| Mcl-1, a member of Bcl-2 family, is capable of blocking apoptosis induced by various apoptotic stimuli, however, the mechanism by which Mcl-1 inhibits apoptosis has not been conclusively determined.In our study, we report that Mcl-1 is able to interact with Puma, a proapoptotic member of Bcl-2 family. Their binding sites are mapped to BH3 (Bcl-2 homology) domain of Puma and BH1 domain of Mcl-1, respectively. Mcl-1 and Puma are shown to coiocalize at the mitochondria by immunostaining. The level of Mcl-1 is increased when coexpressed with Puma, indicating Puma is able to stabilize Mcl-1. Puma binding to Mcl-1 via its BH3 domain is the prerequisite for this effect, which is further supported by the finding that Puma mutant lacking BH3 domain no longer promotes Mcl-1 protein stability. We also show that BH1 domain is essential for Mcl-1 to inhibit Puma-induced apoptosis, since Mcl-1 mutant lacking BH1 domain completely abrogates its protective function. In addition, we conclude that binding of Puma to BH1 domain of Mcl-1 is necessary, but not sufficient to prevent rapid degradation of Mcl-1. In addition to PEST and BH1 domain, some additional degradation signal is expected to reside in the C-terminal region of Mcl-1. In conclusion, our results provide the first evidence that the interaction between Mcl-1 and Puma may represent a novel mechanism by which Mcl-1 prevents apoptosis by increasing its stability through binding to Puma.We also demonstrate that BH3-only protein Noxa is upregulated during Camptothecin-induced apoptosis, which is independent of p53. In addition, we show that PI3k/Akt signaling pathway is responsible for Noxa's induction. Luciferase assay and CREB knock-down experiments further demonstrate that CREB is involved in the transcriptional upregulation of Noxa. More importantly, blocking CPT-induced Noxa using specific siRNA significantly reduces the apoptosis response, indicating that Noxa is an essential mediator for CPT-induced apoptosis. Interestingly, we find that Mcl-1 is also upregulated through PI3k/Akt signaling pathway upon CPT treatment. Using immunoprecipitation assay, we demonstrate that Noxa is able to interact with Mcl-1 to form the complex in CPT treated or untreated cells, suggesting that the balance between Noxa and Mcl-1 may regulate cells' susceptibility to CPT-induced apoptosis. In supporting of this conclusion, knock-down of Mcl-1 is shown to potentiate CPT-induced apoptosis, however, ectopic overexpression of Mcl-1 is able to rescue from apoptosis induced by CPT or CPT and LY294002.In summary, these data suggest that Mcl-1 plays its antiapoptotic role in apoptosis partly by binding to Puma or Noxa. Siah-1 (seven in absentia homolog) is known to cause indirect degradation ofβ-catenin through formation of a complex with SIP, Skp1 and Ebi. Here we report the characterization of a novel splice variant of human Siah-1, designated Siah-1S, which is produced by an alternative splicing mechanism. The novel intron/exon junctions used to generate Siah-1S follow a non-conventional CT-AC rule. Siah-1S exhibits an even shorter half-life than Siah-1 and is able to catalyse self-ubiquitination that results in its subsequent degradation by Proteasome. Siah-1S is shown to upregulateβ-catenin-dependent Tcf/Lef transcriptional activation and antagonize Siah-1's potentiation effect on the apoptosis induced by Etoposide in MCF-7 cells. Additionally, Siah-1S is found to interact with Siah-1 to form heterodimer or with itself to form homodimer. Unlike homodimer Siah-1 ~*Siah-1, neither Siah-1 ~*Siah-1S nor Siah-1S~*Siah-1S is able to bind to SIP (Siah-1-interacting protein), which may explain the underlying mechanism for Siah-1S's dominant negative effect on Siah-1. Importantly, results from in vitro soft agar assay demonstrated that Siah-1S displays a promotion effect on cells tumorigenecity.
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