Signalling mechanisms of growth and division

Question 1

What do adult cells need to divide? What happens if they don’t get this?

Answer 1

Growth signals

• In the absence of growth signals, cells go into G₀ (quiescent/resting phase)
• This is the case with most adult cells - they are not constantly dividing
  
  • e.g. liver hepatocytes

NOTE:

• G₀ is a permanent state for some cells, while others may re-start division if they get the right signals
  
  • Some cells cannot divide once differentiated → G₀ = permanent state
    
    • e.g. cardiac muscle cells
  • Most adult cells resume proliferation as needed to replace cells that have been injured or have died
    
    • e.g. liver hepatocytes

Question 2

What is c-Myc?

Answer 2

• An oncogene
• When this gene is transcribed, the c-Myc protein is a transcription factor
  
  • It stimulates the expression (transcription) of cell cycle genes
    
    • The cell cycle genes refer to the proteins which are required for each phase
    • e.g. DNA polymerase for DNA replication in S phase

Question 3

What is an oncogene?

NOTE: For understanding

Answer 3

Oncogene = a gene which has the potential to cause cancer

• So these are essentially normal genes which promote the proliferation and differentiation of cells
  
  • Known as proto-oncogenes when normal
• These genes become mutated in a way which results in:
  
  • Increased gene expression
  • Increased protein activity due to a change in protein structure
• This mutated genes then become oncogenes as they have the potential to induce tumours and cancer

In the case of c-Myc → these are overexpressed in tumour cells

Question 4

What are the key components of signalling pathways?

Answer 4

• Regulation of enzyme activity by protein phosphorylation
  
  • Phosphorylation is carried out by kinases
• Adapter proteins
• Regulation by GTP-binding proteins

Question 5

Describe generally how growth factors stimulate signalling pathways.

Answer 5

1. Mitogenic growth factor binds to a tyrosine kinase receptor, activating it
   
   • Also known as receptor protein tyrosine kinase (RPTK)
2. The activated receptor then activates a small GTP-binding (G) protein
   
   • EXAMPLE: Ras
3. This triggers a kinase cascade
4. This then triggers the activation of genes that are required for cells to progress through the cell cycle
   
   • Allows cells to come out of the G₀ phase and then proceed through the cell cycle (G₁ → S → etc.)
   • EXAMPLE: c-Myc

Speed:

• The early stage of cell cycle triggering is very fast
  
  • Steps 1-3
• The later stages are slower because it requires transcription and translation to take place
  
  • Step 4

Question 6

What is a mitogenic growth factor?

Answer 6

Growth signals from other cells

• e.g. Hepatocyte growth factor released after liver damage

Question 7

Describe in detail the first thing that happens when the receptor gets activated by a growth factor.

Answer 7

• Once the growth factor dimer binds to the receptor, the receptors come closer together (dimerise)
• This stimulates cross-phosphorylation - the receptors phosphorylate each other
  
  • Tyrosine in the cytoplasmic portion of each receptor monomer is phosphorylated
  • This is done using ATP
• The phosphorylated tyrosines on the RPTK act as docking sites, allowing it to recruit adaptor proteins
• The adaptor proteins contribute to downstream signalling

Question 8

Give an example of a RPTK. Explain its clinical relevance.

Answer 8

HER-2 (human epidermal growth factor receptor 2)

• There is an antibody called herceptin that inhibits HER-2
• The anti-Her2 antibody (herceptin) can be used to block the early stage of growth stimulation
• Therefore, it is used in the treatment of HER-2-positive metastatic breast cancer
  
  • Where HER-2 is overexpressed in the tumour cells

Question 9

What happens when adaptor proteins bind to RPTKs?

Answer 9

Adaptor proteins binding facilitate protein-protein interactions

• Adaptor proteins are modular
  
  • This means that they consist of multiple domains (modules)
  • Domain = functional and structural units that are copied in many proteins
• They have some domains which are important in molecular regocnition
  
  • This allows them to bind to other proteins and bring proteins together
• REMEMBER: No enzymatic function

Question 10

What is an important adaptor protein in growth factor signalling? Explain how it works.

Answer 10

Grb2

It has two types of domains:

• SH2 (one of these)
  
  • Binds to the phosphorylated tyrosines of the receptor
  • Inducible - the level of SH2 binding is dependent on the specific sequence context (the amino acids which are neighbouring/nearby to the phosphorylated tyrosine)
• SH3 (two of these)
  
  • Binds to the proline rich regions of other proteins
  • Constitutive - SH3 always recognises and binds to the same proline-rich sequence

Question 11

What is Ras? How is Ras activated?

