signalling mchanisms of growth and division

Question 1

What is important to remember about the state of most adult cells

Answer 1

Most adult cells are not constantly dividing.In the absence of growth signals they go into the G0, or quiescent, phase (e.g. liver hepatocytes)

Question 2

What is c-Myc

Answer 2

c-Myc - transcription factor - stimulates the expression of cell cycle genes

This genes and proteins are transiently expressed, so a lot of coordination is needed between the cytoplasm and the nucleus to reactivate these genes

Question 3

When is c-Myc expressed

Answer 3

§ Concentration of Myc is low in the quiescent phase and then in triggered cell division, Myc rapidly rises ans the cell progresses to the S phase.

Peaks after 4 hours- prior to the S phase (8 hours).

Triggered by an external signal (i.e growth factor) which goes to transcription of c-Myc gene- which controls entry into the cell cycle.

Question 4

What are the key components of signalling pathways

Answer 4

1. Regulation of enzyme activity by 
	protein phosphorylation (kinases)

Adapter proteins

Regulation by GTP-binding proteins

Question 5

Outline the growth factor stimulation of signalling pathways

Answer 5

Ligand binds to receptor (Mitogenic growth factor
(i.e. growth signals from other cells, e.g. Hepatocyte Growth Factor released after liver damage)
Receptor protein tyrosine kinases activated by phosphorylation

Question 6

Describe two growth factor receptors which are over expressed in many breast cancers

Answer 6

EGFR/HER2, mutationally activated or overexpressed in many breast cancers

Question 7

Compare the speed of phosphorylation to the speed of translation/transcription in the growth factor signalling cascade

Answer 7

1. GF (e.g. hepatocyte GF) binds to the receptor.
2. GF acts via a small GTP-binding protein (Ras) which triggers a kinase cascade.
3. This triggers activation of genes required for progression of cells through the cell cycle.
   a. This process is slower as it requires transcription/translation.

§ Early stages are very fast and late stages are much slower.

Question 8

Describe what happens to tyrosine kinase residues when growth factors bind to them

Answer 8

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 9

What are adapter proteins

Answer 9

The phosphorylation sites on the phosphorylated receptor protein tyrosine kinase act as docking sites for adapter proteins.
They bring proteins in close proximity (i.e the kinases and their substate)
Different phosphorylation sites recruit different adapter proteins
This creates a structure which regulates the kinase cascades more effectively

Question 10

What is the end result of growth factor activation of receptor protein tyrosine kinase

Answer 10

Cross-phosphorylation of cytoplasmic tails
The phosphorylated RPTK (Receptor Protein Tyrosine Kinase) recruits adaptor and signalling proteins (e.g. Grb2).
Downstream signalling- signal relayed to the cell’s interior

Question 11

Name an important adapter protein

Answer 11

Grb2

Question 12

Describe Herceptin as a treatment for HER2-positive metastatic breast cancer

Answer 12

Herceptin is an anti-HER2 antibody
Herceptin – inhibits the Her2 tyrosine kinase receptor (important in many tumours e.g. breast)
Binds to extracellular domain of growth factor receptor- to prevent the ligand from binding and thus activating the receptor.

Question 13

Summarise the function of adapter proteins

Answer 13

Tyrosine phosphorylation provides docking sites for adapter proteins
Protein-protein interactions: protein binding – bringing proteins together

Question 14

Summarise the structure of adapter proteins

Answer 14

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

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

Adaptor proteins are often modular – different domains are mixed and matched to give the protein different properties.

Question 15

Describe the structure of Grb2

Answer 15

It is modular

It has an SH2 domain, which binds to the docking sites (phosphorylated tyrosine residues on the tyrosine kinase receptors)- inducible, specific sequence context- binds to tyrosine and 4 amino acids in front and behind - binds to EGFR and HER2 at the tyrosine phosphorylated regions

It has two SH3 domains, which bind to proline-rich regions of proteins- constitutive

SH= Src homology domains - Src is an oncogene

Question 16

What is Ras

Answer 16

Oncogene- can become mutated in many cancers
It is a GTP binding protein.

