User:Kinkreet/MCBII/Enzyme-coupled receptors

There are three types of cell-surface receptors - ion channel-coupled receptors, enzyme-coupled receptrs and G-protein-coupled receptors. The ligands are usually present at very low concentrations (10^-9-10^-11 M) and generate responses which are immediate (cytoskeleton organisation) as well as delayed (e.g. gene expression). They often not diffusible and are present on surfaces where the cell crawls. The receptors which bind the ligands will either have an intrinsic enzymic activity, or will be able to recruit an enzyme to transduce the signal.

There are 6 types of enzyme-coupled receptors:

1. Receptor tyrosine kinases
2. Tyrosine-kinase-associated receptors
3. Receptor-like tyrosine phosphatases
4. Receptor serine/threonine kinases
5. Receptor guanylyl cyclases
6. Histidine-kinase-associated receptors

When a ligand binds to a G-protein coupled receptor, it changes the relative orientation of the α-helices of the 7-transmembrane (7TM) protein, and thus changing the position of the cytoplasmic loops relative to each other. This is not possible for RTK because it only has one TM α-helix.

For enzyme-coupled receptors, activation is by either oligomerization of the receptors, or by re-orientation of the receptor chains of already-formed oligomers. This rearranges the cytosolic tails in a way which induces signal transduction.

Types of enzyme-coupled receptors

Receptor Tyrosine Kinases

Receptor tyrosine kinases are the most abundant enzyme-coupled receptors. Ligand binding to both receptor tyrosine kinases and tyrosine-kinase-associated receptors will result in the phosphorylation of tyrosine residues on a small set of intracellular signalling proteins. The difference between the two is that receptor tyrosine kinases have the kinase domain as part of the receptor, as opposed to tyrosine-kinase-associated receptors, which do not have any intrinsic kinase activity but must associate with a kinase to have an effect. Both RTK and tyrosine-kinase-associated receptors must oligomerize to function.

Ligands that bind receptor tyrosine kinases are usually growth factors and hormones. Specific examples includes epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factors (FGFs), hepatocyte growth factor (HGF), insulin, insulinlike growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), macrophage-colony-stimulating factor (M-CSF), and all the neurotrophins, including nerve growth factor (NGF).

There are at least 20 different subfamilies of receptor tyrosine kinases; the most abundant receptor tyrosine kinase is the Eph receptors. It binds to the less diffusible, cell-surface bound ephrins; ephrins regulates cell adhesion and repulsion by activating the Rho family which acts on the actin cytoskeleton, and functions to guide cells and axons during development. Ephrins and Eph receptors are different from other receptor-ligand pairs in that the ligand is also a receptor, exhibiting bidirectional signalling. The ephrin-expressing cell will initiate a signalling cascade upon being bound by an Eph receptor. This bidirectional signalling is required in brain development, to keep cells of one types separate from other type.

Receptor tyrosine kinases usually consists of just one subunit, but others can form multimeric complexes, such as the insulin receptor. They all have an N-terminal extracellular ligand-binding domain, a 25-38 AA hydrophobic transmembrane helix and a C-terminal cytosolic tyrosine kinase domain with juxtamemraben regulatory regions. When a ligand is bound to the extracellular domain, it induces the formation of receptor dimers, which allows the one kinase to phosphorylate the other using ATP, and vice versa; this is called cross-phosphorylation. There are 90 unique genes identified for tyrosine kinases, of which 58 are for RTK (split into 20 subfamilies), meaning that it can bind a large variety of ligands. Although the extracellular domains can vary a lot, the tyrosine kinase domain is highly conserved.

It has recently been found that the mechanism for activation of the tyrosine kinase domain, and for dimerization, are very diverse.

RTK are key regulators of critical cellular processes such as proliferation and differentiation, cell survival and metabolism, cell migration and cell-cycle control; all these processes require extracellular signals to be carried out correctly. Mutations in RTK genes, or the activation of the intracellular pathways that they regulate can lead to cancer, diabetes, inflammation, severe bone disorders, arteriosclerosis and angiogenesis.