Answer 11

Ras = small GTP-binding protein (or G-protein)

Ras activation:

• This involves exchange factors - exchange GTP for GDP
  
  • EXAMPLE: Sos
    
    • Sos works by binding to Ras, stimulating a coformational change
    • This causes Ras to release GDP and bind to another guanine nucleotide from the cytosol
    • This guanine nucleotide is usually GTP because GTP is much more abundant than GDP in the cytosol
• ​Grb2 is constitutively (always) bound to Sos via its SH3 domain
• When the RPTK is phosphorylated and forms docking sites allowing Grb2 to bind, this brings Sos close enough to Ras to exchange GDP for GTP, activating Ras
  
  • Ras is anchored to the inner leaflet of the plasma membrane
• Once Ras is activated, it goes on to activate further molecules within the cell - signal transmission

REMEMBER: Ras is a signalling protein but it is not a kinase

Question 12

Describe how Ras is activated and inactivated.

Answer 12

Activated:

• By exchange factors - e.g Sos
• Exchanges GDP → GTP
• Ras bound to GTP → activated

Inactivated:

• By GTP-ase activating proteins (GAPs)
• Ras is small GTP-ase
  
  • REACTION - hydrolysis:
    
    • GDP → GDP + P_i (released)
  • However, this process is very slow
  • Therefore, GAP binds and induces a conformational change in the G-protein (Ras), which allows GTP to be hydrolysed more readily
• Ras bound to GDP → inactivated

Question 13

Describe two mutations involving Ras which could lead to cancer.

Answer 13

Ras can be oncogenically activated by mutations that increase the amount of Ras bound to GTP (active)

NOTE:

• The numbers (12, 61) refer to the position of the AA
• Constitutively active means that Ras is now always active as a result of the mutation

Question 14

As well as being bound to GTP, what does Ras need to be activated?

Answer 14

Ras must bind to the plasma membrane to become activated

Question 15

What does Ras activate?

Answer 15

A protein kinase cascade called:

• Extracellular signal-regulated kinase (ERK) cascade

Question 16

What type of cascade is the ERK cascade?

Answer 16

It is a type of MAPK cascade

• Mitogen-activated protein kinase (MAPK) cascase

Question 17

Describe how Ras activates the ERK cascade.

Answer 17

Ras → Raf (kinase I) → MEK (kinase II) → ERK (kinase III)

IMPORTANT:

• → = activates
• Kinases activate molecules by phosphoryaltion

Question 18

Once activated, what does ERK (kinase III) do?

Answer 18

Phosphorylates several proteins which leads to changes in:

• Protein activity
• Gene expression
  
  • EXAMPLE: Activation of c-Myc gene

The end result of this is to promote cell division

Question 19

Give 3 examples of oncogenes.

Answer 19

• Ras
• B-Raf
  
  • Member of the Raf kinase family - part of the ERK cascade
• Myc
  
  • c-Myc is within the Myc family
  • Myc = transcription factors involved in cell cycle progression, regulation of cell growth and apoptosis

NOTE: B-raf mutations present in malignant melanoma, therefore called an oncogene, but other forms of Raf involved in the ERK cascade all have the potential to be oncogenes​

Question 20

What is cell cycle control based on?

Answer 20

Cell cycle control is based on Cdks

• Cdks = cyclin-dependent kinases

Cdks:

• Present in proliferating cells throughout the cell cycle
• Activity is regulated by:
  
  • Interaction with cyclins
  • Phosphorylation

Question 21

What are cyclins?

Answer 21

Cyclins are proteins which:

• Are transiently expressed at specific points in the cell cycle
  
  • Because they are synthesised then degraded
    
    • Essentially degraded once they have carried out their function
• Have a regulated level of expression
  
  • i.e. The expression of cyclin genes and cyclin concentration varies depending on the stage of the cell cycle

Question 22

What do cyclins do?

Answer 22

Cyclins activate Cdks

• Different cyclin-Cdk complexes trigger different events in the cell cycle.
  
  • EXAMPLE: Cdk1 + cyclin B = M-phase-promoting factor (MPF)
    
    • MPF initiates the M-phase (mitosis)

NOTE: Cdk activation is not only dependent on cyclins, but also on phosphorylation

Question 23

What do activated Cdks do?

