Signalling Mechanisms in Growth and division Flashcards
1
Q
What are some points at which DNA can be damaged?
A
- At certain checkpoints the cell cycle could be arrested if something is broken
- This could be temporary if the damage is reparable
- If the problem is irreversible, then you will get apoptosis
2
Q
Which are the different Cell Cycle Checkpoints and Tumour Progression
A
- The first checkpoint is during G1
- Then the next checkpoint is just before mitosis, to check for DNA damage before entering mitosis
- Then there is also a metaphase-anaphase checkpoint
- Tumours develop means by which they can bypass these checkpoints
3
Q
Describe the De-regulation of the cell cycle during tumorigenesis
A
- Normally, when cells come out of G1 they enter G0 (during this phase they are still being very active)
- Tumours block the ability of the cells to leave the cell cycle and enter G0 - once they finish mitosis, they enter another cell cycle
4
Q
What is the effect of Growth Factor?
A
Growth factor can regulate the entrance of the cell to the cell factor, it will cause a cascade of phosphorylations which will have an effect to the cell
5
Q
Describe the signalling cascade through the cell:
A
- Response to extracellular factors
- Signal amplification
- Signal integration
- Modulation by other pathways
- Regulation of divergent responses
This will lead to metabolic effects, gene expression effects and changes in cytoskeleton
6
Q
Describe and give an example on the binding of the ligand
A
Kinase domains autophosphorylation each other by coming close and therefore activated the receptor
7
Q
Which structures are phosphorylated, and what is their affect?
A
Kinase domain will catalyse the removal of the hydroxyl and therefore provides Negative charge changing the properties and conformation of the proteins and therefore initiate the process of reactivation
The added phosphate group (negatively charged) can alter protein function by:
- causing a change in shape (conformation) leading to change in activity (+ve or –ve)
- creating a docking site for another protein
8
Q
Which are the molecules that have the opposite effect to kinase
A
phosphatases re-add the cleave the phosphate ard restore the hydroxyl group
9
Q
What is the role of c-Myc?
A
- It is an oncogene
- This has a key role for c-Myc
- The concentration of Myc is really low when the cell is in the quiescent G0 phase
- If you trigger cell division (e.g. by adding a growth factor) you get a rapid and dramatic rise in Myc, which then plateaus at an intermediate level
- This correlates with cells moving out of G0 and into G1
- Myc is a transcription factor - it is a protein that controls the expression of other genes
- In the case of Myc, many of the genes it controls are involved in the cell cycle (hence why it is elevated when the cell wants to enter the cell cycle)
10
Q
Which are the Key Components of Signalling Pathways?
A
- Regulation of enzyme activity by protein phosphorylation (kinases)
- Adapter proteins
- Regulation by GTP-binding proteins
11
Q
Describe the Growth Factor Stimulation by Signalling Pathways
A
- The growth factor arrives, and it binds to a receptor (usually tyrosine kinase type receptors) which is the cascade, then some of them go into the nucleus to produced, multiple gens that co-oporate with each other
- It then acts via a small GTP-binding protein (Ras)
- This then triggers a kinase cascade
- The early stage of cell cycle triggering is very fast
- This then triggers the activation of genes that are required for the progression of cells through the cell cycle - this is slower because it requires transcription and translation to take place
NOTE: it takes an hour or so to induce a gene that is required for progression through the cell cycle
- One of the genes that are triggered early in the kinase cascade is c-Myc, which then goes on to regulate the expression of many other genes, hyperactivate
- Example of a mitogenic growth factor = hepatocyte growth factor
12
Q
Signalling by Peptide Growth Factors (GFs)
A
- The phosphorylated RPTK (Receptor Protein Tyrosine Kinase) recruits adaptor and signalling proteins (e.g. Grb2).
- Close proximity of the tail
- Dimeric GF binds to and draws together the RPTK and cross-phosphorylation occurs using ATP.
- The receptors usually sit on the cell membrane as monomers (but growth factor is dimeric)
- When the receptors are close together the tyrosine kinase domain are able to phosphorylate the partner receptor
- Ant-cancer drug: sits at the growth factor receptor and blocks all the cascade reaction
- The phosphorylated domains act as docking sites for adaptor proteins à contribute to signalling downstream.
- Grb2 is an important adaptor molecule that is recruited.
