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

TK receptors are usually present on membranes as monomers
Most GFs are dimers, so on binding, they bring TK receptors close together
Allows TK receptors to cross-phosphorylate (using gamma phosphate from ATP to phosphorylate tyrosine residues in proteins)
Phosphorylated domains on TK 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 HER2 TK receptor (important in many tumours e.g. breast)
Prevents ligand binding + subsequent signalling

Question 4

Name an important adaptor protein.

Answer 4

Grb2

Question 5

Describe the structure of Grb2.

Answer 5

Modular
SH2 domain: binds to docking sites
2 SH3 domains: bind to proline-rich regions of proteins

Question 6

Describe how receptor protein tyrosine kinases can signal to Ras.

Answer 6

Grb2 is constituently bound to an exchange factor: Sos
When TK receptors become active + docking sites become available, Grb2 binds to the docking site
This brings Sos close enough to the membrane + 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 become activated?

Answer 7

Plasma membrane

Interference with membrane binding of Ras can make a good anti-cancer drug

Question 8

How is Ras turned on and off?

Answer 8

On: Exchange factors e.g. Sos exchange GDP on Ras to GTP

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

Question 9

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

Answer 9

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

Question 10

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

Answer 10

V21Ras: glycine replaced by valine. Prevents GAP binding Ras, thus prevents inactivation of Ras.
L61Ras: glutamine replaced by leucine. Prevents GTP hydrolysis 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 the 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 (MAPKKK)
MEK (MAPKK)
ERK (MAPK)

Question 14

What does the last kinase in the cascade phosphorylate?

Answer 14

Gene regulatory proteins (transcription factors e.g. c-Myc), which go on to regulate the expression of genes
Also phosphorylates other proteins + change their activity

Question 15

What are c-Myc and Ras classed as? What would mutation in either result in?

Answer 15

Oncogenes

Mutation causes Cancer

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 + 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 phosphatase

Question 20

What activates MPF at the end of interphase?

Answer 20

Removal of the inhibitory phosphorylation by Cdc25 phosphatase

Question 21

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

Answer 21

Removal of the inhibitory phosphorylation by Cdc25 produces active MPF, which then phosphorylates Cdc25 + 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 end of metaphase, it phosphorylates key substrates + inhibits their action (thus putting mitosis on hold)
Signal from fully attached kinetochores causes cyclin B degradation, Cdk1 is inactivated + the substrates become dephosphorylated + hence 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, substrate availability changes throughout cell cycle

Question 26

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

Answer 26

Cyclin D | Stimulates transcription of other genes

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

Question 28

The Cdk4/6-cyclin D 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

Gives cell cycle direction + timing (because Cdk-cyclin complexes must reach a certain concentration before they can trigger the next stage)

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 | Retinoblastoma protein

Question 31

Describe the role of retinoblastoma in the quiescent G0 state.

Answer 31

Retinoblastoma is unphosphorylated | It binds to + sequesters a group of transcription factors: E2F

Question 32

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

Answer 32

It multiply phosphorylates retinoblastoma On phosphorylation it loses its affinity for E2F + releases E2F E2F transcription factors regulate gene expression + promote progression of the cell cycle

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 (acts a brake on the cell cycle)

Question 35

State some important genes that are regulated by E2F.

Answer 35

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 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 + conc. of E2F increases

Question 37

What is the significance of the increasing concentration of E2F?

Answer 37

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 2 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: displaces cyclin D from Cdk4/6-cyclin D complex CIP/KIP: S phase: binds to Cdk/cyclin complexes + inhibits them These inhibitors need to be degraded at various stages for the cell cycle to progress

Question 40

State 5 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 2 important tumour suppressor genes.

Answer 41

Rb: inactivated in many cancers p27KIP1: under-expression correlates with poor prognosis in many malignancies

Question 42

Describe the presence of Cyclins in the cell cycle

Answer 42

Transiently expressed at specific points Regulated at level of expression Synthesised then degraded quickly