Answer 23

• They are kinases, so they phosphorylate proteins to drive cell cyle progression
• Phosphorylatio of the amino acids:
  
  • Serine
  • Threonine

Question 24

Describe how Cdk activation is regulated by phosphorylation.

Answer 24

EXAMPLE: Cdk1 activation

• Cdk1 binds to cyclin B → form cyclin-Cdk complex (MPF)
  
  • MPF is inactive
• Cdk1 phosphorylated by:
  
  • Cdk-activating kinase (CAK)
    
    • Activating phosphorylation
  • Wee1 inhibitory kinase
    
    • Inhibitory phosphorylation
• Cdc25 (phosphatase) then removes the Wee1 inhibitory phosphate - i.e. dephosphorylation
  
  • ​Activates MPF

More information on Wee1:

• While the Wee1 phosphate is still attached, MPF is inactive
• This is an important checkpoint protein
  
  • You don’t want to activate MPF and initate mitosis until you have made sure the cell is ready for cell division:
    
    • The cell needs enough time for growth
    • Replicated DNA needs to be checked for damage
• So at the end of interphase (at the G₂ checkpoint), once the cell is ready, MPF is activated → mitosis initiated

NOTE:

• Not all Cdks are activated in exactly the same way
• But they all involve some sort of phosphorylation as well as cyclin binding to become fully active

Question 25

Describe the positive feedback effect of MPF.

Answer 25

MPF = Cdk1 + cyclin B

• Once MPF is activated, Cdk1 phosphorylates more Cdc25 enzymes, activating them
• This leads to even more MPF molecules being dephosphorylated and activated
  
  • i.e. Having the inactivating phosphate removed
• This further drives mitosis

Question 26

Describe the role of MPF once mitosis has already started.

Answer 26

• The active MPF (cdk1/cycB complex) phosphorylates various substrates to initiate mitosis
  
  • e.g. Phosphorylates proteins to trigger chromatin condensation
• ​BUT, phosphorylation of certain substrates have an inhibitory effect
  
  • i.e. Prevents those substrates from carrying out their function
• So essentially at the spindle assembly checkpoint (end of metaphase), the active MPF puts mitosis on hold by phosphorylation (i.e. inhibition) of some key substrates
• Kinetochores signal that they are fully attached (→ = leads to):
  
  • → Cyclin B is degraded
  • → Cdk1 is inactivated
  • → Key substrates dephosphorylated
  • → Mitosis progresses (onto anaphase)

SUMMARY:

• MPF in the G₂ checkpoint:
  
  • Once activated, MPF phosphorylates key substrates
    
    • This activates the substrates required for prophase - metaphase
    • This inhibits the substrates required for after metaphase
• MPF in the spindle assembly (M) checkpoint:
  
  • MPF degraded due to degradation of cyclin B
  • The post-metaphase substrates are dephosphorylated and become active again

Question 27

Do all stages of the cell cycle require the same cyclin and Cdk?

Answer 27

No - different cyclins and different Cdks required at different stages of cell cycle

IMPORTANT (about cyclins):

• Different cyclins can bind to the same Cdk at different stages of the cell cycle and give the Cdk a different subtrate specificity
  
  • EXAMPLE:
    
    • Cdk2 activated by both cyclin E and cyclin A but at different stages of the cell cycle
• This is important because substrate accessibility changes throughout the cell cycle
  
  • i.e. There are different molecules that need to be phosphoryated (substrates) at different stages of the cell cycle

Question 28

What does growth factor stimulation of signalling pathways promote? How does this happen?

Answer 28

Promotes G₀ → G₁ transition

• Recap:
  
  • Growth factor → RPTK → Grb2 → Sos → Ras → ERK cascade
• ERK (kinase III in the cascade) stimulates increased expression of immediate early gene transcription factors
  
  • EXAMPLES: c-jun, c-Fos, c-Myc
• ​These stimulate the transcription of other genes
  
  • EXAMPLE: cyclin D
    
    • Stimulated by the transcription factor examples given above

NOTE:

• Immediate early gene = genes which are activated (trasncribed) transiently and rapidly in response to a stimulus
• These genes mainly code for transcription factors which then go on to activate other genes
  
  • Other genes - i.e. the ones which are more relevant to the context (e.g. cell cycle specific)

Question 29

What does cyclin D do? What is important about cyclin D?

