Cancer 5 - Signalling mechanisms of Growth and Division

Question 1

There is a restriction point in G1 - what does it do?

Answer 1

When cell monitors its own size and external signals

Question 2

Which transcription factor is key in controlling entry into G1 from G0?

Answer 2

C-myc

Myc levels rise sharply if growth factor added to cell

C-Myc is over expressed in tumour cells, hence is an oncogene

Question 3

How does arrival of a mitogenic growth factor trigger cells to enter the cell cycle

Answer 3

1. GF arrives and binds to Tyrosine Kinase type receptor
2. Tyrosine kinase type receptor activates Small G-protein (Res)
3. This causes the kinase cascade

(Above stages of the cell cycle occurs quickly)

4. Kinase cascade triggers activation of genes required for progression of cell into cell cycle

(Stage 4 is longer because it requires transcription and translation to occur)

Question 4

Give an example of a mitogenic growth factor

Answer 4

Hepatocyte growth factor

Question 5

Give a gene that is triggered early in the kinase cascade that goes onto regulate other genes

Answer 5

c-Myc

Question 6

Explain how cross-phosphorylation of tyrosine residues works

Answer 6

1. Dimeric GF binds to (2) Receptor Protein Tyrosine Kinase (RPTK), which brings the 2 receptors closer together.
2. When they are closer together, tyrosine kinase domains can cross-phosphorylate the partner receptor - several tyrosine resides are phosphorylated
3. The gamma Phosphate of ATP is used by Tyrosine kinases
4. Phosphorylated domains on RPTKs act as docking sites for adapter proteins

Question 7

What do adapter proteins do?

Answer 7

1. Bind to phosphorylated (tyrosine) domains in RPTKs

2. Contribute to downstream signalling

Question 8

What does herceptin do?

Answer 8

Binds to the her-2 receptor tyrosine kinase ; important in breast cancer

Can be used to block the early stages of growth stimualtion

Question 9

Name an important adapter molecule that is recruited?

Answer 9

Grb2

Question 10

Adapter molecules are modular, meaning?

Answer 10

Various domains are mixed and matched to give the protein different properties

Domains used for molecular recognition

Question 11

Adapter molecules have no enzymatic function. What do they do?

Answer 11

Bring other proteins together

Question 12

What are the 2 protein-protein interactions of GRB-2?

Answer 12

SH2 - which binds to phosphorylated tyrosines of the receptor

SH3 - (2 copies) - binds to proline rich areas of other proteins

Question 13

What type of protein is Res?

Answer 13

GTP binding protein

Question 14

GTP binding proteins are powerful molecular switches (e.g. Ras)
When are they on and when are they off

Answer 14

GTP bound - ON

GTP hydrolysed/GDP bound - OFF

Question 15

What exchange factor catalyses the exchange of GDP for GTP?

Answer 15

Sos (it is also a (proto/)oncogene

Question 16

GTP binding proteins have intrinsic GTP hydrolysis ability. What does this mean?

Answer 16

GTP protein can turn itself off by hydrolysing GTP into GDP - so Ras can turn itself off

Question 17

Hydrolysis of GTP to GDP can also be regulated by?

Answer 17

GTPase Activating Proteins (GAPs)

Question 18

What are the 2 main factors controlling the cycling of GTP proteins?

Answer 18

1. Exchange factors (e.g. Sos) that turn it on - GTP

2. GTPase Activating Proteins (GAPs) that turn it off - GDP

Question 19

GTP binding proteins are not kinases, they simply switch signalling on or off. What happens in cancer with regard to Ras?

Answer 19

Ras = GTP binding protein

Ras mutated meaning that it is always in the GTP bound form

Question 20

What is Sos and what does it activate?

Answer 20

Sos = Exchange factor

Activates Ras

Question 21

Grb 2 (adapter protein) is bound to RPTK via which domain and to Sos (exchange factor) via which domain?

Answer 21

RPTK - SH2

Sos - SH3

(Grb 2 is always bound to Sos)

Question 22

Explain the propogation of the signal

Answer 22

1. RPTK activated via dimeric ligand (e.g. GF)
2. RPTK phosphorylated
3. Grb 2 with Sos attached binds to phosphorylated tyrosine residues
4. Sos is then close enough to Ras to activate it
5. Ras activated as Sos catalyses the exchange of GDP to GTP - we now have the GTP-bound version of Ras (i.e. on) that can signal downstream

Question 23

What is a prerequisite for Ras to work?

Answer 23

Ras must be bound to the plasma membrane

Question 24

There are 2 oncological mutations that cause more Ras to switch to active GTP-loaded Ras. What are the 2 mutations

Answer 24

1. V12-Ras (constitutively active):
   Glycine in position 12 of Ras is changed to Valine - causing hydrogen to be replaced by hydrophobic side chain - means GAPs can’t bind to Ras so Ras can’t switch off easily
2. L61-Ras (constitutively active) - Glutamine in position 61 converted to glycine - causes side chain to go from amide to hydrophobic - inhibits intrinsic GTP-ase activity of Ras protein - Ras constantly in GTP-bound state (active)

Question 25

What is the kinase cascade involved in growth stimulatory signalling called? What generic family of cascades does it belong to?

