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.

Signal Transduction Pathway - Phospholipids and Calcium Ion Signaling

Phosphatidylinositol 4,5-bisphosphate abbreviated as PIP2 is a phospholipid present in the inner leaflet of the bilayer of the plasma membrane. The second messengers are derived from this small component (phospholipid) and the pathway is based on these messengers.

How does hydrolysis of PIP2 takes place? The hydrolysis of PIP2 takes place by the enzyme phospholipase C as can be seen in the adjacent figure. It is interesting to note that the enzyme phospholipase C is ultimately activated by G-protein coupled receptors (GPCRs) or protein tyrosine kinases. This is so because one form of phospholipase C (PLC-β) is stimulated by G proteins while another form of phospholipase C (PLC-γ) contains SH2 domains (as can be seen in the figure shown below) and hence it associates with activated receptor protein tyrosine kinases. This interaction helps PLC-γ to localize to plasma membrane and also leads to its phosphorylation. This tyrosine phosphorylation increases PLC-γ activity, which in turn stimulates hydrolysis of PIP2.

The hydrolysis of PIP2 produces two distinct second messengers as diacylglycerol and inositol 1,4,5-triphosphate which is abbreviated as IP3. Both these messengers stimulate different downstream signaling pathways thereby triggering two distinct cascades of intracellular signaling. Diacylglycerol stimulates protein kinase C mobilization while IP3 stimulates Ca2+(ions) mobilization. The diacylglycerol as second messenger activates serine/threonine kinases which belongs to the protein kinase C family which play an important role in cell growth and differentiation.

IP3, another second messenger is released into the cytosol and it acts to release the  Ca2+(ions) from intracellular stores. The level of the Ca2+(ions) inside the cell is very low and is maintained by pumping through Ca2+(ion) pumps across the plasma membrane.
The Ca2+(ions) are pumped into the ER and hence ER is considered to be the store of intracellular Ca2+(ions). Here, IP3 binds to the receptors in the ER membrane as can be seen in the adjacent diagram. These receptors are ligand-gated ion channels and hence, there is efflux of Ca2+(ions) into the cytosol. This increase of Ca2+(ions) in the cytosol has an effect on variety of proteins like protein kinases. For example, there are some members of protein kinase C (PKC) family that requires Ca2+(ions) as well as diacylglycerol for their functioning. Hence, these PKC family members are regulated by both IP3 and diacylglycerol.

Calmodulin is another very important protein to mention while we are studying about Ca2+(ions). The word 'calmodulin' means - cal(cium) + modul(ate) + in(g). Thus, calmodulin is 'calcium modulating' protein that mediates most of the activities of Ca2+(ions). Calmodulin is dumbbell shaped protein which has four Ca2+(ions) binding sites (figure is shown below). When the Ca2+(ions) concentration in the cell increases, calmodulin is activated. This active Ca2+/calmodulin complex then binds to a variety of target proteins, like Ca2+ion/calmodulin -dependent protein kinases thereby rendering them active. The examples of Ca2+ion/calmodulin dependent-protein kinases are: myosin light-chain kinase and members of CaM kinase family.

Lets understand how the regulation of Ca2+ ions is important in regulating electrically excitable cells? When there is a change in plasma membrane's potential i.e.; when there is membrane depolarization, the voltage-gated Ca2+ ion channels are opened in the plasma membrane. Because of the opening, there is influx of Ca2+(ions) from the extracellular fluid into the cytosol of the cell. This increase in the levels of Ca2+(ions) further triggers the opening of the another receptor called the ryanodine receptor in the plasma membrane which further releases the Ca2+(ions) from the intracellular stores. This increase in the Ca2+(ions) results in triggering the release of neurotransmitter. Hence, we can say that Ca2+ ion plays an important role in converting electric signals to chemical signals.
In muscle cells, the ryanodine receptors on the sarcoplasmic reticulum. These receptors maybe opened directly when there is membrane depolarization.

Signal Transduction Pathway -The cGMP Pathway

In this post, we will discuss about cGMP. Just like cAMP, as we have seen in the previous post, there is another second messenger in animal cells called the cyclic GMP or cyclic guanosine monophosphate, simply written as cGMP. However, its role is not clearly understood as against that of cyclic AMP.