Answer 29

• Cyclin D activates Cdk4 or Cdk6
• This leads to the stimulation of cyclin E synthesis
  
  • Cyclin E required from G₁ → S, which means the cell has committed to divide

IMPORTANT:

• Cyclin D is an oncogene
  
  • Overexpressed in 50% of breast cancers

Question 30

Describe the regulation of cyclins/Cdks?

Answer 30

Cdks become sequentially active and stimulate synthesis of genes required for next phase (i.e. synthesis of the next cyclin)

• e.g. Cyclin D-Cdk4/6 stimulates expression of cyclin E
• This gives direction and timing to cycle
  
  • Direction - keeps the cell cycle going in one direction

The activation of cyclins are cyclical

• This is because they are susceptible to degradation
• Cyclical = goes up and then down

Question 31

Explain the role of Rb on the cell cycle.

Answer 31

pRb = retinoblastoma protein

• pRb acts as a ‘brake’ on the cell cycle.
• Cdks phosphorylate (at multiple sites) & progressively inactivate pRb
• Rb is a ‘tumour suppressor’

Mechanism of action:

• In G₀, pRb protein is bound to E2F (transcription factor), rendering E2F inactive
• Cyclin D-Cdk4/6 phosphorylates pRb
• This results in a conformational change of pRb it releases E2F
• E2F is now active - free to bind to gene promoters to drive transcription
  
  • e.g. E2F promotes transcription of cyclin E (G₁ → S)

REMEMBER:

• c-Myc stimulates cyclin D synthesis
• Cyclin D: G₀ → G₁

Question 32

Describe the progressive inactivation of pRb.

Answer 32

• c-Myc stimulates cyclin D transcription → cyclin D-Cdk4/6
• Cyclin D-Cdk4/6 phosphorylates pRb → E2F released
  
  • Some E2F released
  • This is because there are different isoforms (conformations) of mono-phosphorylated pRb as there are multiple phosphorylation sites on the protein
  • So some isoforms release E2F while others don’t
• E2F stimulates cyclin E transcription → cyclin E-Cdk2
• Cycln E-Cdk2 further phosphorylates pRb → more E2F released
  
  • More phosphorylated isoforms (conformations) release E2F
  • The increase in E2F concentration means that it can now bind to targets with lower affinity
    
    • The cyclin A gene transcription is not activated until the E2F concentration is high enough
• Higher E2F concentration stimulates cyclin A transcription → cyclin A-cdk2
• Cyclin A-cdk2 further phosphorylates pRb → more E2F
• More E2F stimulates phosphorylation of another transcription factor (TF)
  
  • pRb binds differently to different transcription factors
  • So you may need different levels of phosphorylation (i.e. different conformations) to release different transcription factors from pRb
    
    • ​i.e. to inactivate pRb
• TF stimulates cyclin B transcription → cyclin B-cdk1

EXPLANATION:

• Progressive phosphorylation leads to change in conformation
• Phosphorylating pRb more releases more E2F
  
  • Because the more phosphorylated isoforms of pRb will have a conformation which causes it to release E2F
• pRb binds differently to different transcription factors
  
  • So you may need different levels of phosphorylation to release different transcription factors from pRb
    
    • ​i.e. to inactivate pRb

Question 33

What are CKIs? How do they work?

Answer 33

CKI = Cdk inhibitor

• They bind to Cdks or the Cdk/cyclin complex
• Therefore, they prevent the Cdks from carrying out their function

Question 34

What are the two families of CKIs?

Answer 34

These are all proteins

INK4 family

• p15^INK4b
• p16^INK4a
• p18^INK4c
• p19^INK4d

These are G₁ phase CKIs

• They inhibit Cdk4/6 by displacing cyclin D

CIP/KIP family

• p21^CIP1/WAF1
• p27^KIP1
• p57^KIP2

These are S phase CKIs

• They inhibit all Cdks by binding to the Cdk/cyclin complex
  
  • Essentially inhibits all the CKIs involved in the S phase
    
    • ​Cyclin E-Cdk2
    • ​Cyclin A-Cdk2

CKIs must be degraded to allow cell cycle progression

Link to cancer:

• p27^KIP1 is a tumour suppressor
  
  • Reduced expression correlates with poor prognosis in many malignancies
  • Technically they could all be tumour suppressors as CKIs inhibit cell cycle progression
    
    • This helps prevent uncontrollable proliferation, which is essentially what cancer is
    • But this particularly CKI is known as a tumour suppressor as reduced expression has been linked to cancer