Regulation of mTOR by Ubiquitination and Resveratrol

DoctormTOR is a Ser/Thr kinase that functions as a cellular growth and metabolism in response to nutrient, growth factors and stresses. In mammals, mTOR functions through two distinct complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although these two complexes share same mTOR, functions of them are not similar. This feature is due to different composition of two different mTOR complexes. mTOR Complex I (mTORC1) pathway is considered as a master regulator of cell growth and proliferation. This idea is based on the features of mTORC1, which has been reported since its molecular identification. These features include positive effect of mTORC1 on translational upregulation on capped mRNAs, lipid synthesis, nucleotide synthesis and mitochondrial biogenesis. In addition to this anabolic feature, mTORC1 suppresses catabolic pathway through inhibition of autophagy by phosphorylation of autophagy regulating kinase such as ULK1. Additionally, both mTORC1 and mTORC2 are involved in cell survival. In yeast, suppression of mTOR activity by rapamycin treatment induces cell death. In mammals, rapamycin treatment does not induce cell death albeit cellular proliferation is inhibited. Under stress conditions, inhibition of mTORC1 activity is important for cell survival since mTORC1 inhibition prevents waste of energy and turns on stress resistant signals. However, mTORC2 activation promotes cell survival because mTORC2 is a activator of cell survival promoting kinase Akt. Misregulation and malfunction of mTOR is highly associated with many diseases such as cancer, diabetes and neurodegenerative diseases. So, investigation of how mTOR is regulated under various conditions is very important for understanding the pathologic conditions and the development of the cure methods. Chapter two in this thesis suggests how mTORC1 is regulated under mitochondrial stress. To perform its duty properly, mTORC1 became sensitive to stressful signals. Stressful conditions such as hypoxia, genotoxic stress, ER stress and mitochondrial stress inhibit mTORC1 activity. mTORC1 activity is correlated with mitochondrial membrane potential, an indicator of mitochondrial activity. Also, it was reported that pharmacological inhibition of mitochondrial activity suppresses mTORC1. Sensitivity of mTORC1 to cellular energy level led me to hypothesize lack of ATP by mitochondrial damage is major cause of reduced mTORC1 activity under mitochondrial stress, I still don’t know how mitochondria influence mTORC1. It is generally accepted that stress induced mTORC1 inhibition is critical for cell survival. However, in my study, pharmacological inhibition of mTORC1 by rapamycin during mitochondrial stress increased apoptosis evidenced by enhanced PARP cleavage. Along with this result I also observed decreased mitochondria mass in the same experimental condition. This result implicates mTORC1 has pro-survival role plausibly through mitochondria homeostasis. This pro-survival role of mTORC1 under mitochondria stress raises a question of upstream regulator of mTORC1. I found that E3 ligase Parkin is required for pro-survival action of mTORC1. Inhibition of mTORC1 activity was more apparent in Parkin knock down cells and Parkin overexpression restored mTORC1 activity under mitochondrial stress. I also observed Raptor mediated interaction between mTORC1 and Parkin. Additionally, I found Parkin ubiquitinates mTOR on Lys2066 and Lys2306 upon mitochondrial stress. Replacement of these Parkin dependent ubiquitination residues with Arginine to prevent ubiquitination make mTORC1 has lower activity under mitochondrial stress and cell survival to be compromised. Throughout this study I suggest ubiquitination is a key modification for regulation of mTORC1 activity. Furthermore I suggest elaborate regulation, not switch like activity regulation, of mTORC1 is important for cell survival under stress condition. In third chapter, I demonstrated that resveratrol, which is a natural polyphenols, directly targeting mTOR for induction of autophagy. Resveratrol is a natural polyphenol that administration of this compound is beneficial for our health. There has been suggested that resveratrol-induced autophagy is a key process for many effects of resveratrol, like reduction of inflammation and induction of cancer cell death. Various targets of resveratrol have been suggested, however, it is still ambiguous that which molecule mediates resveratrol-induced autophagy formation. Here, I demonstrate that resveratrol induces autophagy through mTOR-ULK1 pathway. In more detail, I found that resveratrol directly inhibits mTOR in AMPK and SIRT1 knock out MEF and AMPK and SIRT1 were pharmacologically inhibited conditions. Additionally, modulation of PDE activity, which is one of the suggested direct targets of resveratrol, did not prevent resveratrol-induced mTOR suppression. These results indicate that resveratrol inhibits mTOR independent of previously suggested resveratrol’s target. Mechanistically, I found that resveratrol inhibits mTOR directly. This is evidenced by an in vitro kinase assay. Furthermore, I verified that resveratrol-induced mTOR inhibition occurs through competition with ATP. In terms of autophagy formation, I found that autophagy formation by mTOR inhibition and resveratrol treatment was induced in the same pathway. This result indicates that resveratrol induces autophagy by inhibition of mTOR. Additionally, I found that ULK1, which is a downstream target of mTORC1, is required for resveratrol-induced autophagy. Therefore, through this study, I provide mTOR as a novel direct target of resveratrol, whose inhibition by resveratrol is required for autophagy induction