- Herceptin – anti-Her2 antibody = blocks early growth stimulation
- EXAMPLE: there is an antibody called herceptin that inhibits the her2 receptor tyrosine kinase - this is important in a number of tumours e.g. breast cancer
13
Q
Adaptor Proteins
A
- Adaptor proteins are often modular - there are different domains that are mixed and matched to give the protein different properties
- These different domains are important in molecular recognition
- The adaptor molecules have no enzymatic function - they don’t do anything other than bringing other proteins together
- An extremely important adaptor molecule in growth factor signalling is Grb2 (only three domains, Sarcohpmpolic regions, they are coping and switching those protein domains,)
- Grb2 only has TWO types of protein-protein interactions:
- SH2 - binds to the phosphorylated tyrosines of the receptor
- SH3 (there are two copies) - bind to the proline rich regions of other proteins
14
Q
Describe the function of Grb2, how it can be turned off and on and what happens to it in cancer.
A
- Ras is a GTP binding protein and they are very powerful molecular switches.
- They are either on or off:
- On - GTP bound
- Off - GDP bound
- Under the influence of appropriate signals, the GTP can replace the GDP to make Ras active
- NOTE: this is NOT phosphorylation - it is merely the exchange of GDP for GTP (catalysed by Sos)
- This is a self-regulating system so Ras can turn itself off
- The GTP binding protein is able to hydrolyse GTP to GDP to turn itself off - there is an intrinsic GTP hydrolysis capability
- The hydrolysis itself can be stimulated by another family of proteins - GTPase activating proteins (GAPs)
- So the cycle of GTP binding proteins is almost always controlled by:
- Exchange factors (e.g. Sos) that turn it ON , it can be easily mutated
- GTPase activating proteins (GAPs) that turn it OFF, Ras can also turn off but it is really sallow
- It is not a kinase
- In the case of growth factor stimulatory pathways, the main G-protein is Ras
- It is very important that you are able to turn off the signalling activity of Ras protein to prevent uncontrolled division
- In cancer, you find that the Ras protein is mutated in ways that cause the Ras protein to constantly be in the GTP bound form
15
Q
Receptor Protein Tyrosine Kinase (RPTKs) signal to Ras
A
- Grb2 is bound to the RPTK via its SH2 domain and it binds to a protein called Sos through its SH3 domains
- NOTE: Grb2 is always bound to Sos
- RAS proteins, how is it activated by mutations and stimulus, it binds to GTP
- Sos is an exchange factor for Ras (a signalling molecule that sits in the membrane of the cell)
- When the RPTK becomes activated, you get phosphorylation of the receptor
- Then Grb2 (with Sos attached) binds to these phosphorylated tyrosine domains and Sos is then close enough to the membrane to activate Ras
- Sos allows the exchange of GDP for GTP in Ras to form a GTP bound form of Ras
- This changes the conformation of Ras, which puts it into an active state that can signal downstream and can allow the propagation of the signal
- It is very important that the Ras protein binds with the plasma membrane to work
- If you can interfere with the membrane binding of Ras, you can make a good anti-cancer therapy
- Importance the spatial activation, only the close by pool will be activated
16
Q
Describe the possible mutations of Ras protein and what they will result in
A
Ras can be oncological activated by mutations that increase the amount of active GTP-loaded Ras
-
V12Ras
- The glycine residue in position 12 of the Ras protein has changed to VALINE due to a Ras gene mutation
- This proteins cannot be diactivated
- This prevents GAPs from binding to Ras, thus meaning that Ras can’t turn of very easily (prevents inactivation)
-
L61Ras
- Glutamine in position 61 is converted to LEUCINE amino acid
- Makes it really difficult to be hydroxylated and therefore can not be deactivated
- This mutation inhibits the intrinsic GTPase activity of the Ras protein
- Ras ends up constantly being in the GTP bounds (on) state and therefore giving growth stimulatory signals
Ras activates a protein kinase cascade
- GTP bound Ras binds to a kinase and then that kinase activates several other kinases
- The Ras then directly initiates the kinase cascade where each kinase activates another kinase.
- The kinase cascade that is involved in the growth stimulatory signalling is known as the ERK cascade (extracellular signal-regulated kinase cascade) (extracellular signal regulated kinase, MAPK same thing)
- ERK is a specific example of this type of cascade - specific to growth stimulatory signalling
- The family of these kinase cascades are called the MAPK cascades(mitogen-activated protein kinase cascade), they will stimuclate mitosis s
17
Q
Describe the ERK cascade
A
- Learn the three kinases that are specific to ERK:
- Raf – MAPKKK.
- MEK – MAPKK.
- ERK – MAPK.
- Protein kinases stimulate changes in cell proteins and gene expression to promote division.
