Tyrosine Kinase receptor signalling 2

Question 1

How do EGF receptors dimerise

Answer 1

1. Binding of an EGF molecule to EGFR induces a conformational change that allow association of two activated receptors.
2. So can undergo transautophosphorylation

Question 2

How do INSR receptors dimerise

Answer 2

1. The INSR has a homodimeric structure in which the intracellular domains are held apart (in a u shape) in the absence of ligand
2. Insulin binding induces a conformational change (inverted U to T shape) that brings the kinase domains together
3. Each receptor can bind up to 4 insulin molecules

Question 3

What happens downstream of activated RTKs?

Answer 3

1. Phosphorylates phosphatidylinositol substrates: cell survival pathway
2. Inactivates Ras/MAP-kinase pathway
3. Activates inositol phospholipid pathway (cross-talk with GPCR signalling)
4. Note that many

Question 4

Give examples of signalling molecules that interact with the activated RTKs that are not protein kinases.

Answer 4

1. PI3-kinase is a lipid kinase,
2. Phospholipase C catalyses hydrolysis of inositol phospholipids
3. while GAP has no intrinsic enzyme activity but through stimulating the Ras GTPase can inhibit or reverse MAP kinase signalling

Question 5

How do some key signalling proteins interact indirectly with RTKs

Answer 5

1. through adaptor proteins such as Grb2
2. Adaptor proteins contain no catalytic domains and serve as a link between receptor and other intracellular signalling proteins
3. Adaptor proteins serve a function similar to scaffold proteins in bringing specific proteins into proximity with the activated RTK.
4. Sub-cellular localisation, or concentration, of proteins at the plasma membrane is important for efficient function of signalling systems.

Question 6

Give an example of a key signalling proteins that interacts indirectly with RTKs

Answer 6

1. e.g. Guanine nucleotide releasing protein (GNRP) or Ras-guanine nucleotide exchange factor (Ras-GEF)
2. GNRP/Ras-GEF activates the Ras monomeric GTPase

Question 7

Give examples of the small monomeric GTPase superfamily (As opposed to trimeric GTPases involved in GPCR signalling)

Answer 7

1. Ras
   a) H-Ras, N-Ras, K-Ras
   b) Relay RTK signals (cell prolif.)
2. Rheb
   a) Rheb
   b) mTOR activation (cell growth)
3. Rho
   a) Rho, Rac, Cdc42
   b) Receptors to cytoskeleton
4. Rab
   a) Rab1-60
   b) Intracellular vesicle trafficking & assembly
5. ARF
   a) ARF1-6
   b) Intracellular vesicle trafficking & assembly

Question 8

What activates the monomeric GTPase Ras

Answer 8

1. GNRP/Ras-GEF activates the monomeric GTPase Ras

Question 9

What does inhibition and mutation of Ras genes lead to

Answer 9

1. Inhibition of Ras function can block cell proliferation.
2. Conversely, mutations in Ras genes leading to production of constitutively active Ras protein occur in around 30% of human cancers and are a key step in tumour progression.

Question 10

How is Ras activated

Answer 10

1. Note that Ras is tethered to the inner leaflet of the plasma membrane by lipid side-chains, where it is needed for interaction with RTKs.
2. Recruitment of Grb2 and Ras-GEF to the activated receptor brings them into proximity with Ras molecules.
3. In its inactive state Ras is associated with a molecule of GDP.
4. Interaction of Ras-GEF with Ras is sufficient to cause a conformational change that promotes release of GDP by Ras
5. instead, Ras acquires GTP molecule (since GTP is more abundant in the cytoplasm than GDP) and undergoes a conformational change that activates it.

Question 11

How does Ras act as a molecular switch

Answer 11

1. Lipid side-chains anchor Ras to plasma membrane
2. GEF/GNRP promotes GDP release (activating Ras)
3. Interaction of inactive RAS with receptor-bound GEF induces a conformational change in Ras that causes GDP release.
4. GAPs promote Ras GTPase activity (inactivating Ras through GTP hydrolysis)
5. Interaction of (receptor-bound) GAP with active Ras stimulates the intrinsic Ras GTPase activity, leading to GTP hydrolysis, converting it to GDP (releasing a phosphate) and returning Ras to the inactive state.

