Lecture notes
mTOR
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- Aston University, Birmingham (Aston)
In depth cell signalling and physiology notes, created using synchronous and asynchronous material.
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mTOR. mTOR stands for mechanistic target of Rapamycin.
Scientists took their soil samples from an island back to their lab and they isolated a
compound from bacteria in one of the soil samples.
The compound showed anti-fungal, anti-tumour and immunosuppressive properties.
This compound was named rapamycin.
They didn’t know the cellular targets of rapamycin at this time- so it is an example of
bottom-up scientific discovery.
They carried out biochemical studies and further analysis of rapamycin showed that
it acted in part by forming a complex with the enzyme FKBP12, which is a peptidyl
pro allele isomerase.
The found that the complex of FKBP12 and rapamycin could inhibit signal
transduction pathways that are required for normal cell growth and proliferation.
It took time to identify the target of rapamycin, but eventually that protein was
isolated, and is named mTOR.
The inhibitor of mTOR is rapamycin, and this was identified before the actual protein
that the drug targets.
mTOR structure.
mTOR is a serine threonine protein kinase.
mTORs structure is closely related to PI3 kinase.
They are members of the same family of kinases.
mTOR, the kinase, forms a central catalytic subunit, of two distinct protein
complexes.
The protein complexes are called mTOR complex 1 and mTOR complex 2.
The two complexes have distinct functions and are regulated differently.
mTOR protein has several functional domains. The functional domains are highly
conserved in mTOR proteins across different species.
mTOR kinases are found from yeasts to humans.
If a protein domain has been conserved and structured through evolution, this
implies that it has a very important function.
The N-terminal region of mTOR, we find 20 tandemly arranged HEAT repeats.
Each one of the HEAT repeats consists of two alpha helices linked by a short loop,
this region of the protein functions as an interaction domain, so it allows mTOR to
form protein-protein interactions.
In the C-terminal region of the protein we have the kinase domain.
Kinases are enzymes that phosphorylate other proteins.
Kinase domain has sequence similarity with the catalytic subunit of PI3 kinases,
which is why these two kinases are considered to be related.
Kinase domain is embedded within a catalytic domain, which contains two FAT
domains (FAT and FATC).
These two domains are a common feature of a particular sub-family of PI3 kinase
related proteins.
Both FAT domains are required for the catalytic activity of mTOR itself.
Next to the first FAT domain is the FRB domain, this is the domain that will bind to
the rapamycin FKBP12 complex.
, Also, within the catalytic domain there is a negative regulatory domain (NRD), this
part of the protein contains specific serine and threonine amino acids that are
phosphorylated by other cellular kinases.
These phosphorylation sites are really important for the regulation of mTOR activity.
mTOR itself is phosphorylated by other cellular kinases.
Threonine 2446 in the human mTOR is targeted by AMPK and a kinase called s6
kinase.
Serine 2448 is a target of AKT.
Serine 2481 is an autocatalytic target of mTOR. So mTOR can phosphorylate its own
negative regulatory domain to inhibit its own function.
mTOR does not work alone in the cell.
It is the catalytic subunit of two distinct but related multi-protein complexes.
mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2).
The distinct feature that we can use to differentiate the two is that mTORC1 is
specifically inhibited by rapamycin, whereas mTORC2 is considered refractory to
rapamycin activity.
This is because the mechanism of action of rapamycin, the way in which mTORC1
activity is inhibited by it, is because rapamycin prevents assembly of the mTORC1
complex.
The specificity of mTORC1 complex inhibition by rapamycin is because the mTORC2
complex, contains distinct proteins, that can still bind to mTOR even in the presence
of rapamycin.
mTOR complexes.
mTOR will form mTOR complex 1 (mTORC1) and the mTOR complex 2 (mTORC2).
mTORC1 vs mTORC2.
mTORC1: is defined by its 3 core components: the mTOR kinase itself, the protein
raptor, and the protein mLST8.
The function of raptor is to facilitate the recruitment of different mTOR substrates,
so the proteins that the mTOR kinase will actually phosphorylate and bring them to
the complex.
They do so by binding to a specific protein motif within those substrates called the
TOR signalling motif.
Raptor is also required within the mTORC1 complex for its correct subcellular
localisation.
mTORC1 shows inhibition by rapamycin, although we do not understand the precise
mechanism by which rapamycin inhibits mTORC1, one of the proposed mechanisms
is that the FKB12 rapamycin complex disrupts the interaction between mTOR and
raptor.
The protein mLST8, is associated with the catalytic domain of mTOR, and it stabilises
the kinase activation loop within the kinase domain of mTOR.
Studies have shown that if you remove mLST8 from within the mTORC1, then the
complex is still functional. mLST8 is not essential for the functions of mTORC1.
mTORC1 also contains inhibitory subunits: pras40 and the deptor protein.
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