mTOR is a highly conserved serine/threonine-protein kinase enzyme, initially found in mammals. The activity of mTOR protein kinase can be inhibited by a drug, rapamycin, to reduce the adverse effects of mTOR signals, hence the name mammalian Target Of Rapamycin (mTOR).
Functions of mTOR
- mTOR is involved in cancer cell proliferation.
- Involved in an enhancement of vascular endothelial growth factor (VEGF) synthesis.
- mTOR complex 1 is sensitive to rapamycin, while complex 2 is resistant to rapamycin.
- mTOR regulates the actin-cytoskeleton association and Akt/PKB phosphorylation.
- mTOR regulates cell growth, migration, metabolism, and survival.
- Regulates synthesis of biomolecules such as protein, lipid, and nucleotide.
- According to this pathway, growth and proliferation should occur when nutrients, growth factors, and energy molecules are available.
- mTOR inhibits the process of autophagy.
- It shows a negative effect on the ubiquitin-proteasome system (ubiquitination).
- The common mechanism of activated mTOR is downstream of the PIP3.
- Rapamycin is an antibiotic produced by the organism, Streptomyces hydroscopicus.
- Rapamycin inhibits mTOR activity, therefore, protein synthesis.
- Insulin hormone, growth factors (insulin-like GF), phosphatidic acid, certain amino acids, mechanical stimuli, and oxidative stress stimulate the mTOR activity.
- Rapamycin is also used as an immunosuppressant drug after organ transplantation.
- Inhibition of mTOR signaling by rapamycin slows down the growth and proliferation of cell that is supposed to increase the lifespan of the organism.
- mTOR exist in two forms, mTOR complex-1 (mTOC1) and mTOR complex-2 (mTOR2).
- mTOR complex-1 induces the protein synthesis by phosphorylating the eIF-4E-binding protein 1 (EIF4EBP1) and ribosomal protein S6 kinase 1 (RPS6K1). (look at the signaling pathway of complex-1 given below).
- mTOR complex-2 enhances the cell survivability and induces the spatial aspects of growth (cytoskeletal organization).
- An interesting fact is that the activated mTOR complex-2 in turn releases and activates the mTOR complex-1 from their inhibitors via phosphorylating Akt/PKB. (look at the pathway).
- The phosphorylated Akt/PKB inhibits the activity of tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR complex 1.
- Dietary restriction inhibits the activity of mTORC1 through both upstream pathways of mTORC1 that converge on the lysosome.
What is mTOR?
The other names of mTOR protein are Mechanistic target of rapamycin (mTOR) and FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1), which regulates a variety of biological reactions in response to multiple environmental signals, including, cell proliferation, cell survival, cell growth, cell motility, protein synthesis, autophagy, transcription, lipid and nucleotide synthesis, ribosome and lysosome biosynthesis, expression of metabolism-regulated genes, autophagy, and cytoskeletal reorganization. The activity induction is not restricted to intracellular factors like nutrients, hormones, growth factors but can also activate by diverse forms of external stresses. In earlier studies, scientists found only mTOR. Interestingly, during genetic studies (DNA sequencing) of yeast, they have found TOR (Target of rapamycin) protein which is the homology of mTOR, that is also inhibited by rapamycin.
mTOR types based on protein interactions
The activated mTOR exists in two forms such as mTOR complex 1 (mTOC1) and mTOR complex 2 (mTOC2), the formation of these two forms strictly depends on the proteins which are associated with mTOR.
1. mTOR complex-1
mTOR complex 1 is a composite of mTOR, regulatory-associated protein of mTOR (RAPTOR), mammalian lethal with SEC13 protein 8 (mLST8), DEP domain-containing mTOR-interacting protein (DEPTOR), and proline-rich Akt/PKB substrate 40 kDa (PRAS40). The function of complex 1 is to control the cellular metabolism by sensing nutrient, energy, and redox status and to enhances protein synthesis. The mTORC1 activity can be inhibited by the drug rapamycin. While the activity of mTORC1 can be induced by insulin, growth factors, phosphatidic acid, certain amino acids and their derivatives such as β-hydroxy β-methyl butyric acid and l-leucine), mechanical stimuli, and oxidative stress.
2. mTOR complex-2
mTOR complex 2 is a composite of mTOR, Rapamycin-insensitive companion of mTOR (RICTOR), mLST8, and DEP domain-containing mTOR-interacting protein (DEPTOR), mammalian stress-activated MAP kinase interacting protein 1 (MSIN1) and protein observed with RICTOR (PROTOR).
Note: mTOR, MSIN1, MLST8, and RICTOR are the important core molecules of the complexes essential for sustaining structural integrity. However, PROTOR and DEPTOR are not essential for mTORC2 activity or structural maintains. However, DEPTOR acts as a regulatory protein, that negatively controls mTORC2 activity, but the function of PROTOR in the complex is unknown.
mTOR signaling pathway (Signaling cascade via mTOR) step by step
1. mTORC1 activation by RTK-Akt/PKB pathway
Insulin-like growth factors activate the mTORC1 via receptor tyrosine kinase (RTK)-Akt/PKB signaling pathway.
