Wednesday, March 6, 2013

Signal Transduction Pathway: The PI3-K/Akt and mTOR Pathway

In this post, I am going to discuss my favorite pathway - the PI3-kinase/Akt and mTOR pathway. I personally like this pathway maybe because I have worked on it for almost two years and studied in-depth and realized how wonderful this pathway works.

In the previous post, we have seen that PIP2 is the source of diacylglycerol and IP3. Here, we will see that how PIP2 also serves as a starting point of another second messenger pathway. PIP2 is phosphorylated on another position 3 of inositol by an enzyme called as phosphatidylinositide 3-kinase (PI3-K). Just like phospholipase C, one form of PI3-K is activated by G-proteins while another form of PI3-K has SH2 domains which is activated by the association with protein tyrosine kinases. Phosphorylation of PIP2 yields the second messenger PIP3 phosphatidylinositol 3,4,5-triphosphate.
A very important target of PIP3 is a protein seine/threonine kinase, called Akt. Akt has a domain called pleckstrin homology domain. PIP3 binds to this pleckstrin homology domain thereby bringing the Akt to the inner face of the plasma membrane. Here, Akt is phosphorylated by another protein kinase called PDK1. This PDK1 also possess the pleckstrin homology domain and binds to PIP3 as can be seen in the adjacent diagram. Thus, we can say that when PIP3 is formed from PIP2, it leads to the association of Akt and PDK1 with the plasma membrane. So, the phosphorylation of Akt is done by PDK1 which activates it. However, recently, it has been found that Akt requires another phosphorylation to get activated. This phosphorylation is done at another site of Akt by a protein called rictor. This rictor protein is complexed with mTOR protein. This mTOR/Rictor complex is itself stimulated by growth factors.

Once, Akt is activated, it has a variety of target molecules which play an important role in cell differentiation, proliferation etc. These target molecules include protein kinases, transcription factors and various regulators of transcription. The important transcription factor that is the target of Akt is FOXO which belongs to the Forkhead family. Active (or phosphorylated) Akt phosphorylates FOXO. Once, FOXO is phosphorylated, it then creates a  binding site for a cytosolic chaperone protein (14-3-3 protein) which then sequesters FOXO in inactive form in the cytoplasm (diagram on the left). Hence doesn't allow the FOXO to go into the nucleus and results in non-expression of FOXO-induced genes. When growth factors and Akt are not present, the FOXO is active and is released from 14-3-3-proteins and gets translocated to the nucleus. In the nucleus, it stimulates transcription of genes that inhibits cell proliferation of induces cell death.

Another target of Akt is another protein kinase GSK-3β. GSK-3β stands for glycogen synthase kinase-3β which is a serine/threonine kinase. It is involved actively in a number of pathways like proliferation, migration, inflammation etc. Just like the FOXO protein, the GSK-3β when phosphorylated is inhibited. Phosphorylation of GSK-3β generally inhibits the activity of its downstream target.  So, what is the target of GSK-3β? The answer is - the translation initiation factor, eIF-2B. When this eIF-2B is phosphorylated by GSK-3β, it is inhibited and there is downregulation of overall translation initiation.

Before going ahead, lets know about mTOR protein. The mTOR is 289kDa protein and is a serine/threonine kinase. mTOR stands for mammalian Target of Rapamycin. The mTOR pathway is the central regulator of cell growth. The mTOR pathway is regulated via multiple pathways including the above mentioned PI3-K/Akt pathway. The interesting part about this mTOR kinase is that it exists in the cell as two different complexes in association with either raptor or rictor. We have discussed above that mTOR/rictor protein kinase phosphorylates and activates Akt. The complex, mTOR/raptor on the other hand, is activated downstream of Akt and functions to control the protein synthesis. How is this mTOR/Raptor regulated? It is by another GTP-binding protein called, Rheb, This Rheb is in turn regulated by another complex called TSC1/2 (Tuberous Sclerosis 1/2) which is a tumor suppressor.  A little confused? Okay! Lets make it easy....(look at the diagram on the right and keep reading) When growth factor binds to the receptor, PIP3 is phosphorylated and activated. This active PIP3 then activates Akt by phosphorylating it. The active Akt then phosphorylates TSC1/2 complex and inhibits it which in turn leads to the activation of Rheb which is known to activate mTOR/Raptor. I hope this is clear now.!

Now, this TSC1/2 is regulated by another protein kinase called AMPK, AMP-activated protein kinase. AMPK is the master metabolic switch and senses the energy state of the cell. That means when the levels of ATP inside the cell is low (AMP being high), AMPK gets activated. We can say that when the ratio AMP:ATP is high, AMPK is activated. This activated AMPK phosphorylates TSC1/2 thereby inhibiting mTOR/raptor pathway. Thus, when the energy levels of the cell are low, AMPK is activated which inhibits protein synthesis.

The active mTOR/raptor complex then further phosphorylates two very well known and well characterized targets as ribosomal protein, S6-kinase and eukaryotic initiation factor-4E (eIF4E) binding protein (4E-BP1). S6-kinase is a protein that controls translation by phosphorylating ribosomal protein S6 and some other proteins involved in translation. When mTOR is active, it phosphorylates S6-kinase which in turn phosphorylates ribosomal protein S6 and hence increases the rate of translation.  Another protein 4E-BP1 controls translation by binding with eIF4E which binds to 5'cap of mRNA. When mTOR is active, 4E-BP is phosphorylated and this active 4E-BP prevents the binding to eIF-4E and leads to increased rates of translation whereas when mTOR is inactive, non-phosphorylated 4E-BPs bind to eIF4E and inhibits translation by interfering with the interaction of eIF4E and eIF4G.