mTOR Signaling Pathway

mTOR is a highly conserved serine/threonine protein kinase initially found in mammals. The activity of mTOR protein kinase can be inhibited by the drug rapamycin in order to reduce the adverse effects of mTOR signals, hence the name mammalian target of rapamycin (mTOR).

 

Key points for competitive exams and research projects:

  • mTOR involved in the cancer cell proliferation.
  • Involved in the 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 are available.
  • mTOR inhibit the process of autophagy.
  • It shows negative effect on ubiquitin-proteasome system (ubiquitination).
  • The common mechanism of activation of mTOR pathway is its activation is downstream to the PIP3
  • Rapamycin is an antibiotic produced by the organism, Streptomyces hydroscopicus.
  • Rapamycin inhibits the mTOR activity, therefore, protein synthesis, while insulin, growth factors (insulin like GF), phosphatidic acid, certain amino acids, mechanical stimuli, and oxidative stress stimulate the mTOR activity.
  • Rapamycin is also used an immunosuppressant drug after organ transplantation.
  • Inhibition of mTOR pathway 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).
  • Interesting fact is that, the activated mTOR complex-2 in turn releases (activates) the mTOR complex-1 from their inhibitors via phosphorylating Akt/PKB. (look at the pathway).
  • The phosphorylated Akt/PKB inhibit the activity of tuberous sclerosis complex 2 (TSC2), which is 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.

 

Introduction

The other names of mTOR protein are Mechanistic target of rapamycin (mTOR) and FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1) regulates a variety of biological reactions in response to multiple environmental signals, including, cell proliferation, cell survival, cell growth, cell motility, protein synthesis, autophagy, and transcription, lipid, and nucleotide synthesis, ribosome and lysosome biosynthesis, expression of metabolism-regulated genes, autophagy, and cytoskeletal reorganization. The activity induction is not restricted intracellular factors like nutrients, hormones, growth factors but that can also activate by diverse forms of external stresses. In earlier studies, scientist only found that Rapamycin has potent antifungal activity but not mechanism of action. Interestingly, during genetic studies (DNA sequencing) of yeast, they have found TOR (Target of rapamycin) protein which is homology of mTOR, that also can be inhibited by Rapamycin.

 

Types of mTOR based on proteins interactions

The activated mTOR exist in two forms such as mTOR complex 1 (mTOC1) and mTOR complex 2 (mTOC2), the formation of two forms strictly based on the proteins which are associated with mTOR.

 

1. mTOR complex-1

mTOR complex 1 is 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 controls the cellular metabolism by sensing of nutrient, energy and redox status and enhances the 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-methylbutyric acid and l-leucine), mechanical stimuli, and oxidative stress.

 

2. mTOR complex-2:

mTOR complex 2 is 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), protein observed with RICTOR(PROTOR).

Important point: mTOR, MSIN1, MLST8, and RICTOR are the important core molecules of the complexes essential for sustaining the structural integrity. However, PROTOR and DEPTOR are not essential for mTORC2 activity or structural maintains, but function as regulatory proteins whereby DEPTOR seems to negatively control mTORC2 activity, whereas the PROTOR function in 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 activates 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 activates the Akt/PKB protein kinase.

Step 3: Akt/PKB then phosphorylates TSC2 on 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 (negative regulator of mTOR complex 1), this leads to disrupting of TSC1/TSC2 dimer.

Step 5: When TSC2 is not associated with TSC1, TSC2 loses its GAP activity and can no longer hydrolyze Rheb-GTP.

Step 6: This results into continues activation of mTORC1, allowing for protein synthesis via insulin signaling.

Step 7: Meanwhile, Akt will also phosphorylate PRAS40 which is bound to mTOR, causing it to fall off of the regulatory-associated protein of mTOR (RAPTOR) located on mTORC1.

Step 8: Since PRAS40 stops RAPTOR protein from recruiting the substrates of mTORC1 such as 4E-BP1 and S6K1, its removal will allow the two substrates to be recruited to mTORC1 and thereby activated in this way.

