Bcl-xL and UVRAG Cause a Monomer-Dimer Switch in Beclin1

Abstract

Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x L binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 ϳ4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.Autophagy is an intracellular vesicular pathway for the degradation of long-lived proteins and organelles (1, 2). The bulk cytoplasm is engulfed in vesicles that mature into autophagosomes and are then targeted to the lysosome. This process is well conserved from yeast to mammals, and many of the autophagy-related (atg) genes that are involved were originally identified in yeast (2). Many have mammalian homologues with conserved functions in autophagy. For example the mammalian protein Beclin1 is a homologue of yeast Atg6, and both form a lipid-kinase complex with class III phosphoinositide-3 kinase (PI(3)KC3 4 ; Vps34 in yeast) that mediates vesicle nucleation.

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

“…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. Our studies with Vps34 did not reveal a difference in the amount of Vps34 that coimmunoprecipitates or colocalizes with Beclin-1 in the absence or presence of Bcl-2 (CL Tan and I Ciechomska, unpublished data), in keeping with the data of Maiuri et al (2007a), but not that of Pattingre et al (2005).…”

The Beclin 1 network regulates autophagy and apoptosis

“…Interestingly, ER-localized Bcl-2, but not mitochondrial-localized Bcl-2, inhibits autophagy, 31 which is consistent with the older notion that ER-associated class III PI3K activity may be crucial in the nucleation of autophagosome formation. Beclin 1 can colocalize with Bcl-XL within mitochondria via its BH3 domain, 14 suggesting a differential role of Bcl-XL in Beclin 1 complex when compared with Bcl-2.…”

“…The CCD, a universal oligomerization domain, mediates Beclin 1 self-interaction and dimer formation in vivo and in vitro. 13,14 The amino terminus binds less effectively than the CCD to contribute to Beclin 1 selfassembly. 13 More specifically, the Bcl-2-binding domain of Beclin 1 is not essential for its self-interaction.…”

The autophagic paradox in cancer therapy

“…Overexpression of Bcl-2 or Bcl-xL has been shown to disrupt hVps34/Beclin 1 interactions by sequestering Beclin 1 (Pattingre et al, 2005). Bcl-2 or Bcl-xL also inhibits the heterodimerization between UVRAG and Beclin 1 (Noble et al, 2008). Accordingly, overexpression of BH3 (Bcl-2 homology domain 3)-only proteins or addition of BH3 mimetics, which bind to antiapoptotic members of the Bcl-2 family through their BH domains, reduce the availability of Bcl-2 and Bcl-xL, thereby disinhibiting Beclin 1-dependent autophagy (Maiuri et al, 2007a).…”