*should know all the types of signal proteins for the exam*

Signal Protein	Receptors	Representative Responses
Epidermal Growth Factors	EGF Receptors	Stimulates cell survival, growth, proliferation or differentiation of various cell types. The mechanism is very complicated
Fibroblast Growth Factors	FGF Receptors	Stimulates proliferation of various cell types; act as inductive signals in development (e.g. for growing limbs)
Nerve Growth Factors	TrkA	Stimulates survival and growth of some neurons
Vascular Endothelial Growth Factors	VEGF Receptors	Stimulates angiogenesis
Insulin	Insulin Receptor	Stimulates carbohydrate utilisation and protein synthesis
Ephrins (contact-dependent, not secreted)	Eph Receptors	Stimulate angiogenesis; guide cell and axon migration.

Drugs can act as antibodies against the RTK to stop it activating its TKD; trastuzumab works as an antibody against the ErbB2 receptor HER2, preventing its unregulated proliferation, and thus is used to treat breast cancer.

Activation

Generally, RTKs are activated by dimerization induced by growth factor binding; but note that some RTKs such as insulin receptor and IGF1 receptors exists as oligomers (disulfide linked (αβ)2 dimers in this case) even in the absence of a ligand. There has also been evidence to suggest that epidermal growth factors (EGF) and the Discoidin domain receptor family (DDRs) binds to pre-existing non-covalent oligomers or receptor, and does not cause the oligomerisation. Other evidence shows that RTKs such as Tie2 and Eph may require the formation of larger oligomers (one dimer is not enough). Some RTKs (FGFRs and MuSK) require co-receptors.

Upon ligand binding, the cytosolic tails interact in such a way that allows the cytosolic tails to phosphorylates each other, a process known as autophosphorylation. This requires at least 2 receptors to be in close proximity to each other. This close proximity is induced when the ligand bind. For dimerization to occur, the ligand must be bivalent, meaning either it has two sites of binding, or it is a complex of two subunits, each with one binding site each. The ligand can be a monomer than binds to both receptors (EGF) or a dimer, with each of the dimer binding one receptor each (PDGF). Fibroblast growth factors (FGFs) exists as monomers which forms multimers on heparan sulfate proteoglycans, which allows it to bind to separate receptors and bring them to close proximity. The cytoplasmic domain of the EGF receptor contains a kinase domain with an activation loop and a C-terminal tail. When ligand is absent, the activation loop is hidden inside the domain; when ligand binds, it comes out and activate the cytoplasmic domain on the neighbouring receptor, causing phosphorylation on the C-terminal tail of both receptors.

This mechanism of activation is supported by crystal structures of the ligand-binding domains with ligand bound. However, the ligand can form all, a portion or none of the dimer interface. The ligand induces dimerization by stabilising the dimer. In a TrkA dimer (the first RTK to be discovered), the nerve growth factor associate with the Ig-C2 domain while binding to each other; the receptors do not make contact at all. In the ErbB receptor dimers, ligand-binding causes a conformational change exposes a previously occluded dimerization site, which would form the dimerization interface. Here the ligand is not part of the dimer interface. Those were the two extremes, most receptor dimers have some portions of the interface mediated by ligand, and some by receptor. The fibroblast growth factor receptor (FGFR) have Ig-like D2 domains that associate with each other; there are heparin or heparin sulphate proteoglycans of which 6 units make contact with FGF, acting as co-receptors. In the KIT dimer, there are regions (D2-D3) where the interface consists only of the ligand, but also regions (D4-D5) where parts of the receptors come together.

Autophosphorylation of tyrosines within the kinase domain increases the activity of the kinase; phosphorylation outside the kinase domain creates docking sites to which intracellular signalling molecules can bind. The intracellular signalling molecules are activated either by simply binding, or by phosphorylation. There can be multiple binding sites on the same cytosolic tail, and thus one receptor can activate multiple pathways. The specificity of binding lies on the residues either side of the tyrosine. One kinase can often phosphorylates on multiple sites of a secondary kinase, and thus amplifying the signal and diversifying the pathways involved.

