Autophagy, an evolutionarily conserved process, has functions both in cytoprotective and programmed cell death mechanisms. Beclin 1, an essential autophagic protein, was recently identified as a BH3-domain-only protein that binds to Bcl-2 anti-apoptotic family members. The dissociation of beclin 1 from its Bcl-2 inhibitors is essential for its autophagic activity, and therefore should be tightly controlled. Here, we show that death-associated protein kinase (DAPK) regulates this process. The activated form of DAPK triggers autophagy in a beclin-1-dependent manner. DAPK phosphorylates beclin 1 on Thr 119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin 1 from Bcl-X L and the induction of autophagy. These results reveal a substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and they provide a new phosphorylation-based mechanism that reduces the interaction of beclin 1 with its inhibitors to activate the autophagic machinery.

Autophagy, a cellular degradation system, promotes both cell death and survival. The interaction between Bcl-2 family proteins and Beclin 1, a Bcl-2 interacting protein that promotes autophagy, can mediate crosstalk between autophagy and apoptosis. We investigated the interaction between anti-and pro-apoptotic Bcl-2 proteins with Beclin 1. Our results show that Beclin 1 directly interacts with Bcl-2, Bcl-x L , Bcl-w and to a lesser extent with Mcl-1. Beclin 1 does not bind the pro-apoptotic Bcl-2 proteins. The interaction between Beclin 1 and the anti-apoptotic protein Bcl-x L was inhibited by BH3-only proteins, but not by multi-domain proteins. Sequence alignment and structural modeling suggest that Beclin 1 contains a putative BH3-like domain which may interact with the hydrophobic grove of Bcl-x L . Mutation of the Beclin 1 amino acids predicted to mediate this interaction inhibited the association of Beclin 1 with Bcl-x L . Our results suggest that BH3 only proapoptotic Bcl-2 proteins may modulate the interactions between Bcl-x L and Beclin 1.

Autophagy, a bulk degradation of subcellular constituents, is activated in several neurodegenerative conditions. Beclin 1, a Bcl2 interacting protein, was found to promote autophagy. The closed head injury model was used to investigate the possible role of autophagy and Beclin 1 after traumatic brain injury. It is demonstrated that levels of Beclin-1 are dramatically increased near the site of injury. Neurons constitute the major population of cells, with the highest Beclin 1 levels near the site of injury at early stages post injury. Elevated levels of Beclin 1 protein in neurons last for at least 3 weeks. In addition, Beclin-1 expression after injury is elevated also in astrocytes starting at 3 days after injury. Confocal microscopy analysis suggests that the high levels of Beclin 1 protein in astrocytes is confined to subcellular organelles, probably lysosomes or autophagosomes. Double staining of Beclin 1 and TUNEL indicate that most of the injured cells that exhibit double staining are neurons and not astrocytes. These findings show that Beclin 1 may play a role in brain responses to head trauma. Overexpression of Beclin 1 may be important for autophagy at the lesion site and may serve as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components.

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Autophagy, a process of self-digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti-apoptotic proteins such as Bcl-2 via its BH3-like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST-Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST-Beclin 1. Further examination by cross linking and co-immunoprecipitation experiments confirmed that Beclin 1 self-interacts and that the coiled coil and the N-terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl-x(L), had no effect on Beclin 1 self-interaction. Moreover, this self-interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full-length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein-protein interactions.