Isolation of Hyperactive Mutants of Mammalian Target of Rapamycin

Abstract

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growthrelated processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1 (mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from lossof-function analyses using rapamycin and (molecular)genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.

Targeted inhibition of mTORC1 and mTORC2 by active-site mTOR inhibitors has cytotoxic effects in T-cell acute lymphoblastic leukemia

“…This resulted in the inhibition of Thr 37/46 4E-BP1 phosphorylation residues, which have been reported to be direct substrates for mTOR catalytic activity. 43 Indeed, phosphorylation of the translation repressor 4E-BP1 is the limiting step for the assembly of the translation initiating complexes. As a consequence of 4E-BP1 dephosphorylation, PP-242 markedly inhibited the assembly of eIF4F-initiating complexes and reduced the recruitment of mRNA molecules to polysomes.…”

The LKB1/AMPK signaling pathway has tumor suppressor activity in acute myeloid leukemia through the repression of mTOR-dependent oncogenic mRNA translation

“…We used pACTAG-2 4E-BP1-HA constructs 28 (4E-BP1 WT , 4E-BP1 E37E46 , and 4E-BP1 A37A46 ) and pcDNA3.1 FLAG-mTOR WT , and -mTOR SL1ϩIT plasmids. 29 …”

“…We next assessed the role of mTOR in the metformin-induced inhibition of 4E-BP1 phosphorylation by comparing the effects of a constitutively activated mTOR catalytic domain mutant, mTOR SL1ϩIT , 29 with its wild-type counterpart (mTOR WT ) in HEK293 cells treated or not with metformin. In mTOR WT -transfected cells, metformin fully inhibited P70S6K T 389 and 4E-BP1 S 65 phosphorylation, whereas the mTOR SL1ϩIT -activated mutant abrogated the inhibitory effects of metformin against mTOR targets ( Figure 2C).…”

Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

“…While only the first of these occurred in an identical amino acid encoded by a described alteration in MTOR [24][25][26], we predicted both to be hyper-activating based on their locations in the kinase and FAT (FRAP-ATM-TRAAP) domains of the mTOR protein, respectively, according to published studies [24][25][26][27][28][29] (Figure 4). However, after transfection of HEK-293 T cells with complementary DNA plasmids encoding wild-type or mutant (L2427P and T1652A) mTOR, only the L2427P mutant induced phosphorylation of S6K (Additional file 6).…”