As mentioned above, autophagy induction correlates with the dissociation of Beclin 1 from Bcl-2 or Bcl-XL. Recent data suggest that multiple distinct mechanisms may account for the release of Beclin 1 from its inhibitory interaction with Bcl-2 or Bcl-XL.
Since the Beclin 1 BH3 domain interacts with the BH3-binding groove of the multi-domain, anti-apoptotic Bcl-2 proteins (see above), one plausible model is that other proteins BH3 domain-containing proteins may competitively displace the Beclin 1 BH3 domain, disrupting the interaction of Beclin 1 and Bcl-2/Bcl-XL
. This would release Beclin 1 from the inhibitory effects of Bcl-2/Bcl-XL
. The first evidence in support of this model was provided by studies using the BH3-mimetic compound ABT-737, an agent designed to bind to the BH3 binding groove of Bcl-2 or Bcl-XL
(but not Mcl-1).43
ABT-737 inhibits the binding of Beclin 1 BH3 peptide to Bcl-XL
in a competitive manner, with an IC50
in the micromolar range, as determined by measuring the binding of synthetic peptides to purified recombinant Bcl-XL
Similarly, ABT-737 pretreatment abolishes the immunoprecipitation between Beclin 1 and Bcl-2 or Bcl-XL
(but not Mcl-1) in cells that are resistant to the pro-apoptotic action of ABT-737. This effect correlates with the induction of high levels of autophagy. ABT-737-induced autophagy cannot be inhibited by Bcl-2 or Bcl-XL
overexpression, yet it is abolished by transfection with Mcl-1 or by the siRNA-mediated knockdown of Beclin 1.18
These results clearly demonstrate that competitive disruption of the Beclin 1 interaction with Bcl-2 or Bcl-XL
suffices to induce autophagy.
To date, EGL-1 is the only pro-apoptotic, BH3-only protein identified in the nematode, Caenorhabditis elegans
. Transgenic expression of EGL-1 causes an increase in baseline autophagy, as measured using an LGG-1::DsRED reporter construct (LGG-1 is the C. elegans
ortholog of Atg8/LC3). In wild-type nematodes, starvation strongly induces autophagy, and this induction is blunted in egl-1
-deficient nematode embryos. This suggests that BH3-only proteins may act as obligate inducers of autophagy, at least in C. elegans
In human cells, upon nutrient starvation, the amount of endogenous Beclin 1 that co-immunoprecipitates with Bcl-XL
or Bcl-2 declines15,18
while the amount of the BH3 protein Bad (whose activation is known to be triggered by serum withdrawal) that co-immunoprecipitates with Bcl-XL
or Bcl-2 increases.18
The knockdown of Bad (in human HeLa cells) or its knockout (in mouse embryonic fibroblasts) results in a partial defect in starvation-induced autophagy that can be restored by the addition of excess ABT-737.18
Enforced overexpression of Bad (but not a Bad mutant with a disruption in the BH3 domain) is sufficient to induce autophagy both in normal conditions and in the setting of caspase inhibition.18
Together, these results indicate that BH3-only proteins may play an evolutionarily conserved role in the activation of starvation-induced autophagy.
A second mechanism that may link starvation to the dissociation of Beclin 1 from its inhibitory interaction with Bcl-2 involves the phosphorylation of Bcl-2 by the stress-activated c-Jun N-terminal protein kinase 1 (JNK1) ().44
Upon nutrient withdrawal, JNK1 is activated and induces phosphorylation of serine and threonine residues (T69, S70 and S87) in the non-structured loop of Bcl-2 that is located between the N-terminal BH4 and BH1 domain. JNK1 inhibition or replacement of wild type Bcl-2 by a non-phosphorylatable Bcl-2 mutant (T69A/S70A/S87A) abolishes the starvation-induced dissociation of Bcl-2 and Beclin 1 and inhibits autophagy. Expression of constitutively active JNK1 leads to Bcl-2 phosphorylation, dissociation of Bcl-2 from Beclin 1, and stimulation of autophagy; however, active JNK1 fails to stimulate autophagy when Bcl-2 is replaced by its non-phosphorylatable mutant. In jnk1-/-
MEFs, starvation fails to induce Bcl-2 multi-site phosphorylation, dissociation of the Bcl-2/Beclin 1 complex, or autophagy. Furthermore, a multi-site Bcl-2 phosphomimetic mutant (T69E/S70E/S87E) fails to co-immunoprecipitate with Beclin 1 and has no autophagy-inhibitory activity. Together, these findings indicate that JNK1-mediated phosphorylation of Bcl-2 causes its dissociation from Beclin 1, thereby inducing autophagy.
Although the two mechanisms for the dissociation of Beclin 1 from Bcl-2/Bcl-XL
described above (competitive disruption by BH3-only proteins or phosphorylation of Bcl-2) appear distinct, they may be functionally linked. Phosphorylation of recombinant Bcl-2 reduces its affinity for Bax as well as for the BH3-only protein Bid, while the non-phosphorylatable Bcl-2 mutant (T69A/S70A/S87A) exhibits enhanced binding to three different the BH3-only proteins (Bid, Bad and Bim) in co-immunoprecipitation assays.45
These observations indicate that multi-site phosphorylation in the nonstructured loop reduces the affinity of Bcl-2 for BH3 domains in general, including that of Beclin 1. Another plausible, but as-of-yet unexplored, possibility is that phosphorylated Bcl-2 might change its profile of selectivity for distinct BH3 domains in a way that Bcl-2-bound Beclin 1 is replaced by other BH3-only proteins, leading to the selective liberation of Beclin 1.
A competing hypothesis is that there are indeed two independent signaling routes that link starvation to the dissociation of Beclin 1 and Bcl-2-like proteins. For example, the dissociation of Beclin 1 and Bcl-XL
could occur preferentially as a result of Bad activation (presumably because Akt becomes inactive, resulting in dephosphorylation of Bad and hence its dissociation from cytosolic 14-3-3σ).11
In contrast, disruption of the Beclin 1/Bcl-2 interaction could be preferentially regulated by the phosphorylation level of Bcl-2 (which increases as a result of JNK1 activation). It is not yet known whether Beclin 1/Bcl-XL
binding, like Beclin 1/Bcl-2 binding, is regulated by JNK1-mediated phosphorylation, although the potential phosphorylation sites in the nonstructured loop are conserved between Bcl-2 and Bcl-XL