Signalling Mechanisms in Growth and Division

Question 1

What transcription factor is stimulated by growth factor signalling and is vital to starting the cell cycle?

Answer 1

c-Myc

Question 2

Describe what happens to tyrosine kinase receptors when growth factors bind to them.

Answer 2

Mitogenic growth factor
(i.e. growth signals from other cells, e.g. Hepatocyte Growth Factor released after liver damage)

Tyrosine kinase receptors are usually present on membranes as inactive monomers

Most growth factors are dimers, so when they bind they bring tyrosine kinase receptors close together

This allows the tyrosine kinase receptors to cross-phosphorylate each other (using the gamma phosphate from ATP to phosphorylate tyrosine residues in proteins)

The phosphorylated domains on the tyrosine kinase receptors act as docking sites for adaptor proteins

Question 3

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

Answer 3

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

Question 4

Name an important adaptor protein.

Answer 4

Grb2

Question 5

Describe the structure of the Grb2 protein.

Answer 5

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

Question 6

Describe how receptor protein tyrosine kinases can signal to Ras.

Answer 6

Grb2 (Growth factor receptor-bound protein 2 ) is bound to RTPK (Receptor protein tyrosine kinase) via SH2 domain and SH3 is bound to Sos

• Sos is an exchange factor for Ras
• Ras sits in the membrane of a cell and is ALWAYS bound to Grb2

When the tyrosine kinase receptors become active and the docking sites become available, Grb2 binds to the docking site ( remember its also attached to Sos )

This brings Sos close enough to the cell membrane and Ras, to allow it to exchange the GDP on Ras for GTP
-GTP bound Ras is active

Question 7

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

Answer 7

It must be bound to the plasma membrane

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

Question 8

How is Ras turned off?

Answer 8

Ras has intrinsic GTP hydrolysis capability

This GTPase activity is stimulated by GTPase-activating proteins (GAPs) turn it off

Question 9

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

Answer 9

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

(Most Ras in a cell is usually in the OFF position)

Question 10

Describe two mutations that lead to an increase in the amount of active Ras.

Answer 10

1. V12Ras
   – glycine is replaced by valine at position 12, which means that a simple hydrogen side chain is replaced by a hydrophobic sidechain.
   - hydrophobic side chain doesn’t allow GAPs to bind to Ras, thus preventing inactivation of Ras.
2. L61Ras
   – glutamine is replaced by leucine (in position 61), which means that an amine side chain is replaced by a hydrophobic side chain.
   - this inhibits the GTPase activity of Ras so Ras remains in the active, GTP bound form.

Question 11

What cascade does Ras activate?

Answer 11

ERK cascade (Extracellular signal-regulated kinase cascade)

Question 12

What is the family that this ERK cascade belongs to called?

Answer 12

MAPK cascade (Mitogen-activated protein kinase cascade)

Question 13

What are the three kinases involved in the ERK cascade?

Answer 13

Raf - generically MAPKKK
MEK- generically MAPKK
ERK - generically MAPK

Question 14

What does the last kinase in the cascade phosphorylate?

Answer 14

It phosphorylates gene regulatory proteins (transcription factors), which go on to regulate the expression of genes involved in the cell cycle
They also phosphorylate other proteins and change their activity

Question 15

What important gene is turned on by the kinase cascade?

Answer 15

c-Myc

both c-Myc and Ras (melanoma) are oncogenes that may be mutated in human tumours

Question 16

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

Answer 16

Serine-threonine kinases

Question 17

What conditions do Cdks require to become activated?

Answer 17

Binding to cyclin
Phosphorylation (activating phosphorylation and removal of inhibitory phosphorylation)
(+ degradation of Cdk inhibitors)

Question 18

What does the mitosis-promoting factor (MPF) consist of?

Answer 18

Cdk1 + cyclin B

Question 19

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

Answer 19

Activating phosphorylation by CAK (Cdk activating kinase)

Removal of the inhibitory phosphorylation (that was placed by Wee1) by Cdc25

Question 20

What activates MPF at the end of interphase?

Answer 20

Removal of the inhibitory phosphorylation by Cdc25

Question 21

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

Answer 21

Removal of the inhibitory phosphorylation by Cdc25 (desphosphorylation of the inhibitory Wee1)
produces active MPF, which then phosphorylates Cdc25 and increases its activity meaning that more MPF can be activated

Question 22

How does MPF put mitosis on hold before progressing to the next stage?

Answer 22

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.

Then the cyclin B is degraded, Cdk1 is inactivated meaning substrates become dephosphorylated and hence become active.

Question 23

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

Answer 23

Cdk2-cyclin E

Question 24

Which Cdk/cyclin is required for S phase?

Answer 24

Cdk2-cyclin A

Question 25

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

Answer 25

Cyclin binding alters the substrate specificity of Cdk

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

Question 26

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

Answer 26

Cyclin D

Question 27

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

Answer 27

Cdk4/6-cyclin D

which stimulates expression of cyclin E

Question 28

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 28

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 29

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

Answer 29

Phosphorylation of nuclear lamins allows breakdown of the nuclear envelope

Question 30

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

Answer 30

Start kinase = Cdk2-cyclin E

-this will phosphorylate Retinoblastoma protein (pRb)

Question 31

Describe the role of retinoblastoma in the quiescent G0 state.

Answer 31

Retinoblastoma is unphosphorylated and binds to and sequesters (hides) a group of transcription factors called E2F
-pRb acts as a brake on the cell cycle

Question 32

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

Answer 32

• phosphorylates retinoblastoma at multiple sites
  – as pRb becomes phosphorylated, it loses its affinity for E2F and releases E2F
• this means that the E2F transcription factors can bind to gene promoters to drive transcription

Question 33

What is one of the main targets of E2F?

Answer 33

Cyclin E (the next cyclin in the cell cycle)

Question 34

What type of gene is retinoblastoma?

Answer 34

Tumour suppressor gene (it acts a brake on the cell cycle)

Question 35

State some important genes that are regulated by E2F.

Answer 35

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

Question 36

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 36

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

Question 37

What is the significance of the increasing concentration of E2F?

Answer 37

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 38

What are the two families of Cdk inhibitors?

Answer 38

INK4

CIP/KIP

Question 39

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

Answer 39

INK4 – 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 40

State some common and important oncogenes.

Answer 40

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 41

State some important tumour suppressor genes.

Answer 41

Rb – inactivated in many cancers

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

Question 42

How is Ras turned on?

Answer 42

Exchange factors e.g Sos turn it on

Question 43

What do protein kinases do?

Answer 43

stimulate changes in cell proteins and gene expression to promote division

Question 44

Describe the order in which Cdks are activated?

Answer 44

Myc turns on cyclin D (which complexes w Cdk4/6)

• Cyclins D - E - A - B
• Cdks 4/6 - 2 - 2 -1

-more and more pRb is phosphorylated to release more E2F (which is what initially created cyclin E)
- cyclin A gene promoter is not activated until there is a high concentration of E2F
ultimately leads to mitosis