OFF when bound to GDP
ON when bound to GTP
Signalling cascades can activate and recruit Ras as part of their signalling pathway.
Ras is bound to GDP and inactive 95% of the time

Question 17

Summarise the activation and inactivation of GTP-binding proteins, such as Ras

Answer 17

Molecular switches- only transiently activated before being inactivated.
Signal in stimulates exchange factors (SOS) to exchange GDP for GTP on the Ras. SOS is mutated in many cancers.
The presence of GTP on Ras activates it and leads to propagation of the signal within the cell.
Ras has intrinsic GTP hydrolysis activity- but this is slow.
So GTPase activating proteins bind to Ras and speed GTP hydrolysis up- removal of inorganic phosphate ion- and Ras is bound to GDP again and inactivated.

Question 18

Describe the signalling of receptor protein tyrosine kinases to Ras

Answer 18

Binding of growth factor to extracellular domain of the receptor
Cross-phosphorylation of the cytoplasmic tails
Recruitment of Grb2 to phosphorylation sites (binds to C-terminus of phosphorylated sites).
Exchange factor Sos
(“constitutively”, i.e. always, bound to Grb2 via SH3 domains)
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

Propagation of the signal within the cell.

Question 19

What is important to remember about the activation of Ras

Answer 19

It must be bound to the plasma membrane to be activated - highlighting the importance of the spatial arrangement of proteins- Ras must be near the receptor protein tyrosine kinase that is activated.

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

Question 20

What is the key difference between SH3 and SH2 domains

Answer 20

SH3- constitutively expressed in Grb2

SH2- expressed at certain times (i.e when tyrosine has been phosphorylated and activated).

Question 21

Describe how Ras can be ontogenically activated by point mutations

Answer 21

V21Ras – glycine is replaced by valine, which means that a simple hydrogen side chain is replaced by a hydrophobic sidechain. This hydrophobic side chain doesn’t allow GAPs to bind to Ras, thus preventing inactivation of Ras.

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

These mean that once Ras is activated, it cannot be switched off- dysregulating cell signalling.

Question 22

What protein cascade does Ras activate

Answer 22

Ras activates ERK cascade (a form of a MAPK cascade).

o Extracellular Signal-Regulated Kinase (ERK) cascade.

o Mitogen-Activated Protein Kinase (MAPK) cascade.

§ The Ras then directly initiates the kinase cascade where each kinase activates another kinase:

Kinase 1 phosphorylates kinase 2 (ATP –ADP)
Kinase 2 phosphorylates kinase 3 (ATP -ADP)
Sometimes a kinase may have two phosphorylation sites added to it.

Question 23

What are the different kinases involved in the ERK cascade

Answer 23

o Raf – MAPKKK.

o MEK – MAPKK.

o ERK – MAPK.

Question 24

What is B-Raf

Answer 24

B-Raf is an oncogene - mutationally activated in melanomas

Question 25

What is the role of ERK

Answer 25

Phosphorylates proteins (transcription factors, cytoskeletal proteins) to change their activity
Phosphorylates gene regulatory proteins to change gene expression- one of these is c-Myc gene- a gene which codes for c-Myc (a transcription factor) which leads to cell proliferation and and starts mitosis. (progression into G1).

Question 26

State some key oncogenes that are often mutated in human cancers

Answer 26

Myc and Ras are key oncogenes that may be mutated in human tumours.

Question 27

Summarise mitosis

Answer 27

Complex process - requires strict regulation!

Checkpoints – need correct timing and sequence

Question 28

Summarise the role of cdks in controlling the cell cycle

Answer 28

§ Cell cycle control is based on Cdks.

§ Cdks:

o Present in proliferating cells throughout the cell cycle.

o Activity is regulated by – interaction with cyclins & phosphorylation.

o NOT tyrosine kinases (Serine-threonine kinases).

Question 29

Describe the role of cyclins

Answer 29

§ Cdks are not activated until they are bound to cyclins.

§ Cyclins:

o Only expressed at certain points of the cell cycle (transiently expressed).

o Regulated at the level of expression.

o Once they activate a Cdks, they degrade.

Question 30

Summarise how different cyclins are active at different parts of the cell cycle,

Answer 30

§ Different cyclin-Cdk complexes trigger different events in the cell cycle.
M-cyclins: produce m-phase promoting factor and hence trigger mitosis
S-cyclins: produce start kinases to trigger DNA replication machinery
once cyclins bind to their cdk, they are degraded immediately and thus they are regulated at the level of their expression.

