PI3K/Akt/mTOR signaling - ALL Chemistry-Custom Chemical Solutions Supplier

PI3K/Akt/mTOR signaling

The PI3K-Akt signaling pathway promotes signal transduction in response to extracellular signals. The core proteins involved are PI3K (phosphatidylinositol 3-kinase) and Akt (protein kinase B).

Initial stimulation with a growth factor causes activation of cell-surface receptors and phosphorylation of PI3K. Activated PI3K further phosphorylates lipids on cell membranes to form a second messenger, PIP3(phosphatidylinositol-3,4,5-triphosphate). Akt, a serine/threonine kinase, is recruited to the cell membrane by interaction with these docking sites for phosphatidylinositol. the Akt mediates downstream responses through the phosphorylation of a range of intracellular proteins, including cell survival, cell growth, proliferation, cell migration, and blood vessel formation. The Akt signaling pathway exists in all higher eukaryotic cells and is highly conserved.

PI3K Activation

There are many classes of PI3K, but only class I phosphorylates lipids in response to growth stimuli. Class I PI3K is a heterodimer with P85 (regulatory) and P110 (catalytic) subunits. The two subunits are named according to their molecular weight.

This signaling pathway can be activated by a variety of signals, including hormones, growth factors, and components of the extracellular matrix. The binding of extracellular ligands to RTKS on the cell membrane promotes receptor dimerization and cross-phosphorylation of tyrosine residues in the intracellular domain. The regulatory subunit P85 binds to phosphorylated tyrosine residues through the SH2 domain. It then recruits the catalytic subunit P110 to form the fully active PI3K. Otherwise, the adaptor molecule Grb2 binds to the phospho-YXN phantom of RTK and recruits P85 via the GAB scaffold protein.

The p110 subunit can also be recruited independent of P85. For example, Brb2 can also bind to RAS-GEF Sos1 and activate Ras. Ras-gtp in turn binds to the p110 subunit of PI3K. Other bridging molecules, such as IRS, also activate P110.

PI3K can also be activated by GPCR via a G-protein βγ dimer. In addition, the Gα subunit activates SrC-dependent integrins, which activate PI3K.

The Formation of Phosphatidylinositol

Activated PI3K catalyzes the addition of phosphate groups to the 3'-OH position on the inositol ring of phosphoinositol. The reaction consists of three lipid products, PI(3)P, PI(3,4)P2 and PI(3,4,5)P3: Phosphatidylinositol (PI)→phosphatidylinositol 3-phosphate(PI(4)P)→phosphatidylinositol 3, 4-diphosphate (PI(4,5)P2)→phosphatidylinositol 3,4, 5-triphosphate.

These phosphorylated lipids are anchored to the cell membrane and can bind directly to intracellular proteins containing PH or FYVE domains. For example, the triphosphate form (PI(3,4,5)P3) binds Akt and PDK1, which leads to their accumulation near the cell membrane.

Phosphatase and tensin homolog (PTEN) in the PI3K pathway regulates phosphatidylinositol. PTEN degrades PIP3 (Phosphatidylinositol 3, 4, 5-Trisphosphate) and converts PIP3 to PIP2 (Phosphatidylinositol 4,5-bisphosphate). Loss or inactivation of PTEN may lead to the generation of malignant tumors, so PTEN is important for maintaining the normal role of PI3K pathway.

Akt Activation

Akt resides in the cytoplasm in a nonactivated conformation until the cell is activated. Akt translocates to the cell membrane. The PH domain of Akt has a higher affinity for the second messenger PI(3,4,5)P3 than for other phosphatidylinositols. Thus PI3K activity is essential for Akt translocation. Interaction with PI(3,4,5)P3 resulted in conformational changes and exposure of phosphorylation site Thr308 to the site and c-terminal residues Ser473. Phosphorylation of PDK1 at T308 results in partial activation of Akt. Complete activation requires phosphorylation at S473, which is catalyzed by a series of proteins, including PDK2, ILK, mTORC, and DNA-PK. Regulation of Ser473 phosphorylation is not fully understood, but may also be affected by daughter phosphorylation following Thr308 phosphorylation. After stimulation, PiP3 is elevated and Akt activity is attenuated by dephosphorylation of serine/ threonine phosphatase.

PI3K-independent Activation

Although PI3K is the primary mode of Akt activation, other tyrosine or serine/threonine kinases can also directly activate Akt in response to growth factors, immunity, or DNA damage. They can even act in the presence of inhibition of PI3K activity. Other studies have shown that Akt can be activated by heat shock or increased intracellular calcium concentration.

Kinase	Akt Phosphorylation Site	Description
Activated CDC42 kinase 1 (Ack1)	Tyr176	Akt tends to bind phosphatidic acid (PA) rather than PIP 3 so that it can translocate to the cell membrane.
Src	Tyr315, Tyr326	Src SH3 scope and Akt are required Interactions in the C-terminal proline-rich region.
Protein tyrosine kinase 6 (PTK6)	Tyr215, Tyr315 and Tyr326	Akt is activated under the action of epidermal growth factor (EGF).
IκB kinase ε (IKKε)	Ser137, Thr308 and Ser473	Independent of the PH domain, PI3K, PDK1 and mTOR.
TANK-binding kinase 1 (TBK1)	Thr195, Ser378 and Ser473	Activation of Toll-like receptors in macrophages.
DNA-dependent protein kinase (DNA-PK)	Ser473	Activated by DNA double-strand breaks caused by ionizing radiation.

mTOR

The downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), and the downstream transcription factors of mTOR include HIF1α, c-MYC, FoxO and other star molecules.