Note: Studies found that RSK can also phosphorylate RAPTOR, which helps it overcome the inhibitory effects of PRAS40.

Furthermore, since insulin is a growth factor that is secreted by pancreatic beta cells upon glucose level increase in the blood, this signaling ensures that there is 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 inhibit its sensitivity to insulin. This has well-significance in diabetes mellitus, which is due to insulin resistance. Upregulation of S6K occurs in cells when mTORC1 signaling is over expressed, that shows a feedback inhibitory effect on Akt.

 

2. mTORC1 activation by Wnt pathway

The Wnt pathway is involved in cell differentiation, proliferation, cellular growth, and cell fate during development of organism; thus, it might be possible way that activation of Wnt pathway also activates mTOR pathway (Buller et al 2008).

Step 1: Wnt is protein molecule act as ligand, binding of this ligand to a 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 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 into 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 into continues activation of mTORC1, thus, it is allowing the protein synthesis via Wnt signaling.

Note:  When the Wnt is not bound to its receptor the pathway will be inactive, then, GSK3 beta is able to 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 on serine residue 1345. It has been found that initially, the AMPK should phosphorylate serine residue 1345 before GSK3 beta can phosphorylate its target serine residues. This TSC2 phosphorylation could activate and stabilize the TSC1/TSC2 complex if GSK3 beta were activated. Since the Wnt pathway inhibits GSK3 signaling, the active Wnt pathway is also play a major role in the activation of mTORC1 pathway. Thus, mTORC1 activates protein synthesis during organism development.

 

3. mTORC1 activation by MAPK/ERK pathway

MAPK/ERK signal transduction pathway also called as Ras-Raf-MEK-ERK pathway involved in the cell differentiation and cell proliferation. This pathway can be activated by both Receptor tyrosine kinase, GPCR trough 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 its 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-bound GRB2 then able to recruit GEF (guanine nucleotide exchange factor) also called Sos.

Step 4: GEF now activates the 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 prevents it from deactivating Rheb. mTORC1 active.

Step 11: The inactivated Rheb, now releases the mTORC1 to get active. Thus, MAPK/ERK signals activates mTOR pathway.

Note: Studies found that RSK can also phosphorylate RAPTOR, which helps it overcome the inhibitory effects of PRAS40.

 

mTOR function:

1. mTORC1 function and Rag complex involvement

mTORC1 is very sensitive to amino acids and its activity strictly depend on amino acid levels in the cell. Even if a cell contains huge energy molecules for protein synthesis, if cell does not contain the proper levels of amino acid molecules for proteins synthesis, then there is no protein synthesis occurs.

Studies have been found that depletion of amino acid levels in the cell inhibits activity of mTORC1 to the point where both amino acids and energy abundance are required for mTORC1 to function.

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 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, the amino acid signaling as well as energy or growth factor signaling converge on endosomes and lysosomes. Thus, the Rag complex recruits mTORC1 to lysosomal surface to interact 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 (ser/thr protein kinase) on serine residue Ser473. Once mTORC2 phosphorylates serine residue Ser473 of Akt (473 indicates Serine amino acid position on Akt protein), that allows PDK1 to phosphorylate threonine residue (Thr308) of Akt that resulted in to 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 into complete activation of IGF-IR and InsR.

 

Important points for research need

  1. PRAS40 and TSC2 are mTOR kinase regulators.
  2. PRAS40 binding inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. Which means, for inhibition of mTOR activity at least either PRAS40 or TSC2 should be expressed in the cell. However low level of amino acids inside the cell inhibit the mTOR activity, which is independent of Rheb, PRAS40 and TSC2 presence.
  3. The cells lacking PRAS40 and TSC2 leads to cancer and quick death.
  4. In normal condition, TSC1/TSC2 complex inhibits mTORC1 by stimulating the GTPase activity of Rheb, converting it active Rheb-GTP to inactive GDP-bound state.
  5. Considering point is, that is not mean that phosphorylation of TSC2 dissociates the heterodimer TSC1/TSC2 complex because phosphorylation of TSC2 by GSK3 beta leads to association of TSC1/TSC2 complex thereby mTOR inactive, while phosphorylation of TSC2 by Erk on serine residue 644 leads to dissociation of the complex thereby mTOR active.
  6. 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.
  7. PRAS40 is a Proline-Rich Akt/PKB Substrate 40kDa, that can be phosphorylated by both Akt and mTORC1 that leads to activation of mTOR pathway.
  8. PRAS40 was first reported as 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.
  9. Any Modifications in activity of Akt and mTOR have been related to development of numerous diseases, including cancer, type 2 diabetes, and hamartoma syndromes.
  10. 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.