Just like how cAMP is formed, cyclic GMP is formed by the action of enzyme guanylyl cyclase on GTP and degraded by a phosphodiesterase. As we have discussed in earlier posts, that guanylyl cyclase is activated by nitric oxide and carbon monoxide and also by peptide ligands. So, when there is stimulation of guanylyl cyclases, there is elevation of cyclic GMP levels. How does cyclic GMP mediates its action? The action of this cyclic GMP is generally mediated when there is activation of various cGMP-dependent protein kinase. However, cyclic GMP is also known to regulate ion channels and phosphodiesterases.

Lets take an example of very well defined activity of cGMP. cGMP serves as a very important second messenger in a vertebrate eye. Isn't it interesting to know how? It converts the visual signals to nerve impulses as light. Hence, we are able to see.
We all know there is retina in the eye which contains the rod cells (studied in school). Now, these rod cells contains a receptor which is a photoreceptor. This photoreceptor is a G-protein coupled receptor (GPCR) called rhodopsin. Lets understand how this rhodopsin is activated.
A small molecule is associated with rhodopsin called 11-cis retinal. This small molecule absorbs light and isomerizes to all-trans retinal. As a result, it induces a conformational change in rhodposin protein. This activated rhodopsin then activates G protein called transducin. Being a G-protein, transducin has three subunits as α, β and γ. Just to recall, the α-subunit in inactive state is bound to GDP. Since, rhodopsin activates G protein transducin, there is a conformational change where α-subunit is now bound to GTP and is in active state. This α-subunit (of transducin) bound to GTP  then stimulates the activity of cGMP phosphodiesterase. Now, being a phosphodiesterase, it reduces the intracellular levels of cGMP. The cGMP has a direct effect on ion channels in plasma membrane and so when there is reduction of cGMP levels in the rod cells of retina, this is translated to nerve impulse as light by the effect of  cGMP on ion channels.

This is how the cGMP functions. In the next post, we will discuss about the phospholipids and Ca2+ signaling.

Signal Transduction Pathway - The cAMP Pathway

cAMP stands for cyclic-adenosine monophosphate and we all know that this is the secondary messenger. The concept as to why cAMP is the secondary messenger was first discovered by Earl Sutherland in 1958, when he discovered that the action of hormone, epinephrine, was mediated by an increase in the concentrations of cAMP. Why it is the secondary messenger? Because the first messenger is the hormone itself (here, epinephrine is the primary messenger) and the cAMP mediates the activity of primary messenger.

How is cAMP formed and then degraded? So, cAMP is formed by the action of an enzyme, adenylyl cyclase which acts on ATP (adenosine triphosphate) as can be seen in the above diagram. This cAMP is further degraded to AMP by cAMP phosphodiesterase.

cAMP has an important function that it mediates the breakdown of glycogen to glucose in muscle cells for energy. But how? This effect and some more effects of cAMP in turn is mediated by action of another enzyme, known as protein kinase A (abbreviated as PKA), also called as cAMP dependent protein kinase. Now, next question is how is PKA regulated? To understand this, first we will have to understand the structure of PKA. The enzyme PKA has four subunits. Two of the subunits are regulatory while the other two are catalytic (figure on the right side). Now, when cAMP binds to regulatory subunits, there is conformational change which leads to the dissociation of the catalytic subunits. These free catalytic subunits are enzymatically active and they phosphorylate the serine residues on the target molecules (proteins).


Now, in effect of glycogen metabolism, how does PKA regulates glycogen metabolism?
The PKA has two enzymes upon which it acts (as can be seen in the adjacent diagram). The first one is another protein kinase called, phosphorylase kinase. This kinase is phosphorylated on serine residue by PKA; which then activates glycogen phosphorylase. The glycogen phosphorylase is an enzyme in glycogenolysis or glycogen breakdown pathway and thus is responsible for breakdown of glycogen to glucose-1-phosphate. Another enzyme which PKA phosphorylates is glycogen synthase. The glycogen synthase is the enzyme which is responsible for glycogen synthesis. The point to note here is that the phosphorylation of glycogen synthase inhibits its enzymatic activity. Hence, from here, we can infer that, the increase in cAMP levels results in the activation of PKA and this stimulates glycogen breakdown and at the same time, also inhibits glycogen synthesis.