Question 31

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

Answer 31

o Mitosis Promoting Factor:

§ Cdk1 + Mitotic cyclin B

Question 32

Describe an experiment that shows the regulated expression of cyclins

Answer 32

Use electrophoresis to show the expression of cyclin A, cyclin B (use ribonucleotide reductase as a control).
Expressed during mitosis (darker band)
Expression reduced (clearer band) during interphase

Question 33

What does the activation of cdks require

Answer 33

Requires activating phosphorylation
AND
removal of inactivating phosphorylation

This is important for reducing the development of cancer- need two processes to go wrong. So this allows for greater control of the cell cycle.

Question 34

Describe the regulation of cdks by phosphorylation

Answer 34

Phosphorylation regulates Cdks:

o Cdk1 binds to cyclinB which is inactive until phosphorylated (by 2 reactions).

o Activation is via Cdk Activating Kinase (CAK) and a balancing inhibitor (still a kinase) called Wee1 (add phosphates).

§ CAK = activating.

§ Wee1 = inhibitor.

o Cdc25 then removes the Wee1 inhibitory phosphate.

So we have inhibitory phosphates and activating phosphates.

Need to add activating phosphates (CAK) and prevent the addition of inhibitory phosphates (Cdc25)

Question 35

When is cdk1 activated

Answer 35

Dephosphorylation activates Cdk1 at the end of interphase

Question 36

Describe the positive feedback loop that is formed by MPF activation

Answer 36

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 37

Describe the role of MPF in mitosis

Answer 37

§ MPF at the end of metaphase (active) phosphorylates key substrates in the mitotic process.

o Puts mitosis on hold whilst substrates are phosphorylated.

§ Once kinetochores are attached, a signal is released to cause cyclin B to be degraded.

§ Once cyclin B is degraded, Cdk1 becomes inactive à means the substrates are dephosphorylated so mitosis proceeds.

Question 38

Describe how different cyclins and different cdks are required at different points in the cell cycle.

Answer 38

o G1/S – Cdk2, Cyclin E.

o S – Cdk2, Cyclin A.

M- Cdk1, cyclin B (MPF)

Question 39

How can the same cdk be used for two different stages

Answer 39

Cyclin binding alters the substrate specificity of Cdk

Also, different substrates are available at different stages of the cell cycle- during synthesis stage- will need to target nucleus and genetic material for protein synthesis.

Different kinases required to activate the different cdk-cyclin complexes.

Question 40

Describe how growth factor stimulation of signalling pathways promotes progression from G0 to G1

Answer 40

GF binds to and activated receptor protein tyrosine kinases
Signalling cascades via Ras
Immediate early gene transcription
factors (e.g. c-jun, c-Fos, c-Myc)
Stimulate transcription of other genes (e.g. cyclin D)
Cyclin D activates Cdk4 and Cdk6 to stimulate synthesis of cyclin E

Question 41

What is the first ckd/cyclin complex that forms once the cell transitions from G0 to G1

Answer 41

Cdk4/6-cyclin D

Cyclin D1 is an oncogene - overexpressed in 50% of breast cancers

Question 42

Summarise the importance of the regulated expression of cyclins/cdks

Answer 42

Cdks become sequentially active and stimulate synthesis of genes required for next phase, e.g. cyclin D/Cdk4/6 stimulates expression of cyclin E – gives direction and timing to cycle

cyclins susceptible to degradation, hence cyclical activation

Question 43

Outline the activity of different cdk/cyclin complexes throughout the cell cycle

Answer 43

GF stimulation –cMyc
Cdk4/6-cyclin D- G1 (this then gets inhibited)
Cdk2- cyclin E - S (this then gets inhibited)
Cdk2-cyclin A- G2 (this then gets inhibited)
MPF- Prophase to metaphase-anaphase checkpoint (this then gets inhibited)
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) and you need degradation of the previous cyclin.

Question 44

Ultimately, what do a activated cdks do to achieve their function

Answer 44

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

Question 45

Give some examples of proteins that are phosphorylated by the activated cdk/cyclin complexes

Answer 45

MPF - Phosphorylation of nuclear lamins allows breakdown of the nuclear envelope
CDK2/cyclin E - e.g. Retinoblastoma protein (pRb)
Tumour suppressor - inactivated in many cancers

Question 46

Describe the regulation of gene expression br Rb

Answer 46

In G0- Retinoblastoma is unphosphorylated and binds to and sequesters a group of transcription factors called E2F (rendering them inactive)
o Cdk4/6-cyclin D kinase phosphorylates Rb and E2F is released and binds to gene promoters to drive transcription of cyclin E (this happens in proliferating cells)
So the transcription factor become active

Question 47

What is the effect of cdk4/6-cyclin D on Rb

Answer 47

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 48

Summarise the retinoblastoma protein

Answer 48

pRb acts as a “brake” on the cell cycle
Cdks phosphorylate (at multiple sites) & progressively inactivate pRb
Rb is a “tumour suppressor”
§ Rb acts as a brake in the cell cycle by holding onto the E2F – this makes it a TUMOUR SUPRESSOR GENE.

o Many tumours have reduced levels of Rb protein so can’t regulate E2F.

