Signal Transduction Pathway - The TGF-β/Smad Pathways

TGF-β/Smad pathway is like the previously mentioned JAK/STAT pathway in a way that there is direct connection between the cell surface (receptors) and the nucleus (transcription factors) instead of having a cascade of proteins.

TGF-β stands for transforming growth factor-beta. It is a protein that is involved in various cellular processes like cell differentiation, proliferation, apoptosis etc. This ligand of TGF-β superfamily is a secreted protein and it includes bone morphogenetic proteins (BMPs), growth factors, differentiation factors, activin etc.
The receptors for TGF-β family are serine/threonine kinases. These receptors are primarily composed of two types of polypeptides as type I receptor and type II receptor.

This pathway is very simple to understand. The first step in this pathway is the binding of the ligand to the receptor. This ligand first binds to a specific TGF-β type II receptor. The binding recruits TGF-β type I receptor. Being a serine/threonine kinase, type II receptor phosphorylates TGF-β type I receptor there by forming a dimer. Type I receptor in turn phosphorylates Smad proteins. The phosphorylated Smad is then translocated to the nucleus where it regulates the gene expression.

In humans, there are 42 members of TGF-β family which elicit different responses. There are seven different types of TGF-β type I receptors and five different TGF-β type II receptors. When there is interaction of TGF-β family members with their respective receptors, it leads to activation of Smads which is a family of  8 member which leads to different responses in target cells.

It is interesting to know that Smad members can also be phosphorylated by ERK and this interaction between TGF-β/Smad and ERK plays an important role in embryonic development.

Signal Transduction Pathway - The JAK/STAT Pathways

The JAK/STAT pathway is one of the pathways where there is a direct connection between cell surface and nucleus. This leads to phosphorylation of transcription factor. directly by receptor-associated proteins. This is unlike the previously described - PI3-kinase and MAP-kinase pathways where there is a  cascade of proteins between cell surface and nucleus that leads to transcription factor phosphorylation. Most of the JAK/STAT pathways are expressed in white blood cells and hence the major role is in the regulation of immune system.

The JAK/STAT pathway mainly comprises of three components:

a. A Receptor - A signal from interferon, interleukin, cytokine, growth factors or chemical messenger activates the  receptor.

b. Janus Protein (JAK) - JAK have tyrosine kinase activity.

c. Signal Transducer and Activator of Transcription (STAT) - STAT proteins are transcription factors that posssess the SH2 domain.

In unstimulated cells, the STAT proteins are inactive and are localized to cytoplasm. The binding of ligand to the receptor leads to the activation of JAK. The function of JAK is activated and being a tyrosine kinase, it phosphorylates the tyrosine residues on the receptor. As a result, the sites for phosphotyrosine-binding of  SH2 domains is created. As mentioned above that STAT proteins have SH2 domains and hence these proteins are recruited to bind to phosphotyrosine residues via SH2 domain. These STATs are now phosphorylated on their tyrosine residues by JAKs. These phosphorylated tyrosine now act as a binding site for SH2 domains of other STATs. This leads to dimerization of STAT proteins. STATs can form homodimers or heterodimers. These dimers then translocates to the cell nucleus where they stimulate the activation of the target genes.

Further studies have shown that the STAT proteins may also be activated downstream of receptor tyrosine kinases where their phosphorylation may be either by the receptors themselves (for example, epidermal growth factor receptor) or by non-receptor tyrosine kinases (for example c-src).

Signal Transduction Pathway - MAP Kinase Pathways (Part 2)

In the last post, we have seen how the ERK pathway gets activated and how the Ras protein gets regulated. Here, we will proceed in continuation with previous post. We will start this post by the mode of Ras activation. The best mode would be that mediated by protein tyrosine kinases. There is autophosphorylation of protein tyrosine kinase receptors. This phosphorylation results in association with Ras guanine-nucleotide exchange factor (GEF). This is mediated by interaction with another protein that possesses the SH2 domain.
Confused? Okay! Let me make it easier to understand by taking an example. Sos is the protein which is a guanine nucleotide exchange factor and Grb2 is a protein in the cytosol which has the SH2 domain in unstimulated cells. The Ras is anchored to the inner leaflet of the plasma membrane with the help of lipids which are attached to the C-terminus of Ras. Now, the mechanism is: The Sos is bound to SH2 domain of Grb2 protein. When there is phosphorylation of the protein tyrosine kinase receptors, it creates a binding site for SH2 domain of Grb2 (as can be seen in the adjacent figure). This association of Grb2 with activated receptors localizes Sos also to the plasma membrane where it interacts with Ras protein. Sos then stimulates the guanine-nucleotide exchange of Ras from inactive Ras-GDP to active Ras-GTP. This active Ras-GTP complex then interacts with a number of proteins, including the Raf, mentioned in earlier post. As mentioned in the earlier post, Raf activates which then leads to activation of ERK.

