The Physiological Function of Beclin, a Novel BCL-2 Interacting Protein in Protein Trafficking
Abstract (Summary)The beclin gene was originally isolated in a yeast two-hybrid screen for Bcl-2 interacting proteins. It encodes a 60-kDa coiled-coiled protein that was reported to play a role in preventing virus-induced cell death. Beclin also has been reported to be a binding partner for mVps34 PtdIns 3-kinase. To identify the normal endogenous partner for Beclin, I conducted a series of co-immunoprecipitation studies. My results show that overexpressed Beclin can interact with the anti-apoptotic proteins Bcl-2 and Bcl-XL. However, endogenous Beclin does not co-immunoprecipitate with endogenous Bcl-2. Interestingly, unlike the situation with Bcl-2, my results show that endogenous mVps34 is co-precipitated with endogenous Beclin. These observations suggest that mVps34 rather than Bcl-2 is the major physiological partner for Beclin in mammalian cells. Previous reports have indicated that Beclin promotes macroautophagy in breast carcinoma cells in response to nutrient deprivation, and that expression of Beclin mRNA is up-regulated during macroautophagy in colon cancer cells. The product of mVps34, PI(3)P, is required not only for macroautophagy, but also for membrane recruitment of PI(3)P-binding proteins involved in endocytosis and sorting of enzymes en route from the trans-Golgi network (TGN) to the lysosomes. Therefore, I determined if the formation of a complex between Beclin and mVps34 is absolutely required for the functions of mVps34 in macroautophagy and normal endocytic trafficking. Stable cell lines with a 90-95% knock-down (KD) of Beclin were generated by using a retroviral vector to produce siRNA in U251 glioma cells. The Beclin KD cells exhibited a substantially reduced ability to initiate macroautophagy in response to nutrient deprivation and C2-ceramide treatment. However, the processing of procathepsin D that occurs during trafficking of the enzyme from the TGN through late endosomes to lysosomes, was not affected. The Beclin KD cells also were not different from controls with respect to EGF-stimulated degradation of the EGF-receptor, indicating that transit of the receptor through early endosomes and sorting in multivesicular endosomes was not disrupted. Taken together, these results suggest that Beclin plays a key role in regulating the function of mVps34 during macroautophagy, but is not required for the normal function of mVps34 in endocytic trafficking.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:beclin bcl 2 vps34 macroautophagy endocytosis
Date of Publication:01/01/2005