Identification of Annexin A2 as a key mTOR target to induce roller coaster pattern of autophagy fluctuation in stress

Autophagy can either be cytoprotective or promote cell death in a context-dependent manner in response to stress. How autophagy leads to autophagy dependent cell death requires further clarification. In this study, we document a nonlinear roller coaster form of autophagy oscillation when cells are subjected to different stress conditions. Serum starvation induces an initial primary autophagic peak at 6 h, that helps to replenish cells with de novo fluxed nutrients, but protracted stress lead to a secondary autophagic peak around 48 h. Time kinetic studies indicate that the primary autophagic peak is reversible, whereas the secondary autophagic peak is irreversible and leads to cell death. Key players involved in different stages of autophagy including initiation, elongation and degradation during this oscillatory sequence were identified. A similar molecular pattern was intensified under apoptosis-deficient conditions. mTOR was the central molecule regulating this autophagic activity, and upon knockdown a steady increase of autophagy without any non-linear fluctuation was evident. An unbiased proteome screening approach was employed to identify the autophagy molecules potentially regulating these autophagic peaks. Our proteomics analysis has identified Annexin A2 as a stress-induced protein to implicate in autophagy fluctuation and its deficiency reduced autophagy. Moreover, we report that mTOR in its phosphorylated condition interacts with Annexin A2 to induce autophagy fluctuation by altering its cellular localization. The work highlights the molecular mechanism of a mTOR-dependent roller coaster fluctuation of autophagy and autophagy dependent cell death during prolong stress.