Question 12

Describe (FRET) experiments used to show activation and inactivation of Ras

Answer 12

1. Tyrosine-specific protein phosphatases reverse RTK phosphorylation
2. Activated Ras hydrolyzes GTP to GDP, rapidly inactivating Ras as shown by fluorescence resonance energy transfer (FRET) experiments
3. Ras-GTP formation and breakdown can be followed in real time using fluorescence microscopy.
4. In this experiment cells are engineered to express Ras tagged with a yellow fluorescent protein, that emits light at a wavelength of 528 nm.
5. The cells are given GTP tagged with a red fluorescent dye and when this comes into very close proximity to the YFP tag, energy is transferred from YFP to the dye such that light is now emitted at 617 nm in a process termed Fluorescence resonance energy transfer (FRET)

Question 13

What are the reversible conformational change switches in signalling pathways

Answer 13

1. Signalling by GTP-binding protein
2. Signalling by phosphorylation
3. Signalling by molecular interaction or contact

Question 14

How does Ras activate a cascade of Serine/Threonine-kinases

Answer 14

1. Ras activates a cascade of Serine/Threonine-kinases through protein-protein interaction with MAP-kinase-kinase-kinase (Raf)
2. MAP= mitogen activated protein

Question 15

Describe the cascade of Serine/Threonine-kinases

Answer 15

1. Raf phosphorylates Mek
2. Mek phosphorylates Erk- specificity
3. Erk phosphorylates many different targets
4. Can vary between different cells
5. Different cellular responses
6. Phosphorylate proteins such as transcription factors- change gene expression

Question 16

What does the Serine/Threonine-kinase cascade allow

Answer 16

1. This kinase cascade allows for signal amplification, sustaining the signal in order to elicit a response.
2. It also allows for cross talk between signalling pathways.
3. Here, the pathway interfaces the cell cycle since many of the protein targets are those involved in the transition from G1 to S-phase of the cell cycle
4. thus, growth factors can stimulate production of cyclins and block/degrade INK and KIP cyclin inhibitory proteins.

Question 17

Describe how fly genetic experiments discovered many components of the Ras-MAPK pathway

Answer 17

1. Experiments from the group of Ernst Hafen,
2. based on the inability of mutant flies to detect UV light,
3. driven by wanting to know which genes regulate eye development.
4. Look for genes which affect eye development
5. Look at eye for developmental defects
6. Comprises ~800 identical ommatidia, each with a lens focussing light onto 8 photoreceptor cells
7. Photoreceptor cells develop sequentially from unspecialized epithelial cells
8. First R8, Then R2 and 5
9. These help to induce differentiation of R3 and 4
10. Then R6 and 1
11. Then R7
12. Sevenless” mutants lack R7
13. The mutation in Sev flies was found to inactivate a RTK (similar to mammalian EGFR).
14. Consequently, the researchers figured there must be other signalling molecules involved, including a ligand.
15. Ligand- Boss- bride of sevenless
16. Ras-GEF- Sos – son of sevenless

Question 18

Give a summary of the RTK downstream signalling

Answer 18

1. MAPK pathway important for cell proliferation in many cells (but also cell fate decisions during development (as in the fly eye).
2. Survival also needs to be instructed and a well known pathway for this involves PI3-kinase signalling.
3. PI3-K was one of the other SH2-domain proteins that interacts with activated RTKs.

Question 19

What is PI3-kinase

Answer 19

1. PI3-kinase phosphorylates phosphatidylinositol substrates
2. PI3-kinase is a lipid kinase that forms products with phosphate groups at the third position on the 6-carbon inositol ring.

Question 20

What does PI3-kinase do

Answer 20

1. In addition to PI3-kinase being rapidly recruited to the activated RTK, PDK1 and PKB are recruited to the membrane through interactions between their PH domains and inositol phospholipid products of PI3-kinase.
2. This brings them into close proximity and PDK1 (activated through conformational change upon binding to PIP3) then phosphorylates and activates Akt (PKB).
3. This alters the conformation of Akt which then dissociates from PIP3 and can phosphorylate the target protein BAD.
4. BAD is normally in association with death inhibitory protein, keeping it inactive.
5. Upon being phosphorylated, BAD undergoes conformational change and releases death inhibitory protein (instead it associates with 14-3-3 protein), to prevent apoptosis.
6. Thus PI3-kinase signalling is required for cell survival which, perhaps surprisingly is an active process, while apoptosis is the default state.

Question 21

What are scaffolding proteins

Answer 21

1. Hold together intracellular signalling components to increase efficiency and ensure specific response to different signals within the same cell