Receptor Tyrosine Kinases Flashcards
1
Q
Enzyme coupled receptors
A
- ligand binding to receptor activates intrinsic catalytic activity
- catalytic activity associated permanently with receptor
- eg. receptor tyrosine kinases
2
Q
Receptor Tyrosine Kinases
A
- RTKs are single span transmembrane proteins with an extracellular ligand binding domain and an intracellular tyrosine kinase domain
- ligand binding results in trans-autophosphorylation of the receptor
- TM monomers are inactive RTKs
- signal protein causes dimerisation and kinase domain activation where each monomer phosphorylates each other
- trans-autophosphorylation of tyrosines generates binding sites for signal proteins
3
Q
Human RTKs
A
- 58 RTKs encoded
- variable binding domain architecture
- conserved cytoplasmic domain
- eg. EGF, NGF, FGF, insulin
4
Q
RTKs in cancer
A
- RTKs frequently over-expressed and mutated in cancer
- Trastuzumab is a monoclonal antibody that interferes with Her2 (EGF receptor)
- treats breast and stomach cancer
- Gefitinib is a tyrosine kinase inhibitor that is specific for EGF receptors
- treat cancers with over-expressed EGFRs
5
Q
Studying RTK Signalling
A
- receptor activation is commonly studied by Western blotting using anti-pTyr antibodies
- this presence is the hallmark of activation (more ligand concentration will give more pTyr)
- cellular responses can be monitored in a number of ways by quantifying DNA synthesis or observation of cellular behaviour
- first growth factors discovered by the fact that transplanted tissue contained ligand causing nerve cell growth to it
6
Q
RTK Activation
A
- RTKs activated by ligand induced proximity of kinase domains
- many RTKs are dimerised by their ligands and some are dimers on their own and proximity of the kinase domains in the RTK dimer favours trans-autophos.
- the phos. generates the binding sites for signalling proteins
7
Q
EGF Receptor
A
- EGF is a monomer
- when binding to monomeric EGF receptor, a dimerisation arm is exposed promoting EGFR dimerisation
8
Q
Insulin Receptor
A
- receptor is a covalent disulphide linked dimer of 4 chains
- insulin brings the intracellular domains together in an active form
- dimer transmits the conformational signal
- some RTKs are non covalently associated in ligand absence, some require higher order clustering, some required co-receptors
9
Q
Heparan sulphate
A
- co-receptor for fibroblast growth factor
- repeating disaccharide of N-acetylglucosamine and glucuronic acid (sulfated sugar abundant on cell surfaces)
- The signalling complex at the cell surface is believed to be a ternary complex formed between two identical FGF ligands, two identical FGFR subunits, and either one or two heparan sulfate chains.
10
Q
Insulin Receptor Kinase Activation
A
Inactive:
- autoinhibited by activation loop with Tyr1162 adopting a pseudosubstrate conformation
Active:
- activation loop no longer blocks the active site and its 3 Tyr residues are phosphorylated.