The metabolism of energy is usually accompanied by the metabolism of substances, and the cycle of substances is also accompanied by the generation and utilization of energy. Such a process needs to be finely regulated according to the material and energy demands of cells and bodies to maintain the stability of life activities.

The star molecule mTOR is a hub molecule that regulates growth and metabolism, so how does it get involved in metabolic regulation?

1. Response to insulin and insulin-like growth factors

PI3K/Akt/mTOR is a classical pathway in response to insulin signaling. After eating, the decomposed glucose enters the blood to promote the release of insulin, which, as a signal in response to nutrient surplus, directs cells to absorb and utilize these nutrients.

Insulin binds cell surface receptors to activate the PI3K-Akt pathway through IRS1. AKT directly promotes glucose absorption, and activates mTORC1 activity through AkT-TSC1/2 -RHEB-MTORC1. MTORC1 further guides the synthesis of glucose and uses enzymes related to biosynthesis for nutrient storage.

2. Response to amino acids and nutrients

Among the three major nutrients, sugars regulate mTORC1 activity indirectly, mainly through insulin and cell-surface receptors. The amino acid absorption can directly activate the activity of mTORC1.

First, the small G-protein Rag is activated in the presence of amino acids and binds mTOR to assist its localization to the lysosomal membrane surface. The small G-protein Rheb, which is localized on the membrane surface, is then activated by signals from growth factors to activate mTOR activity. The modulated mode of direct activation of mTOR by amino acids is mediated by the small G protein Rags, although not by signals originating from the cell surface

Since mTOR can be fully activated only in the presence of both growth factor and amino acid, that is to say, the conditions for activation of mTOR can be met only in the presence of excess matter and energy. This indicates that mTOR, as a hub of metabolic regulation, plays a role in information integration.

3. Effect on autophagy

The regulation of autophagy by mTOR is also essentially a regulation between growth and metabolism. There are nutritional factors and energy factors.

Autophagy is the process when the nutrients or energy of cells are insufficient， in order to maintain their basic survival needs, cells will degrade some relatively minor proteins and some relatively redundant organelles through lysosomes to supply materials and energy.

In addition to the relative insufficiency of ATP, the main carrier of energy, that is, increased AMP/ATP, insufficient nutrient supply, the activity of mTORC1 will also be insufficient, so that mTORC1 releases the inhibition of autophagy initiation regulation.

On the other hand, the regulation of mTORC1 by PI3K-AKT and MAPK cascade signals also reflects the link between growth regulation and metabolism.

In addition, P53/ genome stability also directly regulates mTORC1 activity, adding to the evidence.

4. Implications for the treatment of cancer

Since mTOR has such a large effect on metabolism, and it is well known that tumors are energy consumers, does inhibition of mTOR pathway affect tumor growth?

There are studies. Rapamycin is a classic inhibitor of mTOR, which simultaneously inhibits the activity of mTORC1 and mTORC2, the two big chunks of the pathway. When mTOR is inhibited for a long time, the activity of S6K1 will also decrease, and the feedback inhibition circuit of S6K1 to RTK will be removed. Then, there will be more compensation circuits to compensate for AKT and AGKs, which promote survival. In addition, S6K1 can also inhibit the MEK-ERK signaling pathway. Similarly, rapamycin could relieve the inhibition of MEK-ERK, which showed rapamycin resistance in clinical practice.

Some studies have shown that rapamycin does not completely inhibit the activity of mTORC1, which may be different in different substrates. The phosphorylation of 4E-BP1 is only temporarily or partially inhibited. When the negative feedback loop of S6K1-IRS1 leads to the activation of PI3K-Akt, it may eventually lead to the hyperphosphorylation of 4E-BP1 and other tumor-promoting functions of mTORC1.

Scientists are trying to design inhibitors of mTOR to fight tumors. The inhibitors Torin 1 and PP242, which compete for ATP binding, both have good clinical efficacy, but still cannot avoid the S6K1-IRS1 feedback loop. Now, preserving mTORC1 activity and preserving the integrity of the negative feedback on AKT activity requires only inhibition of mTORC2 activity. In this way, the phosphorylation of Ser473 of AKT can be inhibited to suppress its activity. However, subsequent studies have found that although the phosphorylation of Ser473 of AKT is inhibited, the phosphorylation of Thr308 is theoretically not triggered, but the actual situation is that Thr308 may be compensated by other pathways in this context. The effect of mTORC2 inhibition on AKT activity may affect only some substrates such as FOXO1 and FOXO3. In recent years, strategies have focused on dual inhibitors of mTOR and PI3K, both of which belong to the PI3K family of protein kinases.

The attempt to target mTOR to cancer is also good evidence that cancer is a systemic disease involving the whole system, which is probably the most important reason why cancer is difficult to address. have also well demonstrated that cancer is a systemic disease that engages the whole system, which is probably the most important reason why cancer is difficult to tackle. For tumors with simple causes, treatment is relatively easy.

For tumors caused by complex etiology, it may be initially caused by a single factor that destroyed the self-restraint mechanism of growth and proliferation inside the cell, while the simple damage may be self-repaired, or it may cause the escape of the constraint of chain-positive feedback and trigger the tumor. It is like in a cellular society, if the organism cannot resist the damage from external factors, then the individual cells will try to survive as individuals without relying on overall cooperation.

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