Cancer 5: Signalling mechanisms of growth and division

Question 1

What causes cell to go into the G0 phase?

Answer 1

Absence of growth factors.

Question 2

What is c-Myc?

Answer 2

This is an oncogene - overexpressed in tumours. It is a transcription factor - stimulates the expression of cell cycle genes

Question 3

What are the key components of signalling pathways?

Answer 3

1. Regulation of enzyme activity by protein phosphorylation (kinases)
2. Adapter proteins
3. Regulation by GTP-binding proteins

Question 4

Describe the growth factor stimulation signalling pathway?

Answer 4

1) Mitogenic growth factor (Hepatocyte growth factor released after liver damage)
2) Receptor protein tyrosine kinase
3) Small G (GTP-binding) protein (Ras)
4) Kinase cascade
5) Immediate early genes (c-jun, c-Fos, c-Myc) - control the expression of other genes.

See slides

Question 5

Describe what happens a peptide GF binds to a tyrosine kinase protein receptor

Answer 5

In the presence of a ligand the receptors form dimers. They are then activated by phosphorylation.

When phosphorylated it recruits adaptor and signalling proteins –> Bind to phosphorylated tyrosines.

Herceptin (antibody) is used to treat HER2 positive metastatic breast cancer. In many breast cancers the EGFR/HER2 is mutationally activated or over expressed. Herceptin blocks the binding of GFs.

Question 6

Describe adaptor proteins?

Answer 6

• Tyrosine phosphorylation provides docking sites for adapter proteins
• Protein-protein interactions: protein binding – They bring proteins together so they can signal to each other.

Proteins are modular and contain domains, i.e. functional and structural units that are copied in many proteins. (Kinase domain)

Some domains are important in molecular recognition – have no enzymatic function of their own, simply bring other proteins together

Question 7

What are GTP binding (G) proteins?

Answer 7

G proteins such as Ras bind to GTP to become activated. They are inactivated with the hydrolysis of GTP to GDP with GAP (GTPAse Activating Proteins).

The GDP bound to ras is exchanged with an exchange factor such as Sos.

Question 8

What is the Grb2 protein?

Answer 8

Grb2 (adaptor protein) - contains 3 domains. SH3-SH2-SH3.

SH3 - proline rich (another docking site for other proteins)
SH2 - recognises phosphorylated tyrosines

Main function is to bind to Ras

Question 9

Describe the function of Sos?

Answer 9

Sos is an exchange factor which is constitutively active (always) bound to Grb2 via the SH3 domains.

Exchanges GDP with GTP activating Ras proteins bound to the plasma membrane - essentially the RAS activating protein.

Question 10

What are some mutations in RAS leading it to be oncogenically activated?

Answer 10

V12RAS - consitutatively active (prevents GAP binding - prevents inactivation)
L61RAS - constituatively active (Prevents GTP hydrolysis)

Both forms of mutations effectively ensure that RAS remains active.

See slides

Question 11

What does the activated RAS do?

Answer 11

Activates a protein kinase cascade - by docking
This kinase will activate the protein kinase by phosphorlyating kinase I –> KII –> KIII

These kinases downstream of the EGFR and Ras are called: Extracellular signal-regulated kinase (ERK) cascade = (Generically - Mitogen-activated protein kinase (MAPK) cascades)

Question 12

Describe the ERK cascade

Answer 12

Kinase I = Raf
Kinase II = MEK
Kinase III = ERK

The ERK modifies Myc and Ras (involved in cell proliferation) This means that both of the genes can be oncogenes if mutated or expressed in high amounts.

Question 13

What is b-Raf?

Answer 13

Oncogene - mutationally activated in melanomas

Question 14

What are cyclin-dependent kinases (Cdks)

Answer 14

Control cell cycle - phosphorylate proteins (serine or threonine) to drive cell cycle progression.

Present in proliferating cells throughout cell cycle

Activity is regulated by:

• Interactions with cyclins
• Phosphorylation

Question 15

What are the cyclins?

Answer 15

Transiently expressed at specific points in the cell cycle

Regulated at level of expression

Synthesised, then degraded - highest during the mitosis phase of the cell cycle.

Different pairs of cyclins and Cdks control different parts of the cell cycle.

Question 16

What do activated Cdks do?

Answer 16

They phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression

Cdk1 binds to cyclin B (mitotic cyclin) –> phosphorylates nuclear laminins (causes breakdown of nuclear envelope)

Cdk2 binds to cyclin E (G1 cyclin).
Retinoblastoma protein (pRb) promotes G1 progression –> causes production of cyclin E.
Tumour suppressor - inactivated in many cancers

Question 17

How are Cdks regulatated?

Answer 17

Regulated by phosphorlyation. Just binding of the cyclin to the Cdks will not activate it.

Requires activating phosphorylation (Cdk - activating kinase = CAK)
AND
removal of inactivating phosphorylation (Wee 1 inhibitory kinase)

1) Cyclin binds to Cdk
2) The cyclin Cdk complex is phosphorylated by CAK and Wee1.
3) It is still inactive so dephosporylation by Cdc25 (phosphatase) activates it at the end of interphase.

Question 18

What causes cyclin B to be degraded?

Answer 18

Signals from fully attached kinetochores causes cyclin B to be degraded

• Cdk1 inactivated
• key substrates dephosphorylated
• mitosis progresses

Question 19

What cyclins and Cdks are required at different stages at the cell cycle?

Answer 19

• Cyclins activate Cdks but also alter substrate specificity
• substrate accessibility changes through cell cycle

Mitosis - Cdk1 and cyclin B
G1–>S - Cdk2 and cyclin E
S–>G2 - Cdk2 and cyclin A

Question 20

What do the genes c-jun, c-Fos and c-Myc do?

Answer 20

They stimulate the transcription of cyclins. Therefore GF are directly related to the regulation of the cell cycle promoting growth.

Question 21

How does Rb protein regulate gene expression?

Answer 21

The retinoblastoma protein binds to and holds the inactive E2F transcription factor.

In the proliferating cells Cdk4/6 and cyclin D phosphorylate the Rb protein, inactivating it.

This causes the release of the active E2F transcription factor which transcribes cyclin E

pRb acts a brake on the cell cycle. The Cdks phosphorylate at multiple sites & progessively inactivate pRb. pRb is a tumour suppressor.

See slides

Question 22

See slides for the progressive inactivation of pRb

Answer 22

See slides

Question 23

What are CKIs?

Answer 23

Cdk inhibitors - These are often found mutated in tumours.

Two CKI families:
INK4 family - G1 phase CKIs: Inhibit Cdk4/6 by displacing cyclin D
CIP/KIP family - S phase CKIs: Inhibit all Cdks by binding to the Cdk/cyclin complex.

p27^KIP1 tumour suppressor - reduced expression correlates with poor prognosis in many malignancies.

In normal cell cycle the CKIs have to be degraded at their appropriate step.