Signalling Mechanisms of growth and division

Question 1

When are cells in the quiescent phase

Answer 1

Most adult cells are not constantly dividing

In the absence of growth signals they enter G0 e.g. liver hepatocytes

Question 2

What is c-Myc

Answer 2

Key role in cell cycle entry and G1
Transcription factor that stimulates the expression of cell cycle genes. Once a growth factor binds to a cell, levels of Myc go up.

Question 3

What are the key components of signalling pathways

Answer 3

Regulation of enzyme activity by protein phosphorylation (kinases)
Adapter proteins
Regulation by GTP-binding proteins

Question 4

Describe growth factor stimulation of signalling pathways in the first 15 minutes

Answer 4

(first 15 mins)
1. Mitogenic growth factor binds to a receptor protein tyrosine kinase (RPTK)
2. Phosphorylation of RPTK recruits adaptor and signalling proteins
3. Activation of Ras and docking site formation
4. Ras binding stimulates and mediates early-response gene expression from the
MAPK/ERK cascade, eg c-Myc expression

Question 5

What happens in the second phase (hour) of growth factor stimulation of signalling pathways

Answer 5

Stimulation of expression of delayed-response genes, leading to the activation of the cell-cycle control system

Question 6

What does tyrosine phosphorylation do

Answer 6

Provides docking sites for adaptor proteins e.g. Grb2 (growth-factor receptor binding protein 2)

Question 7

Describe the importance of protein function in protein-protien interactions

Answer 7

Proteins are modular and contain domains

Some domains are important in molecular recognition (have no enzymatic function, simply bring other proteins together)

Question 8

Give examples of mitogenic growth factors

Answer 8

growth signals from other cells, e.g. Hepatocyte Growth Factor released after liver damage

Question 9

What is the effect of a monomer substrate on the receptor tyrosine kinases

Answer 9

Receptors exist in monomers
The ligand is a dimer
Therefore it brings two monomers of tyrosine kinase receptors together

Question 10

Describe what occurs in the FG signalling pathway when adapter proteins bind to the RPTKs

Answer 10

1. Binding of Grb2 recruits inactive Ras protein (associated with a GDP molecule) to the cytosolic surface of the plasma membrane
2. Ras-activating proteins are then able to activate Ras.
3. Use of exchange factor Sos, which exchanges a phosphate from a GTP molecule associated with the ras-activating protein for the GDP molecule associated with Ras
4. The Ras complex with GTP is now active

Question 11

What are the potential drug targets for adapter proteins

Answer 11

Grb2 binding and Ras activation

Question 12

Describe what occurs in the GF signalling pathway after Ras has been activated

Answer 12

G-proteins act as a molecular switch to turn Ras off by GTP hydrolysis (GTP associated with Ras) using GTPase activating proteins (GAP)

Question 13

Describe the structure of Grb2 and what its regions bind to

Answer 13

SH3 - SH2 - SH3
SH3 - proline-rich regions (constitutive)
SH2 - phosphorylated tyrosine (inducible, specific sequence context)

Question 14

Describe the oncogenic activation of c-Myc

Answer 14

Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation
Mutations that increase the amount of active GTP-loaded Ras (either prevent GAP binding, or prevent GTP hydrolysis)

Question 15

Which protein kinase cascade is activated by Ras

Answer 15

Extracellular signal-regulated kinase (ERK) cascade

Generically - Mitogen-activated protein kinase (MAPK) cascades

Question 16

What is the cascade associated with Kinase I (generic and specific)

Answer 16

MAPKKK

Raf

Question 17

What is the cascade associated with Kinase II (generic and specific)

Answer 17

MAPKK

MEK

Question 18

What is the cascade associated with Kinase III (generic and specific)

Answer 18

MAPK

ERK

Question 19

What is B-Raf

Answer 19

Oncogene

Mutationally activated in melanomas

Question 20

What is the role of protein kinases

Answer 20

Stimulates changes in cell proteins and genes expression (e.g. c-Myc) to promote division

Question 21

Describe Ras and c-Myc’s role in tumour formation

Answer 21

Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation

Question 22

Which molecules control the cell cycle

Answer 22

Activated protein kinases, known as cyclin-dependent kinases (Cdks) which are present in proliferating cells throughout the cell cycle

Question 23

What regulates the activity of cyclin-dependent kinases

Answer 23

Regulated by their interaction with cyclins, and their phosphorylation

Question 24

What are cyclins

Answer 24

Cyclins are proteins which activate Cdks
They are transiently expressed at specific points in the cell cycle, and this level of expression is highly regulated
They are synthesized, but then degraded

Question 25

What does cyclins form to trigger events in the cell cycle

Answer 25

They form cyclin-Cdk complexes with different complexes

Question 26

What forms the mitosis promoting factor (MPF)

Answer 26

Mitotic cyclin (B) and Cdk1

Question 27

Describe the activation of the MPF complex

Answer 27

Activating phosphorylation by CAK (cdk activating kinase) Removal of inactivating phosphorylation by inhibitory kinase Wee1 Balance of Wee1 and CAK determines activation

Question 28

When does removal of Wee1 occur and what does this drive

Answer 28

Occurs at the end of interphase using CDC25 phosphatase | Drives progression into mitosis

Question 29

Which cyclins and Cdks are required during G1

Answer 29

Cyclin E and Cdk2

Question 30

Which cyclins and Cdks are required during the S phase

Answer 30

Cyclin A and Cdk2

Question 31

Give an example of how the cell cycle can be controlled by altering cyclins (via c-Myc)

Answer 31

c-Myc targets Cyclin D. Cyclin D is required for re-entry into the cell cycle from G0. Cyclin D activates cdk4 and cdk6, which then stimulate cyclin E production (which is required for progression from through G1 into S phase)

Question 32

What gives direction and timing to the cell cycle

Answer 32

Cdks are sequentially activated by cyclins and then stimulate synthesis of genes required for the next phase

Question 33

Explain how cyclin dependant kinases affect cell cycle progression

Answer 33

Phosphorylation of proteins (on serine or threonine)

Question 34

Give an example of how cdks affect cell cycle progression using MPF

Answer 34

``` MPF targets nuclear lamins; the phosphorylation of which cases breakdown of nuclear envelope (key step in prophase) ```

Question 35

Give an example of how cdks affect cell cycle progression using Cdk4/6-cyclin D

Answer 35

Targets retinoblastoma protein (pRB); the phosphorylation of which inactivates the protein. This releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase

Question 36

What is the role of Cdk inhibitors

Answer 36

Inhibits Cdks to prevent progression through the cell cycle, which is important in ensuring the integrity of the cell cycle is maintained Must be degraded to allow cell cycle progression

Question 37

Give 2 examples of Cdk Inhibitors

Answer 37

INK4 - inhibit Cdk4/6 therefore arresting G1 at the restriction checkpoint (coded by p16 gene) CIP/KIP - all Cdks, especially in S phase (KIP coded by p27)

Question 38

Give examples of how CDK inhibitors can be involved in cancer

Answer 38

Cancers can be caused by: Loss of INK4 inhibition Aver-production of cdk4/cyclin D Loss of retinoblastoma protein (highly prevalent in lung cancer)

Question 39

Give examples of oncogenes

Answer 39

``` EGFR/HER2 Ras Cyclin D1 B-Raf c-Myc ```

Question 40

Give examples of tumour suppressors

Answer 40

Rb | p27KIP1

Question 41

What does Cdk4/6-Cyclin D target

Answer 41

``` Retinoblastoma protein (pRB); the phosphorylation of which inactivates the protein. This releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase ```