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Flashcards in Tyrosine Kinases Deck (72)
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1

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.

2

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.

3

What is the role of EGF

Stimulates proliferation of various cell types

4

What is the role of the insulin receptor

Stimulates carbohydrate utilisation and protein synthesis

5

What is the role of the IGF receptor

Cell growth and survival

6

What is the role of Trk A receptor

NGF binds, stimulating survival and growth of some neurons

7

What is the role of the VEGF receptor

Angiogenesis - therefore receptor antagonists are a good target for cancer treatment.

8

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.

9

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

10

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.

11

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

12

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.

13

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.

14

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

15

What happens to the insulin receptor upon insulin binding

conformational change so cross-phosphorylation can occur because the tyrosine kinases are now close enough.

16

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.

17

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)

18

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.

19

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.

20

Is there an activation loop present on the EGFR

No

21

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.

22

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.

23

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.

24

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.

25

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.

26

What is the result of C terminal tail phosphorylation of the EGFR

Acts an anchor for downstream signalling molecules

27

What is an SH2 domain

Found on signalling proteins, the SH2 domain is capable of recognising and binding to phosphorylated tyrosine residues

28

What is an SH3 domain

A domain capable of binding proline rich regions

29

What part of the SH2 domain is responsible for binding

The phosphate group makes a tight bond with arginine found on the SH2 domain.

30

What is GRB2

Signalling molecule formed of one SH2 and two SH3 domains.

31

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.

32

How was the EGF/RAS activation pathway discovered?

Drosophila used as a model system - specifically the eyes.

33

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.

34

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.

35

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.

36

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.

37

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

38

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.

39

How is a neurite growth assay performed

Add NGF to a ganglion and observe neurite growth

40

What ligands bind to TrkA

NGF, NT3

41

What ligands bind to TrkB

BDNF, NT-3 and NT4/5

42

What ligands bind to TrkC

NT-3

43

What signal transduction is produced by Trk receptor activation

Similar to EGFR pathway - PI3K activates AKT, Ras activates MAPK

44

What do PC12 cells express and respond to

Express both EGFR and Trk receptors and respond to both EGF and NGF

45

What does NGF binding lead to

Differentiation of the cells and the cells shape changes, inducing neurite formation

46

What does EGF binding lead to

Proliferation of PC12 cells

47

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)

48

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.

49

What happens to the EGFR after binding

It is endocytosed and transported to the lysosome

50

What happens to the Trk receptors after NGF binding

It is endocytosed but then recycled to the plasma membrane to be reused.

51

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.

52

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.

53

What is the EGF RAF relationship

Opposite to NGF -short MAPK activation leads to inactivation of RAF

54

What is the result of MAPK in the nucleus

It stabilizes FOS, a TF leading to neurite outgrowth

55

What protein binds to the endocytosed EGFR

ubiquitinated EGFR will be sorted into intraluminal vesicles of the MVB

56

What is the MVB

multivesicular body which will mature to become the lysosome

57

What is the protein that PKC phosphorylates

RKIP (RAF kinase inhibitory protein) RAF can then be phosphorylated leading to a positive feedback loop

58

What does the transient EGF/ERK activation cause

Expression of MAPK specific phosphatases which dephosphorylate ERK and will eliminate the positive feedback loop.

59

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.

60

What enzymes carry out ubiquitination?

E1,2,3

61

What are DUBS

deubiquitinating enzymes - they are proteases

62

What does Ub predominantly conjugate to

Lysine, and rarely methionine

63

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

64

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

65

What is the conformation of K68 linked Ubchains

Extended, open conformation with high conformational freedom

66

What is the conformation of K48 linked chains

Have a closed conformation

67

Which VEGFR is crucial for vascular formation

VEGF-R2

68

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

69

What is synectin

A PDZ domain containing scaffold protein

70

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.

71

What is the effect of a synectin KO on a cells ability to react to VEGF stimulation

Significantly reduced sensitivity to VEGF

72

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.