Cancer: Signalling mechanisms in growth and division

Question 1

Describe the function of c-Myc

Answer 1

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

Question 2

How does c-Myc concentration vary through the cell-cycle?

Answer 2

Low during G₀

Spikes during G₁

Drops a bit through S, G₁ and M

Question 3

Name the three key components of the signalling pathways

Answer 3

Regulation of enzyme activity by protein phosphorylation (kinases)

Adapter proteins

Regulation by GTP-binding proteins

Question 4

Describe the first 3 stages of the growth factor stimulated signalling pathway

Answer 4

GF binds to a receptor (usually tyrosine kinase)

This activated receptor then activates a small GTP-binding protein (Ras) which triggers a kinase cascade

This triggers the activation of genes required for progression through the cell cycle e.g. c-Myc

Question 5

How does GF activate RPTK (receptor protein tyrosine kinase)?

Answer 5

GF is normally dimeric

When it binds to the two monomeric receptors, they are brought close together allowing for kinase domain cross-phosphorylation between the two receptors

Question 6

Give an example of a tyrosine kinase receptor inhibitor

Answer 6

Herceptin - inhibits her2 receptor tyrosine kinase

Question 7

What are adapter proteins?

Answer 7

They contain a variety of protein binding modules, designed to bring other proteins together, facilitating the creation of larger signalling complexes

Question 8

Give an example of an adapter protein and its interactions

Answer 8

Grb2

SH2 region binds to phosphorylated tyrosines of the receptor

SH3 regions bind to the proline rich regions of other proteins

Question 9

What is Ras?

Answer 9

A GTP binding protein and a powerful molecular switch

Question 10

Describe how Ras is activated

Name the catalyst for this

Answer 10

Under appropriate signals, GTP replaces the GDP of Ras.

This is catalysed by Sos

_{note: in cancer, Ras will be in permanent GTP form}

Question 11

Describe how Ras ‘self-regulates’ itself

Answer 11

Ras has a GTPase that hydrolyses GTP to GDP, thus turning itself off (this is mediated by GTPase activating proteins (GAPs)

Question 12

What types of molecule activate GTP binding proteins?

and give an example

Answer 12

Exchange factors

e.g. Sos

Question 13

What types of molecule deactivate GTP binding proteins?

Answer 13

GTPase activating proteins (GAPs)

Question 14

How do RPTKs activate Ras?

Answer 14

Ras has to be bound to the plasma membrane for this to work

Grb2 (bound to RPTK via SH2) binds Sos via SH3

Grb2-Sos binds to phosphorylated tyrosine domains and activates Ras by the GDP–>GTP exchange

Question 15

Describe the two mutations of Ras that can make it oncologically active

Answer 15

V12Ras: Glycine residue in position 12 becomes valine which makes the side chain hydrophibic

this prevents GAPs from binding

L61Ras: Glutamine in position 61 becomes leucine which makes the side chain hydrophobic

this inhibits intrinsic GTPase activity of Ras

Question 16

What does activated Ras do?

Answer 16

It specfically activates the ERK (extracellular signal-regulated kinase) cascade

generically known as the MAPK (mitogen-activared protein kinase) cascade

Question 17

Name the 3 kinases that chain activate each other after activation by Ras

Answer 17

Raf—>MEK—>ERK

Question 18

The protein kinases stimulate changes in what two things?

Answer 18

Protein activity and gene expression

Question 19

Name an important gene turned on by the ERK cascade

and what is special about it?

Answer 19

c-Myc gene leading to c-Myc production

it is an oncogene so it has potential to cause cancer with mutation

Question 20

What are Cdks?

and what do they do?

Answer 20

Cyclin-dependant kinases

They are serine-threonine kinases that trigger different events in the cell cycle

Question 21

What two factors regulate Cdks activity?

Answer 21

Interaction with cyclins

Phosphorylation

Question 22

Describe the 3 stages of Cdk1 activation

Answer 22

Cdk1 binds to cyclin B - this forms an inactive MPF (mitosis promoting factor)

CAK (Cdk activating kinase) and Wee1 kinase (inhibitory) phosphorylate the Cdk1 at activating and inhibitory sites respectively

Cdc25 phosphatase then removes the inactivating phosphate to form an active MPF

Question 23

Briefly describe the feedback loop between active MPF and Cdc25

Answer 23

Active MPF phosphorylates Cdc25 to increase its activity, i.e positive feedback.

Question 24

How is Cdk1 eventually inactivated?

Answer 24

Signal sent from fully attached kinetochores causes cyclin B to be degraded

therefore Cdk1 becomes inactivated

the key substrates are dephosphorylated which signals mitosis to continue

Question 25

Which Cdks and cyclins regulate which stages of the cell cycle? (4)

Answer 25

Start of G1: Cdk4/6 and cyclin D (stimulates synthesis of cyclin E)

G1 to start of S: Cdk2 and cyclin E

S: Cdk2 and cyclin A

Start of M: Cdk1 and cyclin B

Question 26

What does c-Myc do?

Answer 26

Stimulates transcription of cyclin D

this leads to the production of an active Cdk4/6-cycD complex

this complex stimulates synthesis of next cyclin and next and so on

this gives direction

and timing to the cell cycle because it takes time for the conc of the cyclin to build up and activate the appropriate Cdk

Question 27

What do Cdks do?

give 2 examples

Answer 27

They phosphorylate proteins to drive cell cycle progression

e. g. M-phase Cdk (Cdk1-cycB) complex (MPF) phosphorylates nuclear lamins which break down nuclear envelope
e. g. G1-phase Cdk (Cdk2-cycE) complex (start kinase) phosphorylates retinoblastoma protein (pRb) - tumour suppressor

Question 28

What does pRb do?

Answer 28

Retinoblastoma protein acts as a brake on the cell cycle

In resting G0, pRb is unphosphorylated and in this state it binds to E2F - this makes E2F inactive

pRb can be phosphorylated by Cdk4/6-cycD or Cdk2-cycE to make it inactive

This means E2F is then released and active as a transcription factor for genes such as cyclin E

this just leads to even more phosphorylation of pRb and further activation of E2F

Question 29

Name the two families of Cdk inhibitors

and the phases in which they inhibit

Answer 29

INK4 - G1 (inhibit Cdk4/6 by displacing cycD)

CIP/KIP - S (inhibits all Cdks by binding to Cdk/cyc complex)

They must be degraded to allow cell cycle progression

Question 30

Name 5 mutations in oncogenes that can lead to cancer (and possible treatments)

Answer 30

EGFR/HER2 mutationally activated or overexpressed in many breast cancers (herceptin antibody for treatment of HER2-positive metastatic breast cancer)

Ras mutationally activated in many cancers (inhibitors of membrane attachment)

Cyclin D overexpressed in 50% of breast cancers

B-Raf mutationally activated in melanomas (kinase inhibitors)

c-Myc overexpressed in many tumours

Question 31

Name 2 mutations in tumour suppressors that can lead to cancer

Answer 31

pRb inactivated in many cancers

p27^KIP1 underexpression correlates with poor prognosis

Question 32

Nice diagram of useful anti-cancer targets