Tyrosine Kinases Flashcards
What is the similar molecular architecture of all RTKs
A ligand binding region in the extracellular domain, a single transmembrane helix, and a cytoplasmic region that contains the protein tyrosine kinase domain plus additional carboxy terminal and juxtamembrane regulatory regions.
Which amino acids are capable of being phosphorylated and why?
Serine, Threonine and tyrosine - because they have a free hydroxyl group allowing transfer of the phosphate.
What is the role of EGF
Stimulates proliferation of various cell types
What is the role of the insulin receptor
Stimulates carbohydrate utilisation and protein synthesis
What is the role of the IGF receptor
Cell growth and survival
What is the role of Trk A receptor
NGF binds, stimulating survival and growth of some neurons
What is the role of the VEGF receptor
Angiogenesis - therefore receptor antagonists are a good target for cancer treatment.
What role does FGF have and what is the result of knocking it out in mice
Wound healing impaired in knockout mice. In vitro scratching of cells - healing takes longer in the FGF knockout cells.
How does nerve growth factor bind Trk A
It is a dimer and cross-links two TrkA molecules without any direct contact between the two receptors
How does stem cell factor bing KIT
Also cross-links between two KIT molecules, but also two Ig-like domains D4 and D5 which reorientate and interact upon ligand binding. Kit therefore combines ligand mediated as well as receptor mediated dimerization.
How does FGF bind to FGFR
Bind to one part of the receptor and induces a conformational change in the extracellular domain to increase its affinity for the neighbouring receptor/domain - The ligands dont interact
How does EGF bind ErbB/ EGFR
Dimerisation of ErbB is mediated entirely by the receptor. Binding simultaneously to two sites (DI and DIII) within the same receptor molecule. This causes a conformational change allowing for two extracellular domains to bind.
What is the general structure of the intracellular kinase domains
All TKDs have a small N lobe and a C lobe. Key regulatory elements such as the activation loop and the alphaC helix in the N lobe adopt a specific configuration in activated TKDs which is required for catalysis of phosphotransfer.
What is the structure of the insulin receptor
Exists as a pre-assembled receptor dimer composed of four subunits held together by disulphide bridges. However they are too far apart to cross-phosphorylate
What happens to the insulin receptor upon insulin binding
conformational change so cross-phosphorylation can occur because the tyrosine kinases are now close enough.
What is TKD cis-
autoinhibition by the activation loop and which receptors exhibit it
Insulin and FGF receptors: The activation loop of the receptor projects into the active site of its own kinase domain blocking access of both ATP and protein substrates.
How is cis autoinhibition relieved and what is the consequence of this
Insulin binding causes a key tyrosine (Y1162) on the activation loop in one TKD within the dimer to become phosphorylated by its partner. This trans-phosphorylation disrupts the cis-autoinhibitory interactions, the phosphorylated activation loop flips out of the molecule into the active state, freeing the ATP binding site and allowing for phosphorylation of downstream signalling proteins. (IRS)
What is juxtamembrane autoinhibition
sequences in the juxtamembrane region make extensive contacts with several parts of the TKD, including the activation loop, and stabilize and autoinhibitory conformation, where the C lobe is flipped back and blocks the binding site.
what is the result of disrupted juxtamembrane autoinhibition
Mutations in the juxtamembrane domain result in a constitutively active RTKs in KIT/PDGFR families. Frequently resulting in cancer.
Is there an activation loop present on the EGFR
No
What is the result of EGF binding on the TKDs
Extracellular dimerisation means TKDs come into close contact and there is an allosteric effect between the two domains., it is an asymmetric dimer.
What is the activator receiver model of EGFR activation
The C-lobe of one TKD (the activator) makes intimate contact with the N-lobe of the second TKD (the receiver). These contacts induce a conformational change in the N-lobe of the receiver kinase that disrupt cis-autoinhibitory interactions seen in the monomer.
What is an important characteristic of EGFRs as a result of the receiver activator model.
The receiver kinase can adopt the active configuration without activation loop phosphorylation.
How are EGFR/ErbR compromised in cancers.
Oncogenic mutations causing disruption to cis-autoinhibitory interactions means that the receptor can be activated without the need for ligand binding.
What is the role of the phosphorylation sites not found at the activation loop and cytoplasmic domain.
Act as docking sites for cytosolic downstream signalling molecules which become translocated to the plasma membrane.
What is the result of C terminal tail phosphorylation of the EGFR
Acts an anchor for downstream signalling molecules
What is an SH2 domain
Found on signalling proteins, the SH2 domain is capable of recognising and binding to phosphorylated tyrosine residues
What is an SH3 domain
A domain capable of binding proline rich regions
What part of the SH2 domain is responsible for binding
The phosphate group makes a tight bond with arginine found on the SH2 domain.
What is GRB2
Signalling molecule formed of one SH2 and two SH3 domains.
How does EGFR activation lead to activation of the RAS MAPK pathway
Activated EGFR has phosphorylated sites on its cytosolic domain. SH2 domain of GRB2 binds to these sites. GRB2 then interacts with Sos via its SH3 domain is translocated to the plasma membrane. Sos is a guanine exchange factor, RAS which is membrane bound. Guanine exchange causes RAS to change from its GDP bound inactive state, to its GTP bound active state and subsequent MAPK downstream signalling.
