Lecture 11 - Receptor tyrosine kinases Flashcards
What is the difference between RTKs and GPCRs.
Receptor tyrosine kinases, just like the G protein-coupled receptors, are very large
group of receptors including, for example:
* Insulin receptor (glycogen synthesis)
* Vascular endothelial growth factor receptor (new blood vessel growth)
* Platelet derived growth factor receptor (embryonic development, cell proliferation & migration)
* Epidermal growth factor receptor (growth & proliferation & differentiation)
What differentiates these receptors very clearly from other groups, including the G protein coupled receptors, is that they possess intrinsic kinase activity.
G protein-coupled receptors discussed previously are associated with short-term changes in cell function, e.g. metabolism, movement. These receptors don’t have any intrinsic kinase activity or enzyme activity themselves; they recruited and activated G proteins and then the Alpha subunit of the G protein activates downstream effectors which could then activate a kinase
Receptors associated tyrosine kinases are associated with long-term changes in gene expression or development.
Describe the structure and function of the epidermal growth factor receptor.
The Epidermal growth factor receptor is a typical receptor tyrosine kinase (RTK). It is responsible for perceiving and transducing the Epidermal growth factor signal. In humans Epidermal growth factor is a fairly small polypeptide with 53 amino acids which is involved in the regulation of growth, proliferation and Differentiation.
The receptor monomers have just one transmembrane spanning Alpha Helix (instead of
7 in the G protein coupled receptors), an extracellular domain which binds the epidermal growth factor, a cytosolic domain which has intrinsic kinase activity, and a C-terminal tail which is highly enriched in tyrosine residues. The latter are critical for the functioning of these receptors and for the transduction of signals through these receptors.
Describe activation of the epidermal growth factor receptor.
Binding of EGF to the extracellular domain of the receptor monomers causes dimerization of the monomers.
1. Prior to ligand binding dimerisation arm is in the centre of the conformation
2. Ligand binding causes arm to “swing” out
3. Two dimerisation arms can then link together (interact) forming a dimer
Binding of EGF and dimerization of the monomers results in trans autophosphorylation of tyrosine residues on activation lip of the kinase domain (in cytosolic domain).
Phosphorylation of tyrosine residues fully activates tyrosine kinase allowing it to phosphorylate additional tyrosine residues in the RTK cytoplasmic domain. These phosphorylated tyrosine residues create ‘docking sites’ for additional downstream signaling intermediates.
Activation of the EGF receptor by EGF results in the formation of an asymmetric kinase domain dimer
1. Kinase active site blocked by activation loop
2. Asymmetric kinase dimer removes activation loop by a conformational change from the acceptor kinase active site
3. Active kinase phosphorylates tyrosine residues
Activation of the receptor creates docking sites for our downstream signaling intermediates. This allows activation of the MAP signaling cascade. This is a mitogen activated protein kinase signaling pathway
This involves a kinase cascade providing multiple points within the pathway where there can be amplification of a weak signal such as epidermal growth factor. The pathway also involves the second group of G protein, the small monomeric G proteins, in the form of Ras.
Explain how the EGF receptor recruits downstream signalling proteins.
Recruitment of downstream signalling proteins
The EGF receptor exists as monomers in the non-stimulated pathway. Also, the monomeric G protein Ras is inactive with GDP bound to the guanyl nucleotide-binding site of the single subunit G protein.
Stimulation through epidermal growth factor binding to the extracellular domains brings together the monomers to dimerize producing the asymmetric kinase which then phosphorylates many tyrosine residues in the cytosolic tale forming docking sites for downstream signaling intermediates. In this pathway, the next downstream signaling intermediate is GRB2; GRB2 and Sos (Son of sevenless) which are required for activation of the monomeric G protein Ras.
Describe the function of the cytosolic adaptor protein GRB2
Cytosolic adaptor protein GRB2
GRB2 is a very important signal intermediate because it’s what’s referred to as a cytosolic adaptor protein that can interact with two different signaling components (the RTK and Sos). It’s able to do that because it has two different Src Homology domains, SH2 and SH3, which can interact with the different signaling intermediates: SH2 domains can interact with the phosphorylated tyrosine residues on the receptor tyrosine kinase, SH3 domains can interact with Sos. Once Sos has been recruited in that manner it can interact the monomeric G protein Ras and therefore transduce signaling downstream through Ras.
How is Ras activated in the EGF signalling pathway
Recruitment of Sos by GRB2 promotes the exchange of GDP for GTP on the guanyl nucleotide-binding site of the Ras small monomeric G protein activating Ras switching signaling on. In this way, Ras is acting as a molecular switch in the same way as the heterotrimeric G proteins: off - GDP bound, on – GTP bound. Ras also has intrinsic GTPase activity, so as soon GTP is bound it begins to be hydrolyzed back to GDP switching off signaling. Different monomeric G proteins have different levels of GTPase activity such that they also act as molecular timers. This slide also shows the role of GEFs and GAPs in the G protein cycle which affect the rate/easy with which signaling is switch on or off by GDP for GTP:
* GEFs - guanine nucleotide exchange factors; promote the exchange of GDP for GTP switching signaling on.