Another very important role of ERK is the induction of immediate-early genes. So, what are immediate-early genes and how ERK induces? A fraction of activated ERK translocates to the nucleus. Activated ERK fraction regulates the transcription factors by phosphorylation which in turn is stimulated by induction of a family of approximately 100 genes which are called the immediate early genes. Lets take an example to make it more clear. The serum response factor (SRF) and Elk1 are transcription factors which bind to serum response element (SRE) in the promoter region of target sequence. Activated Erk (phosphorylated form) translocates to the nucleus where it phosphorylates and activates Elk1 (adjacent figure). Elk1 binds to SRE in a complex with SRF. Phosphorylation of Elk1 stimulate its activity as a transcriptional activator, as a result the immediate early genes are induced. There are many immediate early genes that themselves encode transcription factors and so there is induction in response to growth factor stimulation that leads to expression of array of downstream genes called secondary response genes. This ERK signalling stimulates cell proliferation.

The mammalian cells (and also yeast cells) have multiple MAPK pathways that control distinct cellular responses. Each cascade consists of three protein kinases: a terminal MAP kinase and two upstream kinases that regulate its activity. In mammalian cells, three major groups of MAP kinase have been identified. These include members of ERK family, JNK and p38 MAP kinase. The JNK and p38 MAP kinase cascade gets activated by members of Rho subfamily (small GTP binding protein) (which includes Cdc42, Rho and Rac) rather than by Ras. Also, the JNK and p38 MAPK leads to inflammation and cell death as against ERK kinases that lead to cell proliferation, survival and differentiation.

There are scaffold proteins which are associated with different components of each MAP kinase cascade as complexes. The specificity of each MAP kinase signalling is maintained in part by this organization in association with scaffold proteins. Example of JNK MAP kinase cascade. JIP-1 is a scaffold protein that organizes the JNK MAP kinase and its upstream molecules into a signalling cassette. Thus, JIP-1 binds MLK, MKK7 and JNK and organizes these components of JNK pathway into a signalling cassette. As can be seen in the diagram, Rac activates MLK which leads to specific and efficient activation of MKK7 which ultimately activates JNK. This interaction with scaffold proteins is thought to play an important role in determining the specificity of signalling pathways within the cell.

Signal Transduction Pathway - MAP Kinase Pathways (Part 1)

This pathway refers to the proteins that are highly conserved in evolution. The main role in this pathway is played by a family of serine/threonine kinases called the MAPK (mitogen activated protein kinases). Sometimes, ERK is also used as a synonym of MAPK. ERK stands for extracellular signal-regulated kinase). However, recently, ERK has been classified for a specific subset of mammalian MAPK.  ERK plays a critical and central role in cell proliferation. ERK is activated by growth factors binding to either protein tyrosine kinases or G-protein coupled receptors (GPCRs).

Lets see how this ERK/MAP kinases get activated? Here are two protein kinases upstream to that of ERK. These kinases are coupled to membrane receptors by Ras-GTP binding protein. When there is stimulation by growth factors, Ras which is a GTP binding protein gets activated. Ras when activated phosphorylates the Raf protein, a serine/threonine kinase. The Raf, then phosphorylates and activates the second protein called MEK (MAP kinases/ ERK kinase). MEK is an interesting protein that activates its downstream members of ERK family by phosphorylating at both threonine and tyrosine residues separated by 1 amino acid (for example, MEK phosphorylates threonine 183 and Tyrosine 185 of ERK2). When this ERK is activated, it phosphorylates a number of targets like protein kinases and transcription factors.

We have discussed Ras protein above as the central player of ERK pathway. Lets understand this protein a bit. We have heard about this protein as “oncogenic protein” i.e.; responsible for causing cancer. So, how it came to be known as oncogenic protein? Ras was first identified as the oncogenic protein of tumor virus that causes sarcoma in rats. The name Ras comes from ‘rat sarcoma virus’. Ras became as the protein of great interest in early 1980s when it was identified that the mutations in Ras can cause cancers in human. Various experiments were performed on normal mammalian cells. These experimental results with Ras showed that when active Ras protein is directly injected in normal mammalian cells, it induces proliferation. On the other hand, when the function of Ras protein is interfered by the injection of anti-Ras protein (antibody), the cell proliferation is blocked. Ras proteins are guanine nucleotide-binding protein. Remember the α-subunit of G-protein? Ras has the function which is analogous to that of the α-subunit of G-protein. Ras in inactive state is bound to GDP while in active state GDP gets exchanged with GTP. This activation of Ras is mediated by guanine-nucleotide exchange factors (GEFs) which helps in release of bound GDP and gets exchanged with GTP. However, there is one difference, Ras acts as a monomer rather than in association with βγ-subunits (as in case of G-protein). This is how the Ras gets activated. Now, it is necessary to terminate its activity when its function is over. So, here comes the role of GTPase-activating protein (GAPs). This GAP interacts with Ras-GTP resulting in hydrolysis of GTP thereby terminating the activity of Ras.

In the next post which will be the continuation of this post, we will see how Ras activation takes place downstream of protein tyrosine kinases; how the genes are induced by ERK and also the pathways of MAP kinase activation in mammalian cells.

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.