Signalling mechanisms of growth and division

Question 1

In the presence of growth signals cells leave the G0 (quiescent phase) and enter the cell cycle. Name a transcription factor that is stimulated as a result of growth factor-mediated signal transduction and is vital to allow starting of the cell cycle.

Answer 1

c-Myc (oncogene over expressed in many tumours) which brings about the expression of cell cycle genes

Question 2

Cross-phosphorylation of tyrosine residues (due to ligand binding e.g. EGF) provides docking sites for what type of protein? Describe their overall function and give an important example of one.

Answer 2

Adaptor proteins

They have no enzymatic function, and are simply responsible for bringing other proteins together.

e.g. Grb2

Question 3

Describe the structure of the Grb2 adaptor protein that allows it to carry out its functions

Answer 3

• It is modular
• One SH2 domain, which binds to the docking sites (phosphorylated tyrosine residues on the tyrosine kinase receptors)
• Two SH3 domains, which bind to proline-rich regions of proteins

Note that SH = Src Homology

Question 4

Describe how receptor protein tyrosine kinases can signal to Ras.

Answer 4

• Grb2 binds to the guanine nucleotide exchange factor SOS (via the SH3 domains).
• When the Grb2-SOS complex docks to phosphorylated receptor, SOS becomes activated.
• Activated SOS promotes the exchange of GDP from Ras (small G-protein) for GTP; GTP bound Ras is now active.

Question 5

What must the Ras protein be bound to for it to work?

Answer 5

It must be bound to the plasma membrane

NOTE: interference with the membrane binding of Ras can make a good anti-cancer drug

Question 6

How is Ras ‘turned off’?

Answer 6

Ras has intrinsic GTP hydrolysis capability which is stimulated by GTPase-activating proteins (GAPs)

Note that Ras is NOT a kinase

Question 7

Broadly speaking, how might Ras signalling be different in cancer?

Answer 7

Ras could be permanently switched on (in the GTP bound form), thus it constantly signals cell division
(Ras gene is therefore an oncogene)

Question 8

State and describe two mutations that lead to an increase in the amount of active Ras.

Answer 8

V12Ras (constitutively active)
=> glycine 12 replaced by valine
=> hydrogen side chain is replaced by a hydrophobic side chain
=> This prevents GAP binding and hence inactivation of Ras.

L61Ras (constitutively active)
=> glutamine 61 replaced by leucine
=> amine side chain is replaced by a hydrophobic side chain
=> prevents GTP hydrolysis

Question 9

What cascade does Ras activate?

Answer 9

ERK cascade (Extracellular signal-regulated kinase cascade)

or more generically,
MAPK (mitogen-activated protein kinase) cascade

Question 10

Describe the kinase cascade, stating the important players. Any relevant mutations?

Answer 10

• Activated Ras activates Raf kinase.
• RAF kinase phosphorylates and activates MEK
• MEK phosphorylates and activates ERK

B-Raf is an oncogene which is mutationally activated in melanomas

Question 11

What does the last kinase in the cascade phosphorylate?

Answer 11

• Phosphorylates gene regulatory proteins (transcription factors), which change expression of genes involved in the cell cycle or proliferation
• Phosphorylates other proteins to change their activity

Question 12

Give examples of important gene regulatory proteins/transcription factors targeting genes in evolved in the cell cycle

Answer 12

c-Myc, c-Jun, c-Fos

Question 13

Give an example of an anti-cancer drug that targets tyrosine kinase receptors.

Answer 13

Herceptin – inhibits the Her2 tyrosine kinase receptor (important in many tumours e.g. breast)

Question 14

What type of kinase are cyclin-dependent kinases (Cdks)? What are they?

Answer 14

Serine-threonine kinases

They are important regulators of the cell cycle that are present in roughly constant amounts in proliferating cells throughout the cell cycle

Question 15

What conditions do Cdks require to become activated?

Answer 15

• Binding to cyclin (which has no enzymatic activity of its own)
• Must be in a particular phosphorylation state — with some sites phosphorylated and others sites dephosphorylated
• (degradation of Cdk inhibitors)

Note that cyclins are transiently expressed at specific points in the cell cycle.

Question 16

What does the mitosis-promoting factor (MPF) consist of? What does it do once it’s activated?

Answer 16

Cdk1 + cyclin B

Drives the cell’s entry into mitosis

Question 17

What are the requirements, in terms of phosphorylation, for MPF to become active?