Post-activation by EGF receptors, the cytoplasmic domain is dephosphorylated by protein tyrosine phosphatases, deactivating it. The EGF receptor cannot be recycled and so is endocytosed and degraded. The E3 ubiquitin ligase c-Cbl is targeted to activated RTKs to ubiquitinize it to target for lysosomal degradation. Other receptors can be recycled.

Inhibitions

Once ligand is bound the receptors dimerized, the intracellular tyrosine kinase domain (TKD) is activated. Every TKD has a C-lobe, an N-lobe and an activation loop. In the activated TKD, the structure of the TKD elements are very similar, but not so in the inactivated TKD. Different inactive TKDs have different conformations, and this affects how it inhibits its kinase activity. In insulin receptor-like TKDs such as FGFR, insulin receptor and IGF1 receptors, the activation loop interacts directly with the active site of the kinase and block its access to protein substrate and ATP (ATP only in insulin and IGF1 receptors).

Even in the inhibited form, the TKD still retains kinase activity, albeit reduced; this is because, like DNA breathing, the loop will dissociate from the active site transciently, allowing it to phosphorylate and be phosphorylated for a short period of time. So if you bring two TKD in close proximity, they might phosphorylate each other. Activation occurs when, a specific tyrosine on the activation loop is phosphorylated and this stops the activation loop from interacting with the active site. ATP can then bind to the active site and be used to phosphorylate intracellular signalling proteins. This is called active loop inhibition, and is the mechanism used in most TKDs.

Other TKDs such as that of KIT, PDFGR and Eph, have a juxtamembrane region that interact with the active site of the kinase, preventing substrate binding; as well as interaction with the activation loop to stabilise the inactive form. This is called juxtamembrane inhibition. In C-terminal tail inhibition, it is the C-terminal tail that interacts with the active site. In EGFR dimers: the C-lobe of one subunit is able to activate the N-lobe of another when brought close together, no prior phosphorylation is required in this mechanism.

Because all activation involves phosphorylation of tyrosine residues, we can monitor the level of activation by Western blotting and then wash with antibodies against phosphotyrosine but not tyrosine, the level of binding would correlate to the level of activation. Antibodies against another protein (such as the receptor and/or cytoskeleton proteins) is used to show that all the lanes are loaded with similar concentration of cell lysate.

We can also monitor its physiological effect, by counting cells to look for proliferation, by monitoring in real-time for migration.

Example of RTK - Discoidin domain receptors

DDRs are a type of RTK, discovered by screening a human placental cDNA library with a³²P-labelled oligonucleotide that code for a sequence conserved in tyrosine kinases. But at the time, the ligand for the DDRs wre not known, and so affinity chromatography with the receptor as the column was used, where every possible substrate for binding is fed through, to see what binds.

Vogel ran the column using different concentrations of Matrigel and then ran the effluent through a SDS-PAGE and western blot. It showed that the higher the concentration of Matrigel, the more substances that was bound, indicating something in the gel binds to the DDRs. Vogel then performed the same experiment with the likely binding partners and found it to be fibrous collagen (not gelatin, or denatured collagen). This activation is slow but sustained.

There are two DDRs - DDR1 and DDR2. Activation of DDR1 induces phosphorylation of a docking site for the Shc phosphotyrosine binding domain; DDR1 have roles in mammary gland development . Activation of DDR2 results in the up-regulation of matrix metalloproteinase-1 expression; DDR2 have roles in proliferation - elimination leads to dwarfism and mutations can cause spondylo-meta-epiphyseal dysplasia (SMED). DDR2 activation requires Src, which phosphorylate 3 tyrosine residues on the activation loop of DDR2.

