Cancer 5 - Signalling mechanisms of Growth and Division Flashcards
There is a restriction point in G1 - what does it do?
When cell monitors its own size and external signals
Which transcription factor is key in controlling entry into G1 from G0?
C-myc
Myc levels rise sharply if growth factor added to cell
C-Myc is over expressed in tumour cells, hence is an oncogene
How does arrival of a mitogenic growth factor trigger cells to enter the cell cycle
- GF arrives and binds to Tyrosine Kinase type receptor
- Tyrosine kinase type receptor activates Small G-protein (Res)
- This causes the kinase cascade
(Above stages of the cell cycle occurs quickly)
- 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)
Give an example of a mitogenic growth factor
Hepatocyte growth factor
Give a gene that is triggered early in the kinase cascade that goes onto regulate other genes
c-Myc
Explain how cross-phosphorylation of tyrosine residues works
- Dimeric GF binds to (2) Receptor Protein Tyrosine Kinase (RPTK), which brings the 2 receptors closer together.
- When they are closer together, tyrosine kinase domains can cross-phosphorylate the partner receptor - several tyrosine resides are phosphorylated
- The gamma Phosphate of ATP is used by Tyrosine kinases
- Phosphorylated domains on RPTKs act as docking sites for adapter proteins
What do adapter proteins do?
- Bind to phosphorylated (tyrosine) domains in RