Beclin 1 self-association is independent of autophagy induction by amino acid deprivation and rapamycin treatment.

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.

Ras inhibition results in growth arrest and death of androgen-dependent and androgen-independent prostate cancer cells.

Prostate cancer is one of the most frequently diagnosed cancers in human males. Progression of these tumors is facilitated by autocrine/paracrine growth factors which activate critical signaling cascades that promote prostate cancer cell growth, survival and migration. Among these, Ras pathways have a major role. Here we examined the effect of the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS), on growth and viability of androgen-dependent and androgen-independent prostate cancer cells. FTS downregulated Ras, inhibited signaling to Akt and reduced the levels of cell-cycle regulatory proteins including cyclin D1, p-RB, E2F-1 and cdc42 in LNCaP and PC3 cells. Consequently the anchorage-dependent and anchorage-independent growth of LNCaP and PC3 cells were inhibited. FTS also induced apoptotic cell death which was inhibited by the broad-spectrum caspases inhibitor, Boc-asp-FMK. Our study demonstrated that androgen-dependent and androgen-independent prostate cancer cells require active Ras for growth and survival. Ras inhibition by FTS results in growth arrest and cell death. FTS may be qualified as a potential agent for the treatment of prostate cancer.

Closed head injury induces upregulation of Beclin 1 at the cortical site of injury.

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.

Differential interactions between Beclin 1 and Bcl-2 family members.

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.

Neurodegeneration induces upregulation of Beclin 1.

Autophagy, a bulk degradation of subcellular constituents, is activated in normal cell growth and development, and represents the major pathway by which the cell maintains a balance between protein synthesis and protein degradation. Autophagy was documented in several neurodegenerative diseases, and under stress conditions the autophagic process can lead to cell death (type II programmed cell death). Beclin 1 is a Bcl-2 interacting protein that was previously found to promote autophagy. We have used Beclin 1 protein as a marker for autophagy following traumatic brain injury in mice. We demonstrated a dramatic elevation in Beclin 1 levels near the injury site. Interestingly Beclin 1 elevation starts at early stages post injury (4 h) in neurons and 3 days later in astrocytes. In both cell types it lasts for at least three weeks. Neuronal cells, but not astrocytes, that overexpress Beclin 1 may exhibit damaged DNA but without changes in nuclear morphology. These observations may indicate that not all the Beclin1 overexpressing cells will die. The elevation of Beclin 1 at the site of injury may represent enhanced autophagy as a mechanism to discard injured cells and reduce damage to cells by disposing of injured components.

Ligand-independent regulation of ErbB4 receptor phosphorylation by activated Ras.

The ErbB family of receptor tyrosine kinases regulates cell growth, differentiation and survival. Activation of the receptors is induced by specific growth factors in an autocrine, paracrine or juxtacrine manner. The activated ErbB receptors turn on a large variety of signaling cascades, including the prominent Ras-dependent signaling pathways. The activated Ras can induce secretion of growth factors such as EGF and neuregulin, which activate their respective receptors. In the present study, we demonstrate for the first time that activated Ras can activate ErbB4 receptor in a ligand-independent manner. Expression of constitutively active H-Ras(12V), K-Ras(12V) or N-Ras(13V) in PC12-ErbB4 cells induced ErbB4-receptor phosphorylation, indicating that each of the most abundant Ras isoforms can induce receptor activation. NRG-induced phosphorylation of ErbB4 receptor was blocked by the soluble ErbB4 receptor, which had no effect on the Ras-induced receptor phosphorylation. Moreover, conditioned medium from H-Ras(12V)-transfected PC12-ErbB4 cells had no effect on receptor phosphorylation. It thus indicates that Ras induces ErbB4 phosphorylation in a ligand-independent manner. Each of the Ras effector domain mutants, H-Ras(12V)S35, H-Ras(12V)C40, and H-Ras(12V)G37, which respectively activate Raf1, PI3K, and RalGEF, induced a small but significant receptor phosphorylation. The PI3K inhibitor LY294002 and the MEK inhibitor PD98059 caused a partial inhibition of the Ras-induced ErbB4 receptor phosphorylation. Using a mutant ErbB4 receptor, which lacks kinase activity, we demonstrated that the Ras-mediated ErbB4 phosphorylation depends on the kinase activity of the receptor and facilitates ligand-independent neurite outgrowth in PC12-ErbB4 cells. These experiments demonstrate a novel mechanism controlling ErbB receptor activation. Ras induces ErbB4 receptor phosphorylation in a non-autocrine manner and this activation depends on multiple Ras effector pathways and on ErbB4 kinase activity.