GAPs - GTPase activating proteins; accelerate the rate at signaling is switch off by hydrolyzing GTP back to GDP.
Binding of Sos to the SH3 domains of GRB2 allows Sos to affect the interaction of the guanyl nucleotide, GDP or GDP, in the guanyl nucleotide-binding region of Ras. There are two regions within Ras referred to a switch I and switch II:
* Ras-GDP: Switch I & II do not interact directly with GDP
* Ras-Sos: Sos “pries” Ras open, GDP to diffuse out
* Ras-GTP: Sos is displaced, Switch I & II interact with GTP
Therefore, Sos is a GEF which promotes the exchange of GDP for GTP switching on Ras and switching on downstream signaling. Ras-GTP diffuses from Sos to activate further downstream signaling intermediates.
Ras is particularly important in the context of colorectal cancer, which is responsible for many deaths, ~50,000 in the USA per year. In 50% of those, tumors are associated with a single mutation in Ras. Therefore, it’s critical to have this signaling pathway functioning correctly to avoid uncontrolled proliferation of cells and the potential development of tumors such as colorectal cancers.
Describe the activation of Raf.
Ras when activated triggers a kinase cascade in this signaling pathway. The first kinase activated by Ras, is a kinase called Raf. In the un-stimulated form Raf is associated with a protein called 14-3-3. 14-3-3 proteins are commonly involved protein-protein interactions regulating the activity of different proteins. When Raf is associated with the 14-3-3 protein through phosphorylation of two serine’s, Raf is not active. However, Ras can then interact with Raf at the N terminal domain causing dephosphorylation of one of the serine residues which bind Raf to the 14-3-3 protein by cytosolic phosphatases. This allows the Ras-Raf complex to dissociate from the regulatory 14-3-3 protein leading to the partial activation of Raf. It’s not completely activated, however, until it dissociates from Ras following the hydrolysis of GTP back to GDP on Ras releasing the active Raf which undergoes dimerization and phosphorylation of one monomer by the other activating the kinase.
* Activated Raf kinase can phosphorylate and activate the next downstream signaling intermediate this pathway. Raf is a serine/threonine kinase which phosphorylates serine and threonine residues on a kinase which is called MEK activating MEK.
* MEK is a tyrosine and serine/threonine kinase which preferentially phosphorylates tyrosine-185 & threonine-183 on the activation loop another kinase which is referred to as MAP kinase. Phosphorylation of these residues causes a conformational change which brings the activation lip out of the catalytic site bringing about activation of the MAP kinase.
The MAP kinase ERK is a serine and/threonine kinase. This kinase cascade is referred to as a MAP kinase signaling pathway:
* ERK – is a MAP (mitogen activated protein) kinase – a MAPK
* MEK – phosphorylates MAPK and is therefore a MAP kinase kinase – MAPKK
* Raf - phosphorylates MAPKK and is therefore a MAP kinase kinase kinase – MAPKKK
Describe the MAP kinase signalling pathway and give an example.
The MAP kinase ERK is a serine and/threonine kinase. This kinase cascade is referred to as a MAP kinase signaling pathway:
* ERK – is a MAP (mitogen activated protein) kinase – a MAPK
* MEK – phosphorylates MAPK and is therefore a MAP kinase kinase – MAPKK
* Raf - phosphorylates MAPKK and is therefore a MAP kinase kinase kinase – MAPKKK
Activation of ERK, the MAP kinase, causes dimerization fully activating the MAP kinase which can then phosphorylate and activate the next downstream signaling intermediate which is another kinase called P90. MA kinase phosphorylates P90 on the activation lip causing at conformational change, removing the occlusion from the catalytic site, and activating the kinase activity. The dimer of MAPK and the active P90 can then diffuse into the nucleus where they phosphorylate two transcription factors: TCF (Ternary Complex Factor) and SRF (Serum Response Factor). Once those transcription factors have been phosphorylated, they combined to a promoter element, the serum response element, in the promoter of genes such as cfos, promoting the transcription of those genes, and therefore ultimately promoting gene expression, cell proliferation, development, differentiation etc. This MAP kinase cascade therefore results in the final end response, changes in gene expression, leading to physiological, biochemical, and developmental changes.
Activation of Raf (MAPKKK):
Activated receptor signals through intermediaries to activate Raf (MAP kinase kinase kinase), often through Ras activation.
Activation of MEK (MAPKK):
Raf phosphorylates and activates MEK (MAP kinase kinase), which is downstream of Raf in the pathway.
Activation of ERK (MAPK):
MEK phosphorylates and activates ERK (MAP kinase), the final kinase in the cascade.
Dimerization and Activation of ERK:
Active ERK molecules form dimers, which are the fully activated forms of the kinase.
Phosphorylation of Downstream Targets:
Active ERK dimers phosphorylate various downstream targets, including other kinases and transcription factors.
Activation of P90:
ERK phosphorylates another kinase, P90, inducing a conformational change that activates its kinase activity.
Nuclear Translocation:
Active ERK dimers and active P90 dimers translocate into the nucleus.
Phosphorylation of Transcription Factors:
ERK and P90 phosphorylate transcription factors such as TCF and SRF.
