L04 - Cell Signalling & Cancer

Question 1

How many times do receptor tyrosine kinases span the membrane?

Answer 1

Once

Question 2

What is the function of the tyrosine-kinase (intracellular) domain of a tyrosine kinase receptor?

Answer 2

To phosphorylate tyrosine residues on target proteins

Question 3

Which class of receptors trigger the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways?

Answer 3

Tyrosine kinase receptors

Question 4

Describe the action of kinases.

Answer 4

Kinases catalyse the transfer of the terminal phosphate group of ATP to specific serine (Ser), threonine (Thr) or tyrosine (Tyr) residues on target proteins

Question 5

What is the effect of phosphorylation on target molecules?

Answer 5

Usually activation, but sometimes inhibition

Question 6

Describe the action of phosphatases.

Answer 6

Phosphatases cleave a phosphate group from the serine, threonine or tyrosine residues on target molecules

Question 7

Upon binding to a ligand, what conformational change occurs in a tyrosine kinase receptor?

Why is this important?

Answer 7

• Dimerisation of two receptor molecules, known as homodimerisation
• This enables kinase domains of the neighbouring receptor molecules to cross-phosphorylate each other on multiple tyrosine residues (autophosphorylation)
• This creates high affinity binding sites for many proteins e.g. Grb-2 & PI 3-kinase

Question 8

What is the outcome of the MAPK pathway?

Answer 8

Cell proliferation

Question 9

Give a brief overview of the MAPK pathway.

Answer 9

1 - Signalling molecule

2 - Tyrosine kinase receptor

3 - Grb-2

4 - Sos

5 - Ras

6 - Raf

7 - Mek

8 - Erk

9 - Cell growth/proliferation

Question 10

In the MAPK pathway, what is the function of Grb-2?

Which domains are important for its function?

Answer 10

• It acts as an adaptor protein using its SH2 and SH3 domains:
• SH2 recognises specific phosphorylated tyrosine residues on RTKs and binds to the high affinity binding site of a dimerised tyrosine kinase receptor
• SH3 binds to a motif in Sos proteins. Binding to SH3 enables Sos to recruit and activate Ras protein

Question 11

What is Ras protein and how is it activated?

Answer 11

• A small G-protein with GTPase activity bound to the plasma membrane by a covalently attached lipid group
• Sos stimulates the exchange of GDP for GTP in Ras to activate Ras

Question 12

To which class of proteins does Sos belong?

Answer 12

Guanine nucleotide exchange factors (GEFs)

Question 13

What is the effect of GTPase-activating proteins (GAPs) on activated Ras?

Answer 13

• They inactivate ras by stimulating its GTPase activity, which cleaves a phosphate group from GTP to form GDP
• This terminates the signalling event

Question 14

What is the role of Ras in the MAPK pathway?

Answer 14

It activates Raf by inducing a conformational change, localised at the cell membrane

Question 15

What is Raf and what is its function?

Answer 15

• A serine threonine kinase

- It phosphorylates Mek

Question 16

What is Mek and what is its function?

Answer 16

• A serine threonine kinase

- It phosphorylates Erk

Question 17

What is Erk and what is its function?

Answer 17

• A serine threonine kinase
• It phosphorylates transcription factors in the nucleus and regulates gene expression such as:

1 - C-jun

2 - C-fos

3 - C-myc

4 - C-myb

5 - Cyclin D

=> cell growth/proliferation

Question 18

In what 4 ways can the MAPK pathway can be turned off?

Answer 18

1 - Removal of the extracellular signal

2 - Switching off activated RTKs by protein tyrosine phosphatases (PTPs) e.g. SHP-1 & -2

3 - Ras GAPs e.g. p120GAP, neurofibromin

4 - Dephosphorylation of target proteins by serine/threonine phosphatases

Question 19

What are the outcomes of the PI3K pathway?

Answer 19

1 - Cell survival

2 - Cell growth/proliferation

Question 20

Give a brief overview of the PI3K pathway.

Answer 20

1 - Signalling molecule

2 - Tyrosine kinase receptor

3 - PI 3-kinase enzyme activation

4 - PI(4,5)P2 to PI(3,4,5)P3

5 - PDK1 & Akt

6a - Protein BAD

6b - Kinase mTOR

7a - Prevention of apoptosis

7b - ↑ ribosome production & protein synthesis, ↓ protein degradation, Akt activation

Question 21

What is PI3 kinase and what is its function?

Answer 21

• A lipid kinase
• It is activated by binding to phosphorylated Tyr residues on RTKs and catalyses the phosphorylation of PI(4,5)P2 to PI(3,4,5)P3

Question 22

What is the source of PIP2?

Answer 22

• PI is a phospholipid in cell membranes
• It is phosphorylated to PI(4)P
• This is phosphorylated to PI(4,5)P2

Question 23

Describe the structure of phosphatidylinositol.

Answer 23

Phosphatidylinositol is classified as a glycerophospholipid that contains a glycerol backbone, two non-polar fatty acid tails, a phosphate group substituted with an inositol polar head group

Question 24

What is the function of PIP3?

Answer 24

• PIP3 acts as a docking site for PDK1 & Akt

- PDK1 = phosphatidylinositol dependent kinase & Akt = protein kinase B

Question 25

Describe the interaction between PDK1 and Akt.

Answer 25

Upon binding to PIP3, PDK1 phosphorylates & activates Akt

Question 26

What is Bad protein and what are its functions?

Answer 26

- Bad is a cytoplasmic protein that complexes with apoptosis inhibitory proteins, inactivating them - Akt phosphorylates BAD, which causes the release of death inhibitory proteins & the prevention of apoptosis

Question 27

How is mTOR activated and what are its functions?

Answer 27

- Akt phosphorylates kinase mTOR - mTOR complex 1 stimulates cell growth by: 1 - Promoting ribosome production & protein synthesis 2 - Inhibiting protein degradation - mTOR complex 2 stimulates cell survival by helping activate Akt

Question 28

In what 4 ways can the PI3K pathway can be turned off?

Answer 28

1 - Removal of the extracellular signal 2 - Switching off activated RTKs by protein tyrosine phosphatases (PTPs) 3 - Dephosphorylation of target proteins by serine/threonine phosphatases 4 - PTEN = inositol lipid phosphatase which removes phosphate from PIP3 (forming PIP2), so it can no longer act as docking site for PDK1 & Akt

Question 29

What is HER2?

Answer 29

- An oncoprotein with an orphan receptor (no known ligand) | - Activated by heterodimerisation with other EGFR family members

Question 30

What is the treatment for HER2 overexpression?

Answer 30

Trastuzumab (Herceptin) – humanised anti-HER2 antibody, which causes HER2 internalisation/ degradation & ADCC

Question 31

What causes chronic myelogenous leukaemia?

Answer 31

- Chronic myelogenous leukaemia (CML) is usually associated with the Philadelphia chromosome, which forms from translocation between chromosomes 22 & 9 - Breakage & re-joining occurs at the sites of BCR & ABL genes, creating a hybrid gene encoding hybrid BCR-ABL fusion protein - BCR-ABL stimulates inappropriate proliferation of haematopoietic precursors & prevents apoptosis, leading to an accumulation of excessive WBCs in the blood

Question 32

What is the treatment for ABL-BCR fusion gene expression?

Answer 32

Imatinib (Gleevec) – a synthetic ABL kinase inhibitor that blocks the ATP binding site of Tyr kinase domain of BCR-ABL

Question 33

Why do patients show response then relapse with imatinib (Gleevec)?

Answer 33

Patients show response then relapse due to resistance via secondary mutations in Tyr kinase domain (preventing the drug binding)