Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis

Abstract

Autophagy is an evolutionally conserved "self-eating" process. Although the genes essential for autophagy (termed Atg) have been identified in yeast, the molecular mechanism of how these Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through UVRAG and acts as a positive mediator of the class III PI3-kinase (PI3KC3). In response to nutrition deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as LC3. Furthermore, loss of Bif-1 suppresses autophagosome formation. While the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI3KC3 and induce autophagosome formation. We also found that Bif-1 ablation prolongs cell survival under nutrient starvation. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumors in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumor suppressor.Autophagy is a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles that appears to be essential for many physiological processes such as cellular homeostasis, development, differentiation, tissue remodeling, cell survival and death, innate immunity, and pathogenesis in various organisms 1-4 . The process of autophagic degradation is initiated when a portion of the cytosolic components are sequestered in cupshaped membrane structures called isolation membranes 1, 2, 5, 6 . The isolation membranes are elongated and eventually sealed to become double-membrane vesicles called autophagosomes, which are then fused with lysosomes resulting in degradation of the enclosed components. Eighteen autophagy-related (Atg) genes have been characterized in S. cerevisiae and can be categorized into four functional groups: (1) the Atg1 protein kinase complex regulating the induction of autophagy, (2) the class III PI3-kinase (PI3KC3) lipid kinase complex controlling vesicle nucleation, (3) the Atg12-Atg5 and Atg8-phosphatidylethanolamine conjugation pathways for vesicle expansion and completion, and (4) the Atg protein retrieval system 2, 7 . Beclin 1, the mammalian homologue of yeast Atg6, is a key component of the PI3KC3 complex, which plays an essential role in autophagosome formation 8-11 . Although the phosphatidylinositol 3-phosphate (PtdIns-3-P) generated by PI3KC3 has been proposed to control membrane dynamics during autophagosome formation 3 , the molecular mechanism underlying this process remains unknown. Results Loss of Bif-1 suppresses caspase-independent cell deathWe have previously reported that Bif-1 localizes to mitochondria and regulates the activation of Bax and Bak during apoptosis induced by intrinsic death stimuli 21 . To examine Bif-1 localization in mouse embryonic fibroblast (MEF) cells during serum deprivation, we added a pancaspase inhibitor, z-VAD-fmk, to ...

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function

“…Different binding interactions for the BH3-domain of Beclin-1 bound to Bcl-xL compared with other BH3-domains were also reported (Feng et al, 2007;Oberstein et al, 2007). Moreover, Beclin-1 is bound to many other proteins in addition to Bcl-2 family members (Vps34 (Furuya et al, 2005), UVRAG (Liang et al, 2006;Takahashi et al, 2007), Bif1 (Takahashi et al, 2007) Ambra (Fimia et al, 2007)), which may further obscure the function of the BH3-domain. It is interesting to note that UVRAG was recently reported to modulate the stoichiometry of Beclin-1 binding to Bcl-2/xL (Noble et al, 2008;Samara et al, 2008), but the impact on apoptosis is not known.…”

Apoptosis and autophagy: regulatory connections between two supposedly different processes

“…This complex contains multiple proteins including another putative tumor suppressor called UVRAG (34) and a protein called Ambra1 (35) , which regulate the activity of the kinase and thus control autophagy induction. Recently it was demonstrated that another protein called Bif-1 binds to UVRAG and that this interaction can also regulate the PI3 kinase activity and induce autophagic vesicle formation (36). The formation and expansion of the autophagosome requires two protein conjugation systems that involve many of the identified Atg proteins (37); Atg8/LC3 and Atg12 are similar to ubiquitin and are activated by Atg7, which acts as the E1 enzyme for both Atg8 and Atg12 leading to their conjugation to the lipid phosphatidylethanolamine (PE) and Atg5 respectively in multi-step processes that also involve Atg3, Atg4 and Atg10.…”

New frontiers in promoting tumour cell death: targeting apoptosis, necroptosis and autophagy

“…While some studies have suggested a tumour suppressive role for autophagy, for example, the tumour-prone nature of autophagy-compromised mice (Beclin 1 þ /À, ATG4CÀ/À and Bif1À/À mice, mice with liver-specific ATG7-deletion or mosaic deletion of ATG5) (Qu et al, 2003;Yue et al, 2003;Marino et al, 2007;Takahashi et al, 2007;Takamura et al, 2011) and the inactivation of autophagy genes in certain human cancers (Liang et al, 1999;Miracco et al, 2007;Kang et al, 2009) It has been suggested that the polarity between the pro-tumourigenic and anti-tumourigenic role of autophagy may be tumour stage dependent (Rosenfeldt and Ryan, 2009). Autophagy is potentially tumour suppressive at the initial stages of cancer development by preventing the toxic buildup of misfolded proteins and damaged organelles, which if persist, can increase oxidative stress, promote genomic instability and predispose cells to malignant transformation.…”

“…Cells lacking atg5 and overexpressing p62 exhibited a significantly higher tumourigenic potential compared with atg5-deficient cells overexpressing the control vector in xenograft studies (Mathew et al, 2009). Additionally, the ability of autophagic proteins, Beclin 1, Ambra 1, UVRAG and Bif-1, to inhibit cell proliferation, promote non-apoptotic cell death and regulate endocytosis (Liang et al, 1999(Liang et al, , 2006Qu et al, 2003;Yu et al, 2004;Fimia et al, 2007;Koneri et al, 2007;Takahashi et al, 2007;Matsunaga et al, 2009) may also contribute to their tumour suppressive effects.…”