Answer 17

• Cdk activating kinase (CAK) and Wee1 inhibitory kinase both phosphorylate CdK in different amino acid positions. CAK adds phosphate that activates Cdk while Wee1 adds phosphate that serves to inactivate Cdk
• Removal/dephosphorylation of the inhibitory phosphate(s) by the phosphatase Cdc25 => actived Cdk1 at the end of interphase

Question 18

Describe the positive feedback loop that is formed by MPF activation.

Answer 18

Removal of the inhibitory phosphorylation by Cdc25 produces active MPF, which then phosphorylates Cdc25 and increases its activity meaning that more MPF can be activated

Question 19

How does MPF put mitosis on hold before progressing to the next stage? What signal then allows mitosis to progress?

Answer 19

At the end of metaphase, it phosphorylates a number of key proteins and inhibits their action (thus putting mitosis on hold) until it is signalled to proceed.

Signal from fully attached kinetochores causes cyclin B to be degraded => Cdk is inactivated and the substrates become dephosphorylated and hence become active.

Question 20

Which Cdk/cyclin is required for G1/S phase?

Answer 20

Cdk2-cyclin E

Question 21

Which Cdk/cyclin is required for S phase?

Answer 21

Cdk2-cyclin A

Question 22

How can the same Cdk be used for two different stages?

Answer 22

Cyclin binding alters the substrate specificity of Cdk

Also, different substrates are available at different stages of the cell cycle

Question 23

What is one of the most important transcription targets of c-Myc?

Answer 23

Gene encoding for Cyclin D

Question 24

What is the first Cdk/cyclin complex that is formed when a cell goes from G0 to G1?

Answer 24

Cdk4/6-cyclin D

Question 25

This Cdk/cyclin complex then stimulates the expression of the next cyclin in the cell cycle. What properties does this system give to the cell cycle?

Answer 25

This gives the cell cycle direction and timing (because the Cdk-cyclin complexes must reach a certain concentration before they can trigger the next stage of the cycle)

Question 26

Give an example of a phosphorylation target of MPF that allows the cell cycle to progress.

Answer 26

Phosphorylation of nuclear lamins allows breakdown of the nuclear envelope

Question 27

What is start kinase and what is one of its most important targets?

Answer 27

Start kinase = Cdk2-cyclin E

Retinoblastoma protein

Question 28

Describe the role of retinoblastoma in the quiescent G0 state.

Answer 28

Retinoblastoma is unphosphorylated and binds to and sequesters a group of transcription factors called E2F

Question 29

What effect does Cdk4/6-cyclin D have on retinoblastoma?

Answer 29

It multiply phosphorylates retinoblastoma – as it becomes phosphorylated it loses its affinity for E2F and releases E2F
This means that the E2F transcription factors can regulate gene expression and promote progression of the cell cycle

Question 30

What is one of the main targets of E2F?

Answer 30

Cyclin E (the next cyclin in the cell cycle)

Question 31

State some important genes that are regulated by E2F.

Answer 31

Proto-oncogenes – c-Myc, n-Myc
Cell cycle – E2F-1,2,3, pRb, cyclin A, cyclin E, CDK4, CDK2
DNA synthesis – thymidine kinase, thymidine synthetase, dihydrofolate reductase, DNA polymerase

Question 32

The initial release of E2F allows transcription of cyclin E leading to the formation of Cdk2-cyclin E. What effect does this complex have on retinoblastoma?

Answer 32

Cdk2-cyclin E further phosphorylates retinoblastoma so more E2F is released and the concentration of E2F increases

Question 33

What is the significance of the increasing concentration of E2F?

Answer 33

This means that E2F can now bind to targets with a lower affinity (e.g. cyclin A gene promoter isn’t activated until the E2F concentration is high enough)

Question 34

What are the two families of Cdk inhibitors?

Answer 34

NK4

CIP/KIP

Question 35

During which phase do each of the families act and how do they inhibit Cdk?

Answer 35

NK4 – G1 phase – it displaces cyclin D from the Cdk4/6-cyclin D complex
CIP/KIP – S phase – it binds to the Cdk/cyclin complexes and inhibits them
NOTE: these inhibitors need to be degraded at various stages for the cell cycle to progress

Question 36

State some common and important oncogenes.

Answer 36

EGFR/HER2 – mutationally activated or over-expressed in many breast cancers
Ras – mutationally activated in many cancers
Cyclin D1 – overexpressed in 50% of breast cancers
B-Raf – mutationally activated in melanomas
c-Myc – overexpressed in many tumours

Question 37

State some important tumour suppressor genes.

Answer 37

Rb – inactivated in many cancers

p27KIP1 – under-expression correlates with poor prognosis in many malignancies