has been identified as a negative prognostic factor in various cancer types, [14][15][16] and is implicated in resistance to apoptosis induction by anti-cancer agents and ionizing radiation.…”