Step 1: Insulin-like growth factor binds and activates receptor tyrosine kinase.
Step 2: The activated receptor tyrosine kinase then activates Akt/PKB protein kinase.
Step 3: Akt/PKB phosphorylates TSC2 on its three positions such as serine residue at 939, serine residue at 981, and threonine residue at 1462 position.
Step 4: These phosphorylated sites will recruit the cytosolic anchoring protein 14-3-3 to TSC2 (a negative regulator of mTOR complex 1), which leads to disruption of TSC1/TSC2 dimer.
Step 5: When TSC2 is not associated with TSC1, then TSC2 loses its GAP activity, so there is no further hydrolysis of Rheb-GTP.
Step 6: This results in continued activation of mTORC1, allowing it for protein synthesis via insulin signaling.
Step 7: Moreover, Akt also phosphorylates PRAS40 which is bound to mTOR, causing a fall off of the regulatory-associated protein of mTOR (RAPTOR) located on mTORC1.
Step 8: Since PRAS40 stops RAPTOR protein from regulating the recruitment of mTORC1 substrates such as p70S6K1 (S6K1) and eIF4E-binding protein 1 (4E-BP1), then its removal will allow the two substrates to be recruited to mTORC1 and thereby activation of mTORC1 in another way.
Note: Studies found that RSK can also phosphorylate RAPTOR, which helps it to control the inhibitory effects of PRAS40.
Since insulin is a growth factor that is secreted by pancreatic beta cells upon the rise of blood glucose levels, then the insulin signaling ensures that there is an energy for protein synthesis to take place.
In a negative feedback loop on mTORC1 signaling, the insulin receptor will be inactivated by S6K1 phosphorylation and reduces its insulin sensitivity. It has well-significance in diabetes mellitus, which is due to insulin resistance.
2. mTORC1 activation by Wnt pathway
The Wnt pathway is involved in cell differentiation, proliferation, cellular growth, and cell fate during the development of an organism; thus, it might be a possible way that activation of the Wnt pathway also activates the mTOR pathway.
Step 1: Wnt is a protein molecule that acts as a ligand. Binding of this ligand to an FRZ receptor (family of G-protein coupled receptors) and co-receptors (lipoprotein receptor-related protein (LRP)-5/6, receptor tyrosine kinase (RTK), or ROR2) activates and passes the biological signal to the Dishevelled (Dsh) protein present inside the cell.
Step 2: Activation of the Wnt pathway inhibits glycogen synthase kinase 3 beta (GSK3B).
Step 3: Inhibition of GSK3 beta leads to no longer phosphorylation of TSC2, which results in dissociation of TSC1/TSC2 complex.
Step 4: TSC1/TSC2 complex dissociation loses the GAP activity of TSC2, now it cannot hydrolyze the Rheb-GTP.
Step 5: This results in continued activation of mTORC1, allowing the protein synthesis via Wnt signaling.
Note: When the Wnt is not bound to its receptor, then the pathway will be inactive. Now, GSK3 beta can phosphorylate two serine residues of TSC2 at the position of 1341 and 1337, along with other conjugation protein kinase AMPK (5′ adenosine monophosphate-activated protein kinase), which phosphorylates serine residue 1345.
It has been found that initially, the AMPK should phosphorylate serine residue 1345 before GSK3 beta phosphorylates its target serine residues.
This TSC2 phosphorylation could activate and stabilize the TSC1/TSC2 complex if GSK3 beta is activated. Since the Wnt pathway inhibits GSK3 signaling, the active Wnt pathway also plays a major role in the activation of the mTORC1 pathway. Thus, mTORC1 activates protein synthesis during the development of an organism.
3. mTORC1 activation by MAPK/ERK pathway
MAPK/ERK signal transduction pathway also called as Ras-Raf-MEK-ERK pathway, is involved in cell differentiation and cell proliferation. This pathway can be activated by both receptor tyrosine kinase and GPCR through G-protein such as Gβγi and phosphoinositide signaling pathway via IP3, DAG, Ca2+.
Step 1: Mitogens, such as insulin-like growth factor-1 binds and activates receptor tyrosine kinase.
Step 2: The activated receptor allows the adaptor protein GRB2 to bind this receptor with its SH2 domains.
Step 3: RTK receptor-GRB2 association then recruits GEF (guanine nucleotide exchange factor) also called Sos.
Step 4: GEF now activates Ras G protein.
Step 5: Ras activates Raf (MAPKKK).
Step 6: Raf (MAPKKK) activates Mek (MAPKK).
Step 7: Mek (MAPKK) activates Erk (MAPK).
Step 8: Erk can go on to activate RSK.
Step 9: Erk will phosphorylate the serine residue 644 on TSC2, while RSK will phosphorylate serine residue 1798 on TSC2.
Step 10: These phosphorylations will cause the heterodimer TSC1/TSC2 complex to dissociate, and this dissociation inhibits Rheb activity.
