Controlling the master - molecular mechanisms of mTOR regulation

Proteins are the functional units of life. Regulation of protein function and activity is a key cellular
mechanism to respond and adapt to changing extra- and intracellular conditions. Evolution of
complex signaling networks based on multidomain proteins and multiprotein complexes enables
higher eukaryotes to sense and integrate signals and to communicate necessary responses by
modulating the activity of target proteins.
An atypical kinase, the mammalian target of rapamycin (mTOR), is the master regulator of cellular
growth and metabolism. Information about the current status of a cell is processed by a signaling
network converging on mTOR, which integrates stimuli to respond by re-balancing anabolic and
catabolic processes. mTOR acts as a component of two functionally and structurally distinct
complexes, mTOR complex 1 (mTORC1) and mTORC2, and phosphorylates a large set of substrate
proteins. More than 80 substrates are known, however the mechanism of their recruitment to mTOR
complexes remains unknown for most of them. The activity of mTOR complexes is regulated by
association with binding partners, translocation or posttranslational modifications. Dysregulation of
mTOR signaling is associated with cancer, obesity and neurodegenerative diseases establishing
mTOR as prime drug target. DEPTOR is an enigmatic regulator of mTOR that may act as tumor
suppressor or oncogene. It is the only protein known to bind and inhibit both, mTORC1 and mTORC2.
The experimental part of this thesis reveals the mechanism of the regulatory interplay of DEPTOR with
mTOR complexes based on structural and biochemical characterization. Using cryo electron
microscopy I determined structures of DEPTOR bound to mTORC1 and mTORC2 at a resolution of
3.7Å and 3.2Å respectively. I obtained a detailed characterization of the mTOR-DEPTOR interaction
by combining cryo-EM data with solving a crystal structure of the DEPTOR DEP domain tandem.
Biochemical analysis of mTOR activity modulated by DEPTOR, and structure-guided mutants of
DEPTOR allowed us to unravel a novel mode of mTOR regulation involving two distinct binding sites
in the FAT domain of mTOR. Contrary to previous hypotheses, DEPTOR is not only an inhibitor of
mTOR, but it allosterically activates or inhibits mTOR depending on cellular lipid signaling.
The second part of this thesis provides a review of mTOR substrates, their function and recruitment.
We analyzed in particular phosphorylation motifs and recognition of substrates in the active site. The
substrate recognition in the active site of mTOR is, in contrast to other PIKKs, only loosely defined.
In summary, the results presented in this thesis provide new structural and mechanistic insights into
DEPTOR function and the regulation of mTOR, and may contribute to the development of novel
therapeutic approaches.