DDR binds to a conserved sequence on collagen - GVMGFO - through three loops of the discoidin domain. This brings two DDRs together and their kinase domains autophosphorylates each other, leading to activation. Using co-immunoprecipitation using Flag and Myc, Noordeen confirmed that without collagen binding, DDRs still associate as a dimer.

Tyrosine-kinase-associated receptors

Tyrosine-kinase-associated receptors functions in much of the same way as RTKs, but instead of having an intrinsic kinase domain, it recruits cytoplasmic tyrosine kinases to associate with it. The largest family of cytoplasmic tyrosine kinases is the Src family, which includes Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk. They are held at the cytoplasmic side of the plasma membrane by binding of its SH2 or SH3 domain to receptors, and also by interaction with the lipid.

T cell activation

T cell activation involves Lck. When the CD4 or CD8 binds to the MHC complex on the APC, it activates Lck which phosphorylates the tyrosines on all CD3 chains. ZAP70 is recruited by docking to these phosphotyrosines, and Lck phosphorylates ZAP70 also.

Normally, T cell activation is balanced by the actions of tyrosine kinases and tyrosine-kinase-associated receptors, against phosphatases. When an APC binds to the T cell, phosphatases with a larger extracellular domain are pushed to the periphery and so only kinases act at the contact region, and thus able to generate a downstream signal.

Integrin

Some integrins are tyrosine-kinase-associated receptors that change cellular behavior depending on its contact with other cells and the extracellular matrix. The cell can bind to the extracellular proteins, such as fibronectin, laminin and collagen, through integrins. The integrins binds to intracellular dimeric talin, which are anchored onto actin filaments through vinculin. Integrins cluster at those interfaces at junctions called focal adhesions zones. Inactive focal adhesion kinase (FAK) are recruited at the FAT site of talin; this brings it close to the plasma membrane and allows it to bing to PIP₂. This releases its FERM domain which normally auto-inhibits it, and activates FAK by autophosphorylation. This creates phosphotyrosine sites to which Src can bind and signal downstream for survival. This type of integrin-dependent signalling allows the cells to grow and survive when in a tissue, but inhibit its growth when removed; likely to prevent embolism of cells.

RTKs and GPCRs can activate talin and thus activate integrins.

Receptor-like tyrosine phosphatases

Receptor-like tyrosine phosphatases act in contrast to tyrosine kinases and remove phosphate groups from tyrosine residues of specific intracellular signalling molecules. They are called receptor-like because the ligand which it binds had not been found yet, and so presumed to be a receptor.

More than 30 tyrosine phosphatases are found in humans, and they can be both cytoplasmic or transmembrane. They each have a high specificity for their substrate, and together can carry out many dephosphorylation reactions at a specific time, ensuring the level of tyrosine phosphate in the cell is very low.

There are two tyrosine phosphatases in vertebrates that has a SH2 domain, called SHP-1 and SHP-2. They can bind to phosphotyrosines and dephosphorylates activated JAKs.

Another common PTP is the CD45 protein, a protein found on the surfaces of all leukocytes, and is essential for B and T cell activation by antigens.

They can act as cell-adhesion molecules, as well as a signalling ligand. For example, protein tyrosine phosphatase ζ/β is expressed on glial cells and binds to contactin on developing nerve cells and signal for the nerve cell to develop long processes.

Receptor serine/threonine kinases

Receptor serine/threonine kinases phosphorylate specific serines or threonines on associated latent gene regulatory proteins of the Smad family (named after the first two members discovered - Sma in C. elegans and Mad in Drosophila), which migrates into the nucleus and activate gene transcription. There are two types of receptor serine/threonine kinases - type I and type II - both have the same general structure of a single-pass transmembrane protein with a serine/threonine kinase domain on the cytosolic domain.