How was the EGF/RAS activation pathway discovered?
Drosophila used as a model system - specifically the eyes.
What role do RTKs have in rhabdomere selection
The RTK sevenless is dedicated to R7 regulation - flies with a sevenless mutation lack R7 cells in their eyes.
How do Boss and Sevenless interact and what is the consequence of this
Boss is found on R8 cells and binds to Sev on the potential R7 cell. Activated Sev activates Sos in a similar way to EGF signalling and also therefore activates RAS. Sev also inhibits the activity of GAP which reverts Ras back to inactive state. Downstream signalling leads to the induction of the R7 cell.
What is the result of a double mutant fly expressing no Sev but expressing RasD
Constitutively active RasD means that even without ligand binding to sev, the cell still produces the downstream Ras signalling cascade so the R7 cell is still formed.
What is the neurotrophic hypothesis
That targets of innervation secrete a limited amount of survival factors which generates a balance between the size of the target organ and the number of innervating neurons.
What is the result of the removal of a limb bud in chicks
Reduced number of sensory and motor neurons in the spinal cord, far away from the limb bud
What could be the cause of reduced neurons in the spinal cord following limb bud removal and how was this tested
Maybe there is a factor secreted from the limb bud that promotes survival. Tested by adding an extra limb bud which caused an increase in motor neurons within the spinal cord.
How is a neurite growth assay performed
Add NGF to a ganglion and observe neurite growth
What ligands bind to TrkA
NGF, NT3
What ligands bind to TrkB
BDNF, NT-3 and NT4/5
What ligands bind to TrkC
NT-3
What signal transduction is produced by Trk receptor activation
Similar to EGFR pathway - PI3K activates AKT, Ras activates MAPK
What do PC12 cells express and respond to
Express both EGFR and Trk receptors and respond to both EGF and NGF
What does NGF binding lead to
Differentiation of the cells and the cells shape changes, inducing neurite formation
What does EGF binding lead to
Proliferation of PC12 cells
Why is it surprising that the binding of NGF and EGF to their receptors has different consequences
Because they both activate the same pathways (MAPK, PLCy and PI3K)
Why is there a difference in the effects caused by EGF and NGF binding
Due to many positive and negative feedback loops interwoven with each other.
What happens to the EGFR after binding
It is endocytosed and transported to the lysosome
What happens to the Trk receptors after NGF binding
It is endocytosed but then recycled to the plasma membrane to be reused.
What is the outcome of EGFR translocation to the lysosome on the cell on MAPK signalling
Both drive MAPK signalling initially, but over time, EGF signalling only transiently activates MAPK whereas NGF activates MAPK much stronger over time. EGF does not enable MAPK to enter the nucleus whereas NGF does, causing phosphorylation of TFs.
What is the NGF PKC relationship
NGF activates PKC creating a positive feedback loop - This phosphorylates a RAF kinase inhibitor leading to its dissociation - therefore a longer lasting RAF activation is seen.
What is the EGF RAF relationship
Opposite to NGF -short MAPK activation leads to inactivation of RAF
What is the result of MAPK in the nucleus
It stabilizes FOS, a TF leading to neurite outgrowth
What protein binds to the endocytosed EGFR
ubiquitinated EGFR will be sorted into intraluminal vesicles of the MVB
What is the MVB
multivesicular body which will mature to become the lysosome
What is the protein that PKC phosphorylates
RKIP (RAF kinase inhibitory protein) RAF can then be phosphorylated leading to a positive feedback loop
What does the transient EGF/ERK activation cause
Expression of MAPK specific phosphatases which dephosphorylate ERK and will eliminate the positive feedback loop.
How does NGF stabilize FOS
ERK is allowed into the nucleus due to its high activation by NGF. ERK in the nucleus phosphorylates FOS and leads to downstream gene expression, leading to neuron outgrowth and differentiation.
What enzymes carry out ubiquitination?
E1,2,3
What are DUBS
deubiquitinating enzymes - they are proteases
What does Ub predominantly conjugate to
Lysine, and rarely methionine
How is polyUb formed
Lysines and amino terminal methionine in the Ub molecule are capable of being conjugated by another Ub molecule leading to the formation of PolyUb chains
How does the specific residue that links the Ub chain affect its structure
The protein will fold differently and can be recognised by specific Ub binding domains with distinct functions
What is the conformation of K68 linked Ubchains
Extended, open conformation with high conformational freedom
What is the conformation of K48 linked chains
Have a closed conformation
Which VEGFR is crucial for vascular formation
VEGF-R2
What tyrosine residue is phosphorylated in VEGF-R2 and what is the result of this
Y1175 - in the cytoplasmic tail of the receptor. Crucial for vascular formation, mutation in Y1175 is embryonic lethal
What is synectin
A PDZ domain containing scaffold protein
What is the role of synectin
Binds to myosin-VI, a retrograde motor involved in endosome transport - by doing so it could lead to the degradation of a newly internalised VEGF-R2 and regulation of its signalling.
What is the effect of a synectin KO on a cells ability to react to VEGF stimulation
Significantly reduced sensitivity to VEGF
What is the result of a mouse KO and a zebrafish KD of synectin
Results in unique vascular phenotype characterised by decreased arterial vasculature size and branching complexity.