- substrate Tyr occupies the former position of Tyr1162 positioned in a way to accept the P from ATP
11
Q
Activation Loop
A
- unphosphorylated activation loop is predominantly in the inactive conformation
- small % of loop in active conformation
- if another kinase with ATP is near it may be activated
- for transphosphorylation to occur two kinase domains with the activation loop in the active conformation have to meet
- once phosphorylated in activation loop, the kinase domain is locked in the active conformation (until P is removed)
eg. FGFR kinase
- FGFR acts as enzyme and substrate
- Tyr of activation loop positioned to be phosphorylated
12
Q
EGF receptor tyrosine kinase
A
- activated allosterically
- inactive monomer with C terminal kinase domain
- activation loop is never phosphorylated (ejected out physically)
- activator kinase domain in the asymmetric EGFR dimer allosterically activates the receiver kinase
- EGFR auto-phos. occurs in the C-terminal tail and not in the activation loop
13
Q
Docking sites
A
- phosphorylated tyrosine residues are docking sites for a variety of intracellular signalling proteins
eg. PLC-y - produces IP3, diacylglycerol
- activates PKC and Ca ion release from ER
eg. Phosphoinositide 3-kinase - phosphorylates lipid head groups that are docking sites for effectors
eg. non-enzymatic adaptors - lead to GTPase Ras activatoin and downstream signalling MAP kinase pathway
14
Q
SH2 Domains
A
- recognise p-Tyr
- Src homology region 2 domains mediate pTyr binding
- SH2 domains have apolar specificity pocket for the residue in the +3 position
- contains both specificity pocket and conserved pocket for pTyr binding
- negative residue binds positive phosphate
- SH2 domains bind to phosTyr (eg. Phospholipase C-y that also activates inositol phospholipid signal pathway to activate PKC)
- modular architecture of signalling proteins enhances specificity and allows formation of multi-protein signalling complexes
15
Q
Src Tyrosine Kinase
A
- non-receptor tyrosine kinase protein that in humans is encoded by the SRC gene
- includes an SH2 domain, an SH3 domain and a tyrosine kinase domain
- c-Src phosphorylates specific tyrosine residues in other tyrosine kinases. It plays a role in the regulation of embryonic development and cell growth
16
Q
Rous Sarcoma Virus
A
- oncovirus
- viral oncogene v-src encodes truncated version (missing 10-20 residues) of normal cellular tyrosine kinase
- constitutively active
17
Q
Src Kinase Activation
A
- inactive Src is autoinhibited allosterically by intramolecular interactions involving the SH2 & SH3 domains
- activation is caused by dephosphorylation of a regulatory pTyr site in the C-terminal tail by the SH2 domain
- SH3 domain is specific for polyproline sequences
- active form has C helix down and the activation loop phosphorylated
18
Q
Ras-MAP Kinase Pathway
A
- MAPK = mitogen activated protein kinase
- pathway central to regulation of cell growth and proliferation
19
Q
Ras
A
- monomeric small GTPase anchored to inner leaflet of membrane via PTM
- 2 states: active GTP bound vs. inactive GDP bound
- Ras-GEFs activate while Ras-GAPs increase GTP hydrolysis
- Indirect coupling of receptor to Ras-GEF drives Ras into active state
- the switch helix is one of the regions that changes most between the GDP and GTP bound state
- in the GTP bound state of Ras the switch helix interacts with Ras effector
20
Q
Rho GTPase
A
- regulate actin dynamics/relays signals to cytoskeleton
- constitutively active forms of the GTPases were microinjected into fibroblasts and stained : showing hyperactive fibres and cytoskeleton
- but this may be artefacts not reflecting 3D cell dynamics
21
Q
SOS
A
- another protein, now called Grb2, which is an adaptor protein that links the Sev receptor to the Sos protein; the SH2 domain of the Grb2 adaptor binds to the activated receptor, while its two SH3 domains bind to Sos. Sos then promotes Ras activation.