The transforming growth factor β (TGF-β) superfamily signal through the serine/threonine kinase pathway. The superfamily includes the TGF-βs themselves, the activins, and the bone morphogenetic proteins (BMPs). The BMPs constitute the largest family. The TGF-β family is a set of secreted, dimeric proteins that signals for pattern formation, differentiation, proliferation, ECM production and cell death; and also act as a cytokine in the adaptive immune system. For members of the TGF-β superfamily to activate the pathway, the ligand usually first binds a type-II receptor homodimer and activates it; then a type-I homodimer (usually bound to an inhibitor) is recruited and phosphorylated to (remove its inhibition and to) form a tetrameric complex. A protein called SARA (Smad anchor for receptor activation) recruits protein complexes (usually trimers) of the Smad family by binding to the receptor, the Smad and also localized to the plasma membrane by binding to phospholipid moieties. Most Smad activation occurs in early endosomes, where the receptor kinases are endocytosed in clathrin-coated pits.

Receptor	Smad phosphorylated
TGF-β or activin receptors	Smad2 or Smad3
BMP	Smad1, Smad5, or Smad8

The receptor-activated Smads then binds to Smad4 and form a complex and moves into the nucleus and associate with other gene regulators and bind to specific DNA sequences, altering expression.

Regulation

Similar to the JAK-STAT pathway, some of the genes which it promotes are inhibitory Smads - Smad6 and Smad7, which provides an atuo-negative feedback mechanism. They are non-functional Smads which binds to type-I receptors and prevent other Smads binding and being activated. The two pathways may also reciprocally negatively-regulate each other, depending on the type of ligand that binds - γ-interferon is a cytokine that activates the STAT pathway that leads to the transcription of the inhibitory Smad7, and inhibits TGF-β signalling. This makes sense because the two ligands have opposite effects - TGF-β inhibits T cell proliferation and effector functions, inhibits B cells and macrophages, promotes class switching to IgE; whereas IFN-γ activates macrophages, increase MHC expression and antigen presentation, promotes the class switching to IgG3.

Negative regulation is also provided by inhibitors of the signalling molecules. Noggin and chordin inhibits BMP, follistatin inhibits activins.

Receptor guanylyl cyclases

Receptor guanylyl cyclases directly catalyze the production of cyclic GMP in the cytosol, typically activated by nitric oxide and natriuretic peptides (NPs). There are several types of NPs - atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) - and generally signal to lower the blood pressure by secreting cations and water, as well as causing smooth muscle relaxation. Upon binding of the ligand on the extracellular side, its cytosolic guanylyl cyclase catalytic domain is activated and produce cGMP. cGMP then binds to cyclic GMP-dependent protein kinase (PKG) and phosphorylates proteins on serine or threonine.

Histidine-kinase-associated receptors

Histidine-kinase-associated receptors first autophosphorylates itself on a histidine residue and then transfer that phosphate onto an intracellular signalling protein. They are dimeric transmembrane receptors which have an extracellular ligand-recognition domain, and an intracellular tail that associates with the adaptor protein CheW that holds the histidine kinase CheA to the receptor. Upon the binding of a repellent, CheA phosphorylates itself on a histidine residue, which is immediately transferred to an aspartate on the messenger CheY. CheY dissociates and move to the motor complex and causes it to rotate clockwise, inducing tumble. The negative regulation of this process is partly provided by the intrinsic phosphatase activity of CheY itself, as well as CheZ, ensuring the phosphorylated moieties only last for 10 seconds. When an attractant binds, it inhibits the phosphorylation of CheA and thus CheY, so the motor continue to rotate anti-clockwise and move forward.

This response to repellent and attractant is not based on the concentration but on changes to the concentration, as the receptors become desensitized or adapt by methylation (catalyzed by a methyl transferase) and demethylation (catalyzed by a methylase).

Pathways activated

The pathways activated depends partly on the type of intracellular signalling molecules that binds to the docking sites. Phospholipase C-γ (PLC-γ) activates the inositol phospholipid signaling pathway, which increases the intracellular Ca²⁺ concentrations. Other common intracellular signalling molecules include cytoplasmic tyrosine kinase Src that phosphorylates other proteins on tyrosines, and phosphatidylinositol 3′-kinase (PI 3-kinase) which generates specific lipid molecules in the plasma membrane to attract other signaling proteins.

