Receptor Tyrosine Kinases

Question 1

Enzyme coupled receptors

Answer 1

• ligand binding to receptor activates intrinsic catalytic activity
• catalytic activity associated permanently with receptor
• eg. receptor tyrosine kinases

Question 2

Receptor Tyrosine Kinases

Answer 2

• 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

1. TM monomers are inactive RTKs
2. signal protein causes dimerisation and kinase domain activation where each monomer phosphorylates each other
3. trans-autophosphorylation of tyrosines generates binding sites for signal proteins

Question 3

Human RTKs

Answer 3

• 58 RTKs encoded
• variable binding domain architecture
• conserved cytoplasmic domain
• eg. EGF, NGF, FGF, insulin

Question 4

RTKs in cancer

Answer 4

• 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

Question 5

Studying RTK Signalling

Answer 5

• 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

Question 6

RTK Activation

Answer 6

• 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

Question 7

EGF Receptor

Answer 7

• EGF is a monomer

- when binding to monomeric EGF receptor, a dimerisation arm is exposed promoting EGFR dimerisation

Question 8

Insulin Receptor

Answer 8

• 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

Question 9

Heparan sulphate

Answer 9

• 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.

Question 10

Insulin Receptor Kinase Activation

Answer 10

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

Question 11

Activation Loop

Answer 11

• 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

Question 12

EGF receptor tyrosine kinase

Answer 12

• 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

Question 13

Docking sites

Answer 13

• 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

Question 14

SH2 Domains

Answer 14

• 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

Question 15

Src Tyrosine Kinase

Answer 15

• 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

Question 16

Rous Sarcoma Virus

Answer 16

• oncovirus
• viral oncogene v-src encodes truncated version (missing 10-20 residues) of normal cellular tyrosine kinase
• constitutively active

Question 17

Src Kinase Activation

Answer 17

• 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

Question 18

Ras-MAP Kinase Pathway

Answer 18

• MAPK = mitogen activated protein kinase

- pathway central to regulation of cell growth and proliferation

Question 19

Ras

Answer 19

• 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

Question 20

Rho GTPase

Answer 20

• 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

Question 21

SOS

Answer 21

• 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

Question 22

MAP Kinase Pathway

Answer 22

• 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

Question 23

Scaffold Proteins

Answer 23

• physically organise MAP kinase modules
• tethers together MAP kinases
• prevents cross talk. between parallel MAP kinase modules
• increases specificity but loses amplification properties

Question 24

Erk

Answer 24

• 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

Question 25

Cell Division and MAP Kinase Path

Answer 25

• mitogen signalling
• activates Erk MAPK
• slow: Fos 1 expression
• fast: phosphorylation and stabilization of Fos1 protein
• only prolonged signal will trigger division

Question 26

Erk Negative Feedback

Answer 26

• phosphorylation of Sos by Erk breaks Grb2-Sos interaction
• Erk turns off Gef leading to Ras activation
• Erk also transcribes a phosphatase dephosphorylating Erk

Question 27

Cytokine Signalling

Answer 27

• 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

Question 28

Transforming growth factor-B

Answer 28

• 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.

Question 29

PIP2 Cleavage

Answer 29

• 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

Question 30

PIP3

Answer 30

• phosphoinositide 3-kinase produces PIP3
• adds a phosphate group onto the C-3 of PIP2
• PIP3 contains 3 phosphates and a phosphodiester linkage

Question 31

Pleckstrin homology domain

Answer 31

• 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

Question 32

PI3K/Akt Pathway

Answer 32

• 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

Question 33

PTEN phosphatase

Answer 33

• reverses action of PI 3-kinase

- removes C3 phosphate off PIP3 to form PIP2

Question 34

Termination of RTK Signalling

Answer 34

1. dephosphorylation
2. endocytosis
   - receptor recycling/degradation

Question 35

EGF Receptor Ubiquination

Answer 35

• E3 ligase Cbl ubiquinates activated EGF receptor

- signal for internalisation

Question 36

Crosstalk of GPCR/RTK Signalling pathways

Answer 36

• both activate phospholipase C
• affect same target proteins
• GPCR arrestin affects Ras
• RTK pTyr affects G proteins

Question 37

Cell proliferation vs. Differentiation

Answer 37

• 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