As already mentioned, there are many other effects of cAMP. Lets have a look at another example. When there is an increase in cAMP levels, there is activation of transcription of various target genes in many animal cells. These target genes are known to contain the specific regulatory sequence called as the cAMP response element, generally abbreviated as CRE (diagramatic representation on the right side). Again, as described above, when cAMP binds to regulatory subunit of PKA, the catalytic subunit is released which carries the signal from cytoplasm to nucleus. Within the nucleus this activated PKA phosphorylates a transcription factor called CREB (CRE binding protein) at serine residue. This in turn recruits various co-activators and transcription of cAMP inducible genes takes place. This regulation of gene expression plays an important role in various processes like proliferation, differentiation, survival etc.

It is important to note that protein phosphorylation needs to be balanced which is done by the activity of protein phosphatases. Some protein phosphatases are transmembrane receptors while others are cytosolic. These phosphatases remove the phosphate group from tyrosine or serine or threonine residues in the substrate proteins thereby terminating the responses which is initiated by the activity of protein kinases. Lets make it more clear by taking the example of protein kinase A. The serine residues of the target proteins (phosphorylase kinase, CREB) which are phosphorylated by PKA are usually dephosphorylated by phosphatase called as protein phosphatase 1. Thus, the levels of phosphorylation of the target proteins which are phosphorylated by PKA is counter-balanced by the activities of protein phosphatases (diagram of regulation of phosphorylation by PKA and protein phosphatase 1 shown below).

Just note that although most of the effects of cAMP are mediated by PKA. However, cAMP can also directly regulate ion channels with no requirement of protein phosphorylation. 

Types of Receptors - Part 4

As we have seen in the previous post that the majority of the receptors are enzyme linked that stimulate the protein-tyrosine phosphorylation. Here, in this last post regarding the types of receptors, we will discuss about the other types of receptors that are associated with some other enzymatic activities. These are: protein tyrosine phosphatases, protein serine/threonine kinases and guanylyl cyclases. Lets understand briefly about each of these classes one by one below.

Protein Tyrosine Phosphatases:

Here, the name suggests the function i.e; phosphatases which is to remove phosphate group from phosphotyrosine residues. Thus, these receptors play a role opposite to that of protein tyrosine kinases thereby counterbalancing the kinases (just to recall, the kinases, add phosphate groups to the tyrosine residues). In some cases, the protein tyrosine phosphatases play a negative role in cell signaling pathways by stopping the signals which were initiated by protein-tyrosine phosphorylation. Whereas in some cases, the protein tyrosine phosphatases play a positive role in cell signaling by their enzymatic activities.

Serine/Threonine Kinases:

The receptors for some polypeptides phosphorylates serine or threonine residues instead of tyrosine residues on their substrate molecules. An example of such receptor is Transforming Growth Factor-β (abbreviated as TGF-β) which falls under this class of receptors i.e.; serine/threonine receptors. TGF-β is a family of growth factors which is involved in the control of proliferation and differentiation of various cell types. When the ligand binds, there is an association of two different types of polypeptides (both of which are encoded by different TGF-β receptor family) and hence forms the heterodimers. Here, one of the receptor kinases phosphorylates another one. This activated TGF-β receptors then phosphorylates another group of transcription factors called Smads, which then translocates to nucleus and causes the expression of targeted genes. The entire TGF-β signaling will be explained in the further post.

Guanylyl Cyclases:

Another class of receptors is guanylyl cyclases. Guanylyl cyclases have the cytosolic domain which catalyzes the formation of cyclic GMP. As we have seen earlier, that the signaling molecule, nitric oxide, also stimulates the guanylyl cyclase. However, the target of NO is intracellular enzyme as against the transmembrane receptor.
The receptor guanylyl cyclases have extracellular domain that binds the ligand, a single transmembrane α-helix and a cytosolic domain which has the catalytic activity. Thus, when the ligand binds at the extracellular domain, cyclase activity is stimulated which results in the formation of cyclic GMP - a second messenger.

So, finally, her we finish almost all the classes of receptors. Now, from the next post, we will proceed to the pathways of intracellular signal transduction.