Although the structure of the intracellular signalling proteins may vary a lot, the phosphotyrosine binding domain is highly conserved. It takes form mostly as the Src homology region (SH2) domain, or less commonly the phosphotyrosine-binding (PTB) domain. The SH2 domain contains a pocket for binding the phosphotyrosine, as well as a pocket for binding a specific amino acid in the flanking region of the phosphotyrosine; it is these pockets will allows the protein to bind to specific activated tyrosine kinases, as the flanking amino acid would be different.

The primary signalling protein then may also have other binding domains, such as the SH3 domain that binds proline-rich motifs, to transduce the signal to secondary signalling molecules, such as Ras. These secondary molecules can have a positive or negative effect. c-Cbl can dock on some activated phosphotyrosine and catalyze its conjugation with ubiquitin, tagging it for degradation in the proteasome; Ras transduce the signal as well as bifurcate the signal to activate multiple downstream signalling pathways, such as the serine/threonine phosphorylation cascade, that leads to a change in gene expression that promotes differentiation and proliferation. Mutations in the Ras protein will cause inappropriate activation of differentiation and proliferation, leading to increased cell division, causing cancer (Ras was first found as the primary mutation which causes rat sarcoma). About 30% of all human exhibits a hyperactive Ras mutation.

Ras

Ras is both a family of proteins, as well as a superfamily of monomeric GTPase (to which the family is part of). The Ras superfamily is defined by homology with the Ras protein, and includes the Rho and Rab family. The Rho family includes Rho, Rac, Cdc42, and is involved in transducing the signal to alter actin cytoskeleton. Rho activation causes the arrangement for stress fibres, Rac activation leads to lamellipodia formation and cdc42 activation leads to filopodia formation. The Rab family is involved in regulating vesicle transport; the Ras family includes H-Ras, K-Ras, N-Ras, Rheb and Rep1, and is involved in broadcasting and amplifying signals, activates mTOR to stimulate cell growth, influences cell adhesion by activating integrins.

Similar to other GTPases, the Ras superfamily of proteins have two states: active state when GTP is bound; and an inactive state when GDP is bound. Guanine nucleotide exchange factors (GEFs) dissociates GDP from Ras and mediate uptake of GTP (which is typically 10 times more abundant than GDP), activating Ras; GTPase-activating proteins (GAPs) promotes the hydrolysis of GTP, inactivating Ras.

The activation of GEF is therefore essential for the activation of Ras. GAP can bind directly to tyrosine kinases, whereas GEF can only bind through an adapter protein, or primary signalling protein. This is also the case for the Rho and Rab families. In the case of the Ras family, Grb-2 protein binds to the activated tyrosine kinase through its SH2 domain, and then transduce this signal to a GEF through its SH3 domain binding to a proline-rich region, called Sos, on the GEF. Some phosphotyrosines on the tyrosine kinase do not form a correct docking site for Grb-2, and thus must recruit an adaptor protein called Shc, to bridge between the kinase and Grb-2. Although this is the main GEF that activates Ras, there are also others Ras-specific GEFs which do not require a signal from RTKs.

MAPK/ERK pathway

Phosphotyrosines and activated Ras only exists for a short period of time before it becomes inactive again, by phosphatases and GAP, respectively. As the signals intends to signal for proliferation and differentiation for a longer period of time, activated Ras must activate other longer-lasting signalling pathways, which involves serine/threonine phosphorylations. There are many serine/tyrosine kinases, but three main ones constitute the whole cascade - Ras activates mitogen-activated protein (MAP) kinase-kinase-kinase (MAP3K), which activates MAP2K, which activates MAPK. To activate the MAPK, a specific threonine and tyrosine residue (separated by one amino acid), must be phosphorylated by MAP2K. In mammals, the specific MAP3K is called Raf, the MAP2K is called MEK, and the MAPK is called Erk. MAPK enters the nucleus and phosphorylates one or more of the transcription factors (such as SRF and TRF) that promotes the expression of immediate early genes (c-Fos), genes which are switched on within minutes of signal detection. These genes may encode for proteins which activates further gene expression, but these require more time. Genes which are activated includes G₁ cyclins, which is required for proliferation.

