Fine-Tuning of mTORC1-ULK1-PP2A Regulatory Triangle Is Crucial for Robust Autophagic Response upon Cellular Stress

Abstract

Autophagy-dependent cellular survival is tightly regulated by both kinases and phosphatases. While mTORC1 inhibits autophagy by phosphorylating ULK1, PP2A is able to remove this phosphate group from ULK1 and promotes the key inducer of autophagosome formation. However, ULK1 inhibits mTORC1, mTORC1 is able to down-regulate PP2A. In addition, the active ULK1 promotes PP2A via phosphorylation. We claim that these double-negative (mTORC1 –| PP2A –| mTORC1, mTORC1 –| ULK1 –| mTORC1) and positive (ULK1 -> PP2A -> ULK1) feedback loops are all necessary for the robust, irreversible decision making process between the autophagy and non-autophagy states. We approach our scientific analysis from a systems biological perspective by applying both theoretical and molecular biological techniques. For molecular biological experiments, HEK293T cell line is used, meanwhile the dynamical features of the regulatory network are described by mathematical modelling. In our study, we explore the dynamical characteristic of mTORC1-ULK1-PP2A regulatory triangle in detail supposing that the positive feedback loops are essential to manage a robust cellular answer upon various cellular stress events (such as mTORC1 inhibition, starvation, PP2A inhibition or ULK1 silencing). We confirm that active ULK1 can up-regulate PP2A when mTORC1 is inactivated. By using theoretical analysis, we explain the importance of cellular PP2A level in stress response mechanism. We proved both experimentally and theoretically that PP2A down-regulation (via addition of okadaic acid) might generate a periodic repeat of autophagy induction. Understanding how the regulation of the cell survival occurs with the precise molecular balance of ULK1-mTORC1-PP2A in autophagy, is highly relevant in several cellular stress-related diseases (such as neurodegenerative diseases or diabetes) and might help to promote advanced therapies in the near future, too.

Oscillation of Autophagy Induction under Cellular Stress and What Lies behind It, a Systems Biology Study

“…As the stress increases in the cell, an unstable steady state is formed surrounded with a stable limit cycle (see unfilled circle and grey trajectories in Figure 2B). Computer simulations also confirm that although mTOR is knocked out of the network (directly or indirectly via AMPK activation), oscillations of autophagy initiation are observed through the delayed negative feedback loop of AMPK -> "regulator" -> ULK1 -| AMPK (Figure 2C), which is consistent with our previous experimental results [33,34]. We claim that, in this way, the system might have an opportunity to release energy and utilise the building blocks produced from more complex biological elements via autophagy.…”

“…Our goal here is to explain the dynamical features of both negative and doublenegative feedback loops of ULK1-controlled autophagy induction. By using systems biology methods, we recently confirmed that autophagy induction has an oscillatory characteristic upon various cellular stress events [33][34][35]. We proved both experimentally and theoretically that nutrient deprivation or mTOR (here, mTOR refers to mTORC1) inhibition via rapamycin treatment resulted in the periodic repeat of ULK1 activation and inactivation, and created an oscillatory characteristic of autophagy [33].…”

“…Since periodic autophagy induction has also been experimentally demonstrated during rapamycin treatment [33,34], the regulatory triangle of AMPK-ULK1-mTOR requires further extension.…”

The Dual Role of Sulforaphane-Induced Cellular Stress—A Systems Biological Study

“…Previously, our group has made some mathematical models to analyze the ER stress-response mechanism [ 17 , 18 ], focusing on the crosstalk between autophagy and apoptosis [ 19 , 20 ] and the autophagy-dependent survival mechanism under various types of cellular stress [ 21 , 22 , 23 , 24 ]. We have found that autophagy can be periodically activated due to the delayed negative feedback loop between AMPK and ULK1 [ 22 , 25 ].…”

“…We have found that autophagy can be periodically activated due to the delayed negative feedback loop between AMPK and ULK1 [ 22 , 25 ]. Furthermore, we have claimed that a robust autophagy response mechanism requires double-negative and positive feedback loops in the system [ 24 ]. We have previously confirmed that the mutual antagonism between AUTA effector (autophagy effector) and APOA effector (apoptosis effector) guarantees that autophagy and apoptosis cannot be active at the same time with respect to ER stress [ 18 , 19 , 20 ].…”