5: Signalling & Mechanisms in Growth/Division

Question 1

How is entry into cell cycle controlled?

Answer 1

c-Myc is an oncogene overexpressed in tumours
Its a transcription factor that stimulates expression of cell cycle genes
GFs increase Myc levels

Question 2

What are the key components of signalling pathways?

Answer 2

1. Kinases - regulation of enzyme activity
2. Adapter Proteins
3. GTP-binding proteins (G-proteins)

Question 3

Describe the GF stimulation of signalling pathways

Answer 3

1. Mitogenic growth factor
2. RPTK
3. Small G-Protein (Ras)
4. Kinase cascade
5. Immediate early genes (e.g. c-Myc which is a TF) control expression of other genes

Question 4

What are adaptor proteins?

Answer 4

Proteins that recognise and bind to specific phosphorylated tyrosines

Question 5

What GFs are overexpressed in breast cancers and how do we treat it?

Answer 5

EGFR/HER2

Use Anti-HER2 antibody (Herceptin) to block binding of ligand to RPTK

Question 6

Describe the structure of Grb2

Answer 6

SH2/SH3 regions
SH2 recognises phosphorylated tyrosines
SH3 is proline-rich region, docking site for other proteins

Question 7

What does Grb2 do?

Answer 7

Recruits Exchange Factor Sos, which is a Ras activating protein

Question 8

How is Ras activated?

Answer 8

Exchange Factor (like Sos) exchanges GDP on Ras to GTP

Question 9

What is the name of proteins that inactivate G proteins like Ras?

Answer 9

GTPase Activating Proteins (GAP)

Question 10

What are common oncogenic mutations of Ras?

Answer 10

V12Ras (glycine -> valine at position 12) prevents GAP binding

L61Ras (glutamine -> Leucine at position 61) prevents GTP hydrolysis

Question 11

What happens when Ras is activated?

Answer 11

Ras activates ERK cascade (Extracellular signal Regulated Kinase)

Question 12

What kinases are involved in the ERK cascade?

Answer 12

1. Raf
2. MEK
3. ERK

Question 13

Give an example of a type of cancer originating from the ERK cascade and state its cause

Answer 13

Melanoma caused by activation of the oncogene B-Raf

Question 14

What are the two main effects of ERK cascade?

Answer 14

Altered gene expression

Altered protein activity

Question 15

How do growth factors/ERK cascade affect cell cycle?

Answer 15

Expression of proteins that go inside the nucleus and upregulate Myc, which regulates cell cycle and proliferation. Myc is also an oncogene

Question 16

What proteins primarily regulate the cell cycle?

Answer 16

Cdks (Cyclin-dependent Kinases)
Present in proliferating cells throughout the cell cycle
CYCLICAL activity regulated by cyclins and phosphorylation

Question 17

What are cyclins?

Answer 17

Proteins that bind to and activate Cdks triggering different events in the cell cycle
Transiently expressed at specific points of cell cycle (i.e. not there all the time - up/downregulated)

Question 18

What are cyclins?

Answer 18

Proteins that bind to and activate Cdks triggering different events in the cell cycle
They also alter substrate specificity in Cdks
Transiently expressed - synthesised and quickly degraded at specific points of cell cycle, hence cyclical

Question 19

Give 2 examples of Cdk/Cyclin complexes and the proteins that they can phosphorylate

Answer 19

Cdk1 + cyclin B - phosphorylates Nuclear Lamins causing breakdown of nuclear envelope

Cdk2 + cyclin E - phosphorylates Retinoblastoma proteins (tumour suppressor)

Question 20

Describe how Cdk activation is regulated

Answer 20

When cyclin binds to Cdk it is still inactive

2 phosphorylations occur:
activating phosphorylation (by Cdk-activating Kinase) AND inactivating phosphorylation (Inhibitory Kinase)

Dephosphorylation of inactivating phosphate by Cdc25 phosphatase activates Cdk1 at the end of interphase (before mitosis starts)

There are also Cdk inhibitors (CKIs)

Question 21

How do Cdks regulate mitosis?

Answer 21

• Cdk1/cycB is active during first stages of mitosis
• It phosphorylates key substrates and puts mitosis on hold
• At the mitotic checkpoint (before anaphase) signals from fully attached kinetochores causes degradation of cyclin B
• This inactivates Cdk1 and dephosphorylates the key substrates
• Mitosis continues

Question 22

What Cdk/cyc complexes are required in the different stages of the cell cycle?

Answer 22

Mitosis: M-Cdk (Cdk1/cycB)
G1 phase: G1-Cdk (Cdk4/6/cycD)
G1/S phase: G1/S-Cdk (Cdk2/cycE)
S phase: S-Cdk (Cdk2 cycA)

Question 23

How can GF stimulation of signalling pathways promote G0 to G1 transition?

Answer 23

GF activates immediate early gene transcription factors such as c-jun, c-Fos and c-Myc
c-Myc stimulates transcription of cyclin D
Cyclin D activates Cdk4/6 (G1-Cdk) which stimulates production of cyclin E
Cyclin E activates Cdk2 (G1/S-Cdk)

Question 24

State why regulated expression of Cdks/cyclins is important in the cell cycle

Answer 24

Cdks become sequentially active and stimulate synthesis of genes/proteins required for the next phase
This gives DIRECTION and TIMING to the cycle

Question 25

How do Retinoblastoma cells regulate the cell cycle?

Answer 25

In G0, activated Rb protein binds to E2F transcription factor and inactivates it (holds it), preventing expression of cyclins like cyclin E

Cdk4/6/cycD PHOSPHORYLATES the Rb protein at multiple sites and INACTIVATES IT, releasing the E2F and resulting in expression of cyclin E which is required for progression of cell cycle

Therefore Rb is a TUMOUR SUPPRESSOR

Question 26

Summarise the sequence of interactions that occur between Cdk, cyclin and Rb proteins throughout the cell cycle

Answer 26

c-Myc makes cyclin D
Cyclin D activates Cdk4/6 which phosphorylates Rb
Rb releases E2F which makes Cyclin E
Cyclin E activates Cdk2 which phosphorylates Rb more
Rb -> E2F -> Cyclin A
Cyclin A -> Cdk2 -> phosphorylates Rb more -> E2F -> Cyclin B -> Cdk1 -> Mitosis

Question 27

What are the two families of Cdk inhibitors and how do they work?

Answer 27

INK4 (G1 phase CKIs): Inhibit Cdk4/6
CIP/KIP (S phase CKIs): Inhibit ALL Cdks

CKIs MUST be degraded for cell cycle progression

Question 28

What is an example of a CKI tumour suppressor?

Answer 28

p27KIP1