The length of time the MAPK remains activated depends on the ligand that is bound. When EGF activates the receptor, MAPK remain activated only for 5 minutes, after which its activity declines; this causes the cells to divide. When NGF activates its receptor, the MAPK remains active for hours, and this promote differentiation.

The three-component MAPK/ERK pathway is observed in all animal cells and yeast cells. Many responses are mediated by these pathways. To avoid cross-talk between parallel pathways that uses some common components, scaffolding proteins are used, each with a different specificity for the three components. This strategy provides precise signalling, but prevent amplification. In reality, some components are bound to scaffolds, while some are diffusible, allowing for amplification to produce many MAPK, while still maintaining a reasonably level of specificity.

Phosphatidylinositol 3-kinase (PI 3-kinase) Pathway

As mentioned before, Ras activates more than one pathway. Apart from the serine/threonine series, it can also activate PI3-kinase, which signals for survival and growth. Cells need this pathway alongside the MAPK/ERK pathway, because without it, the cell will continue to divide without increasing in size, and so the daughter cells will shrink with each subsequent division.

Sometimes, one ligand can activate both the growth and mitotic pathways, but can each pathway can also be activated separately - by a mitogen for division and growth factors for growth.

One of the major pathways for growth is the PI3K pathway, which phosphorylates inositol phospholipids. Phosphatidylinositol (PI) can be phosphorylated by activated PI3K at three different sites to give PIP, PI(3,4)P₂ or PI(3,4,5)P₃. PI(3,4)P₂ or PI(3,4,5)P₃ can then act as docking sites for Pleckstrin homology (PH) domain (first identified in the platelet protein Pleckstrin)-containing proteins (much like the docking site of phosphotyrosine with SH2). Over 200 proteins contain the PH domain, including Sos which activates Ras. An example of the PI3K pathway is observed in B cells. When B cells recognizes antigens, it can activate its PI3K, which creates docking sites for a tyrosine kinase called BTK, as well as PLC-γ. BTK phosphorylates PLC-γ and activates it, phospholipase C (PLC-β for GPCR or PLC-γ for RTK) can cleave PI(4,5)P₂ to produce the two small signaling molecules diacylglycerol and inositol 1,4,5-trisphosphate (IP₃). The IP₃ increases the release of Ca²⁺ from the ER, while diacylglycerol activates PKC along with the cytosolic Ca²⁺ released from the effects of IP₃. The effect of PI3K is countered by phosphatase and tensin homolog (PTEN), of which mutation causes over-activation of the PI3K pathway and leads to cancer. The PI3K pathway does not produce PI(4,5)P₂ but PI(3,4)P₂ or PI(3,4,5)P₃, both of which are not cleaved. But specific inositol phospholipid phosphatases, including PLC-γ, can removes phosphates at the 3-position, giving rise to PI(4,5)P₂ which can be cleaved as mentioned to promote downstream signals. Mutations in the BTK means the B cell cannot respond to antigens, and exhibit deficiency in antibody production.

Protein kinase B (PKB, a.k.a. Akt) and phosphoinositol-dependent kinase (PDK1) both have a PH domain which binds to PI(3,4)P₂ or PI(3,4,5)P₃. PDK1 phosphorylates PKB, which allows PKB to return to the cytosol and phosphorylates target proteins, often cell death factors. One of these factors is BAD, when dephosphorylated, holds onto certain proteins that inhibit apoptosis, thereby allowing apoptosis or cell death. When BAD is phosphorylated, it binds to a 14-3-3 proteins instead, releasing the inibitory proteins which it was bound before, and these can inhibit the induction of apoptosis, leading to cell survival.