 

mTOR inhibitors

Rapamycin: Rapamycin activity on mTOR signaling-inhibition stimulated the clinical and research interests due to its antiproliferative and immunosuppressive properties in not only eukaryotes but also prokaryotes.

Rapamycin is potent antifungal compound originally isolated from the Rapa Nui soils, commonly called as Easter Island.

Everolimus and Temsirolimus are rapamycin derivatives, they also can 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 for the treatment of only RCC due to emerging of some side effect on other cancer treatment.

 

How can Rapamycin inhibit the mTOR signaling?

Initially, Rapamycin Was found as an antifungal compound obtained from Streptomyces hygroscopicus. Subsequently, rapamycin was found to have immunosuppressive activity as well as anti-proliferative activity in mammalian cells, encouraging an interest in identifying the rapamycin’s mode of action. Rapamycin was shown to be a potent inhibitor of 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. Rapamycin obtain a gain-of-function form when complex with the 12-kDa FK506-binding protein (FKBP12), and this complex can bind and specifically act as an allosteric inhibitor of mTOR complex 1.

 

References

  1. Buller CL, Loberg RD, Fan MH, Zhu Q, Park JL, Vesely E, Inoki K, Guan KL, Brosius FC 3rd. A GSK-3/TSC2/mTOR pathway regulates glucose uptake and GLUT1 glucose transporter expression.Am J Physiol Cell Physiol. 2008 Sep;295(3):C836-43. doi: 10.1152/ajpcell.00554.2007. Epub 2008 Jul 23.
  2. Wiza C, Nascimento EB, Ouwens DM. Role of PRAS40 in Akt and mTOR signaling in health and disease.Am J Physiol Endocrinol Metab. 2012 Jun 15;302(12):E1453-60. doi: 1152/ajpendo.00660.2011. Epub 2012 Feb 21.
  3. Vander Haar E1, Lee SI, Bandhakavi S, Griffin TJ, Kim DH. Insulin signaling to mTOR mediated by the Akt/PKB substrate PRAS40.Nat Cell Biol. 2007 Mar;9(3):316-23. Epub 2007 Feb 4.
  4. Bryan A. Ballif, Philippe P. Roux, Scott A. Gerber, Jeffrey P. MacKeigan, John Blenis, and Steven P. Gygi. Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors. PNAS January 18, 2005 102 (3) 667-672. org/10.1073/pnas.0409143102.
  5. Edward W. Arvisais, Angela Romanelli, Xiaoying Hou, and John S. Davis. Home Current Issue Papers in Press Editors’ Picks Minireviews AKT-independent Phosphorylation of TSC2 and Activation of mTOR and Ribosomal Protein S6 Kinase Signaling by Prostaglandin F2α. doi: 10.1074/jbc.M605371200 September 15, 2006 The Journal of Biological Chemistry 281, 26904-26913.
  6. Ma L, Chen Z, Erdjument-Bromage H, Tempst P, Pandolfi PP. Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer Cell. 2005 Apr 22;121(2):179-93.
  7. Kwiatkowski DJ. Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis. Cancer Biol Ther. 2003 Sep-Oct;2(5):471-6.
  8. Nobukini T1, Thomas G. The mTOR/S6K signaling pathway: the role of the TSC1/2 tumor suppressor complex and the proto-oncogene Rheb. Novartis Found Symp. 2004;262:148-54; discussion 154-9, 265-8.

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