Lecture 6 - Receptor Tyrosine Kinase Siganlling

Question 1

Examples of enzyme linked receptors?

Answer 1

TGF beta receptors and receptor tyrosine kinases.

Question 2

There are different classes of tyrosine kinase receptors. What do they all have in common?

Answer 2

They all have an intracellular kinase domain, which is activated by dimerisation of the receptor. The acyl domains very but all allow ligand binding.

Question 3

What activates the phospholipase C signalling pathway?

Answer 3

Activated by both GPCRs and RTKs. Therefore generates cross-talk between the two receptors.

Question 4

Enzymatic function of PLC?

Answer 4

1. Once activated, PLC hydrolyses the phospholipid phosphatidylinositol 4,5 -bisphosphate (PI4,5P2).
2. Hydrolysis products = 1,2 diacylglycerol and inositol triphosphate.
3. DAG remains in the membrane. IP3 is soluble and moves away, acting as a secondary messenger.
4. IP3 moves to the ER and activates IP3 gated calcium channels. They are opened, and calcium enters the cytosol.
5. Target of calcium and DAG is protein kinase C (PKC).

Question 5

PKC has 3 subgroups. Group 1 = conventional.

Answer 5

Alpha, beta and gamma. Activated by calcium and diacylglycerol.

Question 6

PKC has 3 subgroups. Group 2 = novel.

Answer 6

δ, ε, η, Ө. Activated by DAG only.

Question 7

PKC has 3 subgroups. Group 3 = atypical.

Answer 7

ζ. Activated by ceramide only.

Question 8

PKCs form complexes with (and phosphorylate) a range of proteins. Give an example.

Answer 8

RAF1, which is involved in the MAP kinase signalling pathway.

Question 9

How are RTKs activated?

Answer 9

1. Ligand binding causes dimerisation of the receptor.
2. Intracellular tyrosine kinases are activated through their phosphorylation.
3. Act as docking sites for various intracellular signalling proteins.

Question 10

Proteins that bind to phosphotyrosine have a shared phosphotyrosine domain. Name the two types of domain.

Answer 10

SH2 domain (src Homology domain) and PTB domain (phosphoptyrosine domain).

Question 11

The SH2 binding site shows differences in its sequence. Why?

Answer 11

Phosphotyrosine is only part of the recognition site –> also depends what is left and right of the pTyr in the AA sequence.

Question 12

In this lecture 4 proteins with SH2 domains are provided as examples as to how the structure of the SH2 domains differs. Explain how.

1. Shc
2. Lck
3. PLC-gamma1
4. Syp

Answer 12

1. Shc - needs a leucine or valine at the 3rd position after pTyr.
2. Lck - needs an isoleucine at the 3rd position after pTyr.
3. PLC - gamma1 - needs a hydrophobic residue at the 2nd position after pTyr.
4. Syp - needs specific residues up to 5 AA’s away from pTyr.

Question 13

Explain how the binding of insulin to its receptor generates phosphotyrosine docking sites.

Answer 13

Most of the phosphotyrosine docking sites generated by insulin are not found on the receptor itself, but on a specialised docking protein called Insulin Receptor Substrate-1 (IRS-1).

Question 14

What does IRS-1 stand for?

Answer 14

Insulin Receptor Substrate-1 (IRS-1).

Question 15

How is IRS-1 activated?

Answer 15

The activated receptor first phosphorylates its kinase domains, which then phosphorylate IRS-1 on multiple tyrosines, creating many docking sites.

Question 16

IRS-1 has a PTB domain. Why?

Answer 16

Uses it to specifically bind to pTyr of the IR.

Question 17

Explain the relationship between phosphoinositide kinase and IRS1.

Answer 17

• One of the signalling molecules recruited by IRS1 is phosphoinositide-3-kinase. Binds to IRS1 using its SH2 domain.
• Act on phosphatidylinositol through the addition of another phosphate.
• Commonly acts on phosphatidylinositol 4,5 - bisphosphate (PI(4,5)P2), forming phosphatidylinositol 3,4,5 -triphosphate.
• Also acts on phosphatidylinositol 4 phosphate, forming phosphatidylinositol 3,4 biphosphate.
• Catalytic subunit of PI3K = p110 = adds a phosphate to the 3 position.
• PI(3,4,5)P3 is a binding site for protein kinase B (PKB).

Question 18

How is PKB generated and activated?

Answer 18

Binds to the phosphatidylinositol using its PH domain. This partially activates the molecule. PKB is fully activated when it is phosphorylated by PDK1.

Question 19

Why is PKB a major downstream signalling target for insulin signalling?

Answer 19

PI3K is recruited to IRS-1, resulting in the formation of PI(3,4,5)P3, which acts as the binding site for PKB.

Question 20

mTOR is a kinase. What does mTOR stand for?

Answer 20

mammalian Target of Rapamycin.

Question 21

What does mTOR regulate?

Answer 21

It acts on basic metabolic functions: stimulates protein synthesis and mitochondrial function.

Question 22

mTOR receives inputs from IRS-1. What is it inhibited by?

Answer 22

1. Type 2 diabetes.
2. ER stress.
3. Inflammation.
4. Hypoxia.
5. Mitochondrial dysfunction.

Question 23

The drug rapamycin inhibits mTOR. It therefore has a strong influence on growth and can be used as a drug for…

Answer 23

1. Immunosuppression.
2. Cancer.
3. Delaying the ageing process.