JAK-STAT Pathway

The JAK-STAT pathway was first discovered in interferon signalling. When interferon binds to its receptor, it activates a kinase called Janus kinase (JAK). Now more than 30 cytokines and hormones bind to receptors which activates the JAK-STAT pathways.

The structure of Janus kinase is made up of 7 Janus homology domains, JH 1-7. JH1 is the kinase domain, JH2 is a kinase-like domain, likely to be a duplication of the kinase gene which has undergone mutations. The JH3 and JH4 domains form the SH2 domain which can bind to the phosphotyrosines on the adapter. JH4-JH7 makes up the FERM domain, which mediates receptor binding in the focal adhesion zone.

Signalling Ligand	Receptor-associated JAKs	STATs activated	Responses (not complete)
IL-6			stimulates immune response during infection (fever)
γ-interferon	Jak1 and Jak2	STAT1	activates macrophages; increases MHC protein expression
α-interferon	Tyk2 and Jak2	STAT1 and STAT2	increases cell resistance to viral infection
Erythropoietin	Jak2	STAT5	stimulates production of erythrocytes
Prolactin	Jak1 and Jak2	STAT5	stimulates milk production
Growth hormone	Jak2	STAT1 and STAT5	stimulates growth by inducing IGF-1 production
GM-CSF	Jak2	STAT5	stimulates production of granulocytes and macrophages
IL-3	Jak2	STAT5	stimulates early blood cell production

The signal transducer and activator of transcription (STAT) proteins are transcription factors which lay dormant in the cytoplasm and are activated only on detection of signal. As the name suggests, it is involved both in the signal transduction and activation of transcription.

JAK is a kinase which, in the absence of a ligand, non-covalently bind to the receptor. The ligands which binds are usually cytokines and growth factors that binds specifically to receptors; upon binding, JAK is phosphorylated and the receptors form oligomers. This attracts JAK to move closer to the tip of the cytoplasmic tail. JAK transphosphorylates onto tyrosine residues on the cytoplasmic domain of the receptor and these phosphorylated tyrosine residues forms docking sites for the Src-homology-2 (SH2) domain on STAT (as well as other) proteins. The binding of STAT to receptor brings the STAT proteins in close proximity to JAK, and JAK further transphosphorylates STAT proteins at a single tyrosine residue near residue 700 (of a 750-850 residue protein). Immediately, two phosphorylated STAT proteins dimerize by using its SH2 domain to reciprocally binding to the other's phosphorylated tyrosine. This dimerization is thought to underlie the mechanism by which the STAT proteins detach from the receptor. Activated STAT accumulate in the nucleus and activates or increase the level of transcription of quiescent or less-active genes. There are four types of JAKs - Jak1, Jak2, Jak3, and Tyk2 - which binds to different cytokine receptors and activates different STAT proteins have different binding affinity to specific DNA sequences, and also recruits different sets of transcription factors and co-activators, and so different STAT proteins activate different genes.

Negative regulation is provided by cytoplasmic tyrosine phosphatases and suppressors of cytokine signalling (SOCS) proteins that blocks the activation of STAT; nuclear phosphatases and proteins that inhibit activated STAT proteins (PIAS) can inhibit activated STATs. Naturally truncated STATs, which are non-functional, can compete with activated STATs in binding to DNA and other STAT proteins.

Other receptors with tyrosine kinase activity, such as those for epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), can activate STAT proteins. This activation may be direct (e.g. PDGF receptor) or indirect, through the recruitment of non-receptor tyrosine kinases (NRTKs), such as JAK and Src. In C. elegans, JAK is not produced but STATs were still used in this manner, suggesting the STATs evolved before JAK, and the purpose of JAK is only to phosphorylate STATs.

Notes

• Tyrosine kinases uses Ras for signal transduction, and mainly activates MAPK pathway or PI3K pathway - usually slow and involves many steps
• Cytokines activates the JAK-STAT pathway mostly through tyrosine-kinase associated receptors - Direct and fast-acting
• TGF-β activates the serine/threonine kinase pathway