Step 11: The inactivated Rheb, now releases the mTORC1 to get activity. Thus, MAPK/ERK signals activate the mTOR pathway.
Note: Studies found that RSK also phosphorylates RAPTOR, which helps it overcome the inhibitory effects of PRAS40.
Functions of mTOR types
1. mTORC1 function and Rag complex involvement
mTORC1 is very sensitive to amino acids and its activity strictly depends on amino acid levels in the cell. Even if a cell contains huge energy molecules for protein synthesis and if the cell does not contain the proper levels of amino acid molecules for protein synthesis, then there is no protein synthesis.
Studies have been found that depletion of amino acid levels in the cell inhibits the activity of mTORC1.
When amino acids are supplemented to a deprived cell, then higher levels of amino acids cause Rag GTPase heterodimers to shift to their active conformation. The activated Rag heterodimers now interact with RAPTOR, this leads to the localization of mTORC1 to the surface of late endosomes and lysosomes where the Rheb-GTP is anchored.
This localization allows the mTORC1 to physically interact with Rheb. Thus, amino acid signaling and energy or growth factor signaling converge on endosomes and lysosomes. Thus, the Rag complex recruits mTORC1 to the lysosomal surface to make the association with Rheb-GTP.
2. mTORC2 function
mTORC2 is an important regulator for actin-cytoskeleton organization inside the cell by stimulating the F-actin stress fibers, Cdc42, RhoA, Rac1, paxillin, and protein kinase C α (PKCα).
mTORC2 affects the metabolism and cell survival by phosphorylating the Akt/PKB (serine/threonine protein kinase) on serine residue Ser473. Once the mTORC2 phosphorylates serine residue Ser473 of Akt (473 indicates Serine amino acid position on Akt protein), then that allows PDK1 to phosphorylate threonine residue (Thr308) of Akt that resulted in complete activation of Akt.
mTORC2 also has tyrosine-protein kinase activity that phosphorylates the amino acid, tyrosine at the position Tyr1131/1136 and Tyr1146/1151 present on the insulin-like growth factor 1 receptor (IGF-IR) and insulin receptor (InsR), respectively, resulting in complete activation of IGF-IR and InsR.
The mTORC2 signaling process induces proliferation of hepatic cell carcinoma (HCC) and energy-associated lipid synthesis.
Important points for research need
- PRAS40 and TSC2 are mTOR kinase regulators.
- PRAS40 binding inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. This means, for inhibition of mTOR activity at least either PRAS40 or TSC2 should be expressed in the cells. However low levels of amino acids inside the cell inhibit the mTOR activity, which is independent of Rheb, PRAS40, and TSC2 presence.
- The cells lacking PRAS40 and TSC2 lead to cancer and quick death.
- In normal conditions, TSC1/TSC2 complex inhibits mTORC1 by stimulating the GTPase activity of Rheb, converting it active Rheb-GTP to an inactive GDP-bound state.
- Activation of mTOR in response to growth factors and nutrients leads to phosphorylation of several substrates, including the phosphorylation of S6 kinase by mTORC1 and Akt by mTORC2.
- PRAS40 is a Proline-Rich Akt/PKB Substrate 40kDa, that can be phosphorylated by both Akt and mTORC1, which leads to activation of the mTOR pathway.
- PRAS40 was first reported as a substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity.
- Any modifications in the activity of Akt and mTOR have been related to the development of numerous diseases, including cancer, type 2 diabetes, and hamartoma syndromes.
- The inhibition of mTOR kinase activity occurs upon binding of PRAS40 to mTORC1, which occurs when conditions are unfavorable, which includes nutrient or serum deficiency or mitochondrial metabolic inhibition.
Rapamycin: Rapamycin shows antiproliferative and immunosuppressive activities in both eukaryotes and prokaryotes.
Rapamycin is a potent antifungal compound originally isolated from the Rapa Nui soils, commonly known as Easter Island.
Everolimus and Temsirolimus are rapamycin derivatives, they also inhibit the mTOR protein kinase activity.
Everolimus has been used in the treatment of subependymal giant cell astrocytoma, Renal cell carcinoma, and neuroendocrine tumors of pancreatic origin.
While Temsirolimus has been used only for the treatment of RCC due to emerging of some side effects when it is used against other cancer.
How does rapamycin inhibit mTORC1?
Initially, rapamycin was found as an antifungal compound extracted from Streptomyces hygroscopicus. Subsequently, rapamycin was found to have immunosuppressive activity and anti-proliferative activity on mammalian cells.
Rapamycin was shown to be a potent inhibitor for S6K1 activation, a serine/threonine kinase activated by a variety of agonists, and an important mediator of PI3 kinase signaling. Concurrently, the target of rapamycin (TOR) was identified in yeast and animal cells. It is activated only when it complexes with 12-kDa FK506-binding protein (FKBP12), and this complex can bind and act specifically as an allosteric inhibitor for mTOR complex 1.
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