Nutrient Sensing (mTOR) Flashcards
The Target of Rapamycin (TOR) Pathway:
- rapamycin was originally tested as an immunosuppressant, but in fact it inhibits growth of all eukaryotic cells, including yeast
- rapamycin was shown to bind to a protein, called FKBP12
- Mike Hall screened for yeast mutants resistant to rapamycin
- interestingly, he isolated mutations not only in the gene encoding FKBP12, but also in two novel genes, TOR1 and TOR2
- their sequences were related to lipid kinases such as PI3K
- others looked for proteins in mammals that bound FKBP12 in the presence of rapamycin and found a single protein related to TOR1/2, called mammalian target-of-rapamycin (mTOR)
- the rapamycin:FKBP12 complex binds to and inhibits both yeast TOR and mTOR
mTOR forms 2 complexes:
mTORC1 and mTORC2
mTOR
mTOR is a large protein (>2,500 AAs.) with a kinase domain (~300 AA) near the C-terminus
biochemical analysis showed that mTOR exists as two multiprotein complexes called mTOR complexes (mTORC) -1 and -2, with mTOR bound either to Regulatory Associated Protein of TOR (Raptor) or Rapamycin-Insensitive Companion of TOR (Rictor):
- Both complexes also contain other subunits
- Only mTORC1 is directly inhibited by the rapamycin:FKBP12 complex
- Although mTOR is related to lipid kinases, mTORC1 and mTORC2 are in fact protein kinases
- Raptor and Rictor target mTORC1 and mTORC2 to different downstream target proteins
- Because both mTORC1 and cell growth and are sensitive to rapamycin, seemed likely that downstream proteins phosphorylated by mTORC1 would be involved in cell growth control
Downstream Targets of mTORC1:
2 known targets of mTORC1 are S6K1 and eIF4E-binding protein 1 (4E-BP1)
ROLE OF S6K1:
- ribosomal protein S6 kinase-1 (S6K1) selectively promotes translation of certain mRNAs
- although named for its ability to phosphorylate ribosomal protein S6, S6K1 is now thought to promote mRNA translation by phosphorylating other targets such as eIF4B
- phosphorylated eIF4B activates eIF4A, an RNA helicase that unwinds secondary structures in the 5’-untranslated regions of certain mRNAs, which otherwise inhibit their translation
ROLE OF 4E-BP1:
- eIF4E binds to the 7-methyl-guanosine cap located at the 5’ end of most mammalian mRNAs, recruiting ribosomes and other components to allow their translation
- 4E-BP1 binds to and inhibits eIF4E, but phosphorylation of 4E-BP1 by mTORC1 relieves this
- this promotes translation, particularly of mRNAs encoding proteins required for rapid cell growth, such as ribosomal proteins and translation factors
thus mTORC1 promotes mRNA translation, and hence cell growth, by multiple mechanisms
Downstream Targets of mTORC2:
- S6K1 is phosphorylated by mTORC1 at a threonine residue within an FLGFTY sequence that occurs just after the kinase domain (F and Y both have hydrophobic side chains)
- interestingly, mTORC2 phosphorylates and activates protein kinases other than S6K1 at the equivalent serine residue within similar FXXFSY motifs (known as hydrophobic motifs):
Regulation of mTORC1:
In mammalian cells:
mTORC1 was found to be activated by growth factors, such as insulin and insulin-like growth factor-1 (IGF1), which activate the PI3-PKB pathway
however, activation of mTORC1, which is of course blocked by rapamycin, also required the presence of amino acids in the medium:
Regulation of mTORC1 by Amino Acids:
- the sensor appears to detect intracellular, rather than extracellular, amino acids
- addition of AA to cells causes mTOR to co-localize with Rab7, a lysosomal marker:
- mTORC1 sense amino acids at lysosomes
Regulation of mTORC1 by Growth Factor:
- the growth factors insulin and IGF1 activate PI 3-kinase to produce PIP3 , the 2nd messenger for the protein kinase PKB/Akt
- in a screen that searched for novel substrates of PKB/Akt, it was found to phosphorylate TSC2, a protein that formed a complex with TSC1
- mutations in the genes encoding TSC1 or TSC2 were already known to cause a human disorder, tuberous sclerosis complex
TOR Pathway in Drosophila:
- genes for TOR, TSC1, TSC2, PKB/Akt, S6K1 are all found in the fly, Drosophila melanogaster
- a genetic screen in Drosophila identified Rheb as a novel gene that promoted cell growth
- the order of genes in a pathway can be determined by combining mutations by crossing
- over-expression of Rheb in the compound eye of flies causes cells within eye facets (and hence the whole eye facets) to become larger
- gene knockout of S6K1 or PKB/Akt caused facets to become smaller; knockout of S6K1 reversed the effects of Rheb over-expression, but knockout of PKB/Akt did not
- thus, the effect of over-expressing Rheb is dependent on S6K1, but independent of PKB/Akt, which suggests the following order in the pathway: PKB/Akt Rheb S6K1
- similar experiments suggested that TSC1 and TSC2 acted between PKB/Akt and Rheb
Rheb is a G Protein and TSC1:TSC2 is a Rheb-GAP:
- Drosophila Rheb was so-called because it is related to the human protein Ras homologue expressed in brain, a small G protein of the Ras family whose function was unknown
- interestingly, the TSC2 sequence contained a region related to sequences found in GTPase activator proteins (GAPs), which promote GTP hydrolysis by small G proteins like Ras
- this suggested that TSC1:TSC2 might be a Rheb-GAP that converts active Rheb.GTP to its inactive Rheb.GDP form:
- Rheb has lipid modifications that cause its association with the lysosomal membrane
- GTP binding to Rheb causes it to bind mTORC1, thus recruiting the latter to the lysosomal surface, where it is then available for activation by amino acids
- thus, both Rheb.GTP and lysosomal amino acids are required for mTORC1 activation