- Sos (a Ras-GEF) activates Ras by promoting GTP exchange
- Biochemical and cell biological studies have shown that Grb2 and Sos also link activated RTKs to Ras in mammalian cells, revealing that this is a highly conserved mechanism in RTK signaling
- Once activated, Ras activates various other signaling proteins to relay the signal downstream, as we discuss next
22
Q
MAP Kinase Pathway
A
- Ras GTP allosterically activates Raf
- Raf is a MAP kinase,kinase,kinase
- Raf phosphorylates Mek
- Mek is a MAP kinase,kinase
- Mek phosphorylates Erk
- Erk is a MAP kinase
- Erk phosphorylates effector proteins to change protein activity/gene expression
23
Q
Scaffold Proteins
A
- physically organise MAP kinase modules
- tethers together MAP kinases
- prevents cross talk. between parallel MAP kinase modules
- increases specificity but loses amplification properties
24
Q
Erk
A
- MAP kinase regulating gene transcription
- phosphorylates TF turning on relevant genes
- active dimeric MAP kinase and active p90 (ribosomal kinase)
- kinases enter nucleus and activate TF
- transcribe c-fos (immediate early gene) that forms AP-1 transcription factor
- AP-1 dependent genes are involved in cell growth/differentiation
25
Cell Division and MAP Kinase Path
- mitogen signalling
- activates Erk MAPK
- slow: Fos 1 expression
- fast: phosphorylation and stabilization of Fos1 protein
- only prolonged signal will trigger division
26
Erk Negative Feedback
- phosphorylation of Sos by Erk breaks Grb2-Sos interaction
- Erk turns off Gef leading to Ras activation
- Erk also transcribes a phosphatase dephosphorylating Erk
27
Cytokine Signalling
- kinase is a separate protein permanently associated with the receptor
- Cytokine binding alters the arrangement so as to bring two JAKs into close proximity so that they phosphorylate each other, thereby increasing the activity of their tyrosine kinase domains. The JAKs then phosphorylate tyrosines on the cytoplasmic tails of cytokine receptors, creating phosphotyrosine docking sites for STATs
- STAT proteins form dimers and enter nucleus to control gene expression
28
Transforming growth factor-B
- Act via receptor serine/threonine kinases and smads
- The TGFβ dimer promotes the assembly of a tetrameric receptor complex containing two copies each of the type-I and type-II receptors. The type-II receptors (ser/threo kinases) phosphorylate specific sites on the type-I receptors, thereby activating their kinase domains and leading to phosphorylation of R-Smads such as Smad2 and Smad3. Smads open up to expose a dimerization surface when they are phosphorylated, leading to the formation of a trimeric Smad complex containing two R-Smads and the co-Smad, Smad4. The phosphorylated Smad complex enters the nucleus and collaborates with other transcription regulators to control the transcription of specific target genes.
29
PIP2 Cleavage
- cleaved by PLC-y
- generates IP3 second messenger
- equivalent to the action of phosC-B downstream of GPCR
- PLC-y contains SH2 domains and is recruited to the activated RTK
- IP3 opens Ca gated ER channels
- Ca activated PKC
30
PIP3
- phosphoinositide 3-kinase produces PIP3
- adds a phosphate group onto the C-3 of PIP2
- PIP3 contains 3 phosphates and a phosphodiester linkage
31
Pleckstrin homology domain
- PIP3 recognised by PH domains (similar to SH2 domains/pTyr)
- becomes docking sites to recruit species
- uses the C3 phosphate to create specific binding interaction with Arg
32
PI3K/Akt Pathway
- regulates cell survival/growth
- survival signal input needed to keep living
1. RTK is activated and activates PI 3-kinase
2. kinase phosphorylates PIP2 to PIP3
3. PH domains of Akt/PDK bind to PIP3
4. phosphorylation and activation of Akt
5. active Akt dissociates and phosphorylates Bad
6. Bad is an inhibitory protein binding to apoptosis inhibitory protein
7. active apoptosis inhibitory protein promotes survival
33
PTEN phosphatase
- reverses action of PI 3-kinase
| - removes C3 phosphate off PIP3 to form PIP2
34
Termination of RTK Signalling
1. dephosphorylation
2. endocytosis
- receptor recycling/degradation
35
EGF Receptor Ubiquination
- E3 ligase Cbl ubiquinates activated EGF receptor
| - signal for internalisation
36
Crosstalk of GPCR/RTK Signalling pathways
- both activate phospholipase C
- affect same target proteins
- GPCR arrestin affects Ras
- RTK pTyr affects G proteins
37
Cell proliferation vs. Differentiation
- EGF and NGF both activate the Raf-Mek-Erk pathways
- negative feedback loop of EGFR produces transient activation of the MAPK pathway : cell proliferation
- ERK deactivates SOS to turn off Raf
- positive feedback loop of NGFR creates a bistable system : cell differentiation
- NGF activates PKC to activate Raf
- cutting the loops results in switching the outcome of the receptor
