Conversion of Bfl‐1, an anti‐apoptotic Bcl‐2 family protein, to a potent pro‐apoptotic protein by fusion with green fluorescent protein (GFP)

Ko, Jae‐Woong; Choi, Kyoung-Han; Kim, Heejung; Choi, Hyeyoung; Yeo, Dong-Jun; Park, Sue-O; Yang, Wan Seok; Kim, Yong-Nyun; Kim, Chulwoo

doi:10.1016/s0014-5793(03)00872-x

Cited by 12 publications

(28 citation statements)

References 38 publications

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“…41 These observations with Bfl-1 agree with the capacity of other Bcl-2 family proteins like Bcl-2 and Bcl-xL to either suppress cell death (fully or partially) or promote apoptosis depending on the stimulus and/or the cell type. 9,10,31 Contrary to a recent report indicating that GFP-Bfl-1 is proapoptotic in transiently transfected 293T cells in the absence of a death-inducing signal, 42 we found no evidence that GFP-Bfl-1 is by itself proapoptotic, as FL5.12 cells stably expressing GFP-Bfl-1 were viable and proliferated normally. Moreover, GFP-Bfl-1 showed no toxicity when transiently expressed in other cell lines such as MCF-7 or HeLa cells (data not shown).…”

Section: Discussioncontrasting

confidence: 99%

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

et al. 2005

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Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti-versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by l-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1-3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.

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Section: Discussioncontrasting

confidence: 99%

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

et al. 2005

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“…Previous studies have demonstrated a bifunctional property of Bfl1, with the full-length protein containing the recognized anti-apoptotic activity and the N-terminus deletion mutants displaying potent pro-apoptotic activities (Ko et al, 2003a;Yang et al, 2005). A recent study by Kucharczak et al (Kucharczak et al, 2005) showed that TNF␣ receptor activation results in in-vivo proteolysis of Bfl1 by the proteosome and calpain-like protease, thus converting Bfl1 into a pro-apoptotic molecule.…”

Section: Discussionmentioning

confidence: 99%

“…The ATAP sequence has been implicated in targeting of Bfl1 to the mitochondrial membrane, because deletion of 17 amino acids at the C-terminus caused mistargeting of Bfl1 from the mitochondria (Ko et al, 2003a). Moreover, Bfl1s, an alternative splice variant of Bfl1 with a different C-terminal sequence, is found to localize to the nucleus (Ko et al, 2003b).…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, Bfl1s, an alternative splice variant of Bfl1 with a different C-terminal sequence, is found to localize to the nucleus (Ko et al, 2003b). Recent studies from our laboratories and others have revealed the bi-functional nature of Bfl1; proteolysis or deletion of the N-terminus of Bfl1 converts this molecule from having an antiapoptotic function into a pro-apoptotic function (Ko et al, 2003a;Kucharczak et al, 2005;Yang et al, 2005). Thus, it has been suggested that the TA domain of Bfl1 contains proapoptotic activity.…”

Section: Introductionmentioning

confidence: 99%

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The tail-anchoring domain of Bfl1 and HCCS1 targets mitochondrial membrane permeability to induce apoptosis

Choi

Pan

et al. 2007

Journal of Cell Science

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Many Bcl2 family proteins target intracellular membranes by their C-terminal tail-anchor domain. Bfl1 is a bi-functional Bcl2 family protein with both anti- and pro-apoptotic activities and contains an amphipathic tail-anchoring peptide (ATAP; residues 147-175) with unique properties. Here we show that ATAP targets specifically to mitochondria, and induces caspase-dependent apoptosis that does not require Bax or Bak. Mutagenesis studies revealed that lysine residues flanking the ATAP sequence are involved in targeting of the peptide to the mitochondrial membrane, and charged residues that contribute to the amphipathic nature of ATAP are critical for its pro-apoptotic function. The ATAP sequence is present in another tumor suppressor gene, HCCS1, which contains an additional mitochondria-targeting signal (MTS) close to the ATAP. We propose that both ATAP and MTS could be used as therapeutic peptides to induce cell death in the treatment of cancer cells.

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“…In A375 cells, even the presence of the bulky YFP tag did not disrupt the ability of the protein to associate with the mitochondria. There have been suggestions that internal sequences in the protein may also have some responsibility for the mitochondrial localisation of Bfl-1 406,489 .…”

Section: Chapter 5 Conclusionmentioning

confidence: 99%

Role of the pro-survival molecule Bfl-1 in melanoma

Hind

Carter

Harris

et al. 2015

The International Journal of Biochemistry & Cell Biology

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Melanoma, the cancer derived from the melanocytes of the skin, is becoming an ever more common cancer in the ageing population of the developed world and displays an intrinsic resistance to current therapies. This chemo resistance makes metastatic melanoma an extremely difficult cancer to treat leading to a 5 year survival rate of just 20%. As such, it is important to unearth new potential drug targets to increase the prospects for melanoma patients.Bfl-1 is a pro-survival protein of the Bcl-2 family of apoptosis regulating proteins. It has been found to be over-expressed in a small subset of chemo resistant tumours, including melanoma. Accordingly, I characterised the molecule in melanoma cells and determined its role in protecting these cells from chemotherapy-induced apoptosis. I elucidated the expression profile and half-lives of the protein and mRNA across a panel of melanoma cell-lines and healthy melanocytes. I also confirmed, using pathway specific inhibitors, that Bfl-1 was transcriptionally regulated by the NFB pathway and concluded through pulsechase experiments that Bfl-1 protein was degraded, at least in part, by the proteasome. Subcellular fractionation and indirect immunofluorescence techniques elucidated that Bfl-1 was located mostly at the mitochondria in both resting and apoptotic cells, with a diffuse level present throughout the cytoplasm. As well as characterising the protein in both melanoma cells and healthy primary melanocytes, I determined its role in the resistance of these cells to apoptosis. Over-expressed Bfl-1 was found to protect melanoma cells from apoptosis caused by a range of chemotherapeutic agents in A375 melanoma cells, which naturally expressed very low levels of Bfl-1. Further, knock down of Bfl-1 using siRNA technology in melanoma cells revealed the dependence of these cells on Bfl-1 for their resistance to certain chemotherapeutic agents currently used in melanoma treatment, including the MEK inhibitor PD901. All together, this research contributes to our understanding of Bfl-1 and highlights it as a potentially important therapeutic target in metastatic melanoma.

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Conversion of Bfl‐1, an anti‐apoptotic Bcl‐2 family protein, to a potent pro‐apoptotic protein by fusion with green fluorescent protein (GFP)

Cited by 12 publications

References 38 publications

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor

The tail-anchoring domain of Bfl1 and HCCS1 targets mitochondrial membrane permeability to induce apoptosis

Role of the pro-survival molecule Bfl-1 in melanoma

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