The role of Beclin 1 interacting proteins and Beclin 1 post-translational modification in autophagy regulation.

2765 The process of autophagy is a complex catabolic program for lysosomal degradation of proteins and other subcellular constituents. It is important for normal growth control and is defective in a number of cancers. Beclin 1 gene is the first mammalian gene identified involved in autophagy. Mono-allelic deletions of Beclin 1 gene are identified in 40-75% of human sporadic breast, ovarian and prostate cancers. Beclin 1 knockout mice were recently generated. Homozygous Beclin 1 knockout mice are embryonic lethal, while the heterozygous Beclin 1 knockout mice develop various tumors. These mouse genetic results provide compelling evidence that autophagy plays an important role in preventing tumorigenesis. This work also defined a novel class of tumor suppressor genes which are involved in autophagy and its regulation. However, exactly why the Beclin 1 gene and autophagy are critical for tumor suppression remains unknown. In order to understand the molecular mechanism of how Beclin 1 is involved in autophagy, we created a cell line expressing a flag-tag Beclin 1 protein. Western blot analysis revealed that after the induction of autophagy resulting from starvation, Beclin 1 underwent a molecular weight shift. These results suggest that the protein undergoes a post-translational modification when autophagy is activated. We then opted to purify the Beclin 1 protein and its interacting partners by immunoprecipitation. Two proteins that interact specifically with Beclin 1 were purified and further identified by mass spectrometry. They are two kinases homologous to yeast Vps34 and Vps15. Furthermore, the region of the post-translational modification site was defined with a monoclonal antibody that specifically recognizes the non-modified form of Beclin-1, but not the modified one. This post-translational modification is inhibited by okadaic acid, a specific inhibitor of protein phosphatase 2A and known inhibitor of autophagy. Site-directed mutagenesis revealed the region of the post-translational modification of Beclin 1. Expression of a subset of the mutants of Beclin 1 resulted in the constitutive activation of autophagy. Current experiments are underway to address the role of Beclin 1 on autophagy activation. In addition, these mutants provide a molecular tool to study the impact of autophagy activation on cancer therapy and cancer prevention.