Question 49

State some important genes that are regulated by E2F

Answer 49

Proto-oncogenes – c-Myc, n-Myc, B-Myb, IGF-1

Cell cycle – E2F-1,2,3, pRb, cyclin A, cyclin E, CDK4, CDK2

DNA synthesis – thymidine kinase, thymidine synthetase, dihydrofolate reductase, DNA polymerase

Question 50

Describe the role of retinoblastoma throughout the entirety of the cell cycle

Answer 50

c-myc ---cyclin D/cdk4/6
Phosphorylation of pRB- E2F release
cyclin E-cdk2
phosphorylation of pRB- E2F release
cyclin A-CDK2
phosphorylation of pRb- TF release
cyclin b- cdk1- mitosis 

§ Cyclin A gene promoter is not activated until there is a HIGH concentration of E2F.

Question 51

How are cdk/cyclin complexes inhibited

Answer 51

By CDK inhibitors (CDKI)

CKI must be degraded to allow cell cycle progression

Question 52

Describe the INK4 family of CDKIs

Answer 52

INK4 family:
	p15INK4b	
	p16INK4a
	p18INK4c
	p19INK4

G1 phase CKIs
Inhibit Cdk4/6 by displacing cyclin D

Question 53

Describe the CIP/KIP family of CDKIs

Answer 53

p21CIP1/WAF1
	p27KIP1	
	p57KIP2
S phase CKIs
Inhibit all Cdks by binding to the Cdk/cyclin complex

Question 54

Describe p27KIP1

Answer 54

p27KIP1 tumour suppressor – reduced expression correlates with poor prognosis in many malignancies

Question 55

What is needed for progression from G1-S

Answer 55

Degradation of 
G1 CKIs (INK4)

Question 56

What is needed for progression from S

Answer 56

Degradation of S phase

CKIs (CIP/KIP)

Question 57

Describe some genes that are commonly lost in cancers

Answer 57

§ Genes that are commonly lost in cancers are tumour suppressor genes.

o Rb – inactivated in many cancers.

o P27KIP1 – under expression correlates with poor prognosis.

Question 58

Describe some genes that are commonly over-expressed in many cancers

Answer 58

§ Genes that are commonly over-expressed in cancers are oncogenes.

o EGFR/HER2 – mutation activated in breast cancer – treated with Herceptin antibody (in HER2+ cancer).

o Ras – mutation activated – treated with membrane attachment inhibitors.

o Cyclin D1 – overexpressed in 50% of breast cancer.

o B-Raf – mutation activated in melanomas – treat with kinase inhibitors.

o cMyc – overexpressed in many tumours.

The higher up the mutation in the signalling pathway- the harder it is to treat- due to subsequent dysregulation of downstream pathways.

Question 59

Describe how different cancers have mutations in different pathways

Answer 59

small cell lung cancer- 80% loss of pRb
Pancreatic cancer- loss of P16INK4a
Mantle cell lymphoma- 90% overproduction of cyclin D

Question 60

Outline the different circuits involved in changing gene expression and thus hallmark capabilities

Answer 60

Motility circuits
Proliferation circuits
Viability circuits
Cytostasis and differentiation circuits.

Question 61

Summarise the different drugs that target the different stages of carcinogenesis

Answer 61

Telomerase inhibitors- prevent the enabling of replicative immortality
EGFR inhibitors target the constant proliferating signalling
cdk inhibitors target the evasion of growth suppressors
aerobic glycolysis inhibitors target the deregulating of cellular energetics
proapoptotic BH3 mimetic target the resisting of cell death
PARP inhibitors target the genome instability
inhibitors of VEGF signalling target angiogenesis
Inhibitors of HGF/c-met target invasion and metastasis
selective anti-inflammatory drugs target tumour-promoting inflammation.