Answer 25

Extracellular signal-regulated kinase (ERK) cascade specifically Belongs to generic Mitogen-activated Protein Kinase (MAPK) cascades

Question 26

What are the kinases involved in ERK cascade

Answer 26

Kinase 1 = Raf (MAPKKK) Kinase 2 = MEK (MAPKK) Kinase 3 = ERK (MAPK) Must learn these

Question 27

B-raf is the first kinase in the ERK cascade. It is an oncogene which is mutationally activated in?

Answer 27

Melanomas

Question 28

What does the last kinase in the ERK cascade do? What are the knock on effects

Answer 28

Last kinase phosphorylates number of proteins called Gene Regulatory proteins (aka Transcription factors) These TFs go onto regulate gene expression (switches on c-Myc gene)

Question 29

Myc and Ras are both?

Answer 29

Oncogenes

Question 30

Cyclin Dependent Kinases (CDKs) are not tyrosine kinases. What are they

Answer 30

Serine-threonine kinases

Question 31

Cyclins are present in proliferating cells throughout the cell cycle, but have 2 overarching control mechanisms, which are?

Answer 31

1. Cdks only activated when they bind to cyclin | 2. Cdks also controlled by phosphorylation

Question 32

What do cyclins do?

Answer 32

They activate Cdks Cyclins are transiently expressed during the cell cycle Cyclins are degraded once they have activated Cdks Cyclins are regulated at the level of expression

Question 33

How is Cdk activation regulated by phosphorylation | MPF = Mitosis promoting factor

Answer 33

1. Cdk (1) binds to cyclin (B) 2. CDK-cyclin complex is inactive 3. Cdk activating kinase (CAK) puts an activating phosphorylation on CDK1 4. Wee1 puts an inhibitory phosphorylation on CDK1. 5. Inhibitory phosphate must be removed before CDK1 is activated 6. CDC25 removes inhibitory phosphate 7. Active MPF now present

Question 34

What is the positive feedback that drives mitosis?

Answer 34

Activated MPF phosphorylates CDC25 - which means that more inhibitory phosphates are removed

Question 35

In addition to activating Cdks, what else do cyclins do?

Answer 35

They alter the substrate specificity and accessibility of CDK This means that the same CDK can be used at different stages of the cell cycle

Question 36

Myc is a TF - one of its jobs is to activate?

Answer 36

Cyclin D - cyclin D then goes onto activate Cdk 4/Cdk 6 which which stimulates synthesis of cyclin E

Question 37

What do Cdks do?

Answer 37

Phosphorylate proteins in Serine or Threonine to drive cell cycle progression

Question 38

What does Mitosis Promoting factor (MPF) do?

Answer 38

(Cdk 1 and mitotic cyclin/ cyclin B) Phosphorylates nuclear lamins - causing breakdown of nuclear envelope

Question 39

What is start kinase a complex of and what does it do?

Answer 39

CDK2 and G1 cyclin (/Cyclin E) Most important protein it phosphorylates is Retinoblastoma protein (pRB)

Question 40

pRB is a TSG. How does is act as a brake in the cell cycle

Answer 40

In G0, pRB is unphosphorylated - it therefore binds to and sequesters TFs called E2F. When E2F is held by pRB, everything is off. CDK4/6 - Cyclin D kinase complex phosphorylates pRB, meaning E2F affinity is lost. Therefore, E2F can act as TF for genes involved in cell cycle progression

Question 41

Which is one of the targets of E2F TF?

Answer 41

Cyclin E

Question 42

E2F Tfs regulate proto-oncogenes including which proteins?

Answer 42

Myc proteins

Question 43

How is there a positive signalling effect with E2F?

Answer 43

E2F released, and transcribes Cyclin E Cyclin E forms Cdk2 complex This complex also targets pRB (which sequesters E2F) So phosphorylation of pRB causes more release of E2F

Question 44

How is cyclin A activated?

Answer 44

Through positive signalling of E2F, the conc of E2F builds in the cytoplasm, so E2F begins to bind to things that it has a lower affinity for Cyclin A gene promotor is not activated until sufficiently high conc of E2F in cytoplasm

Question 45

What are the 2 families of Cdk inhibitors (CKIs) that regulate Cdks?

Answer 45

1. INK4 family - inhibits Cdk4/6 by displacing cyclin D - so no functional complex can be formed - G1 phase CKI 2. CIP/KIP family - inhibit all Cdk/cyclins by binding to the Cdk/cyclin complex - S phase CKIs CKIs must be DEGRADED to allow CELL CYCLE PROGRESSION

Question 46

Name 5 oncogenes commonly activated/overexpressed in cancers

Answer 46

1. HER2/ EGFR 2. Ras 3. Cyclin D1 4. B-Raf 5. c-Myc

Question 47

Name 2 TSGs inactivated/underexpressed in cancers

Answer 47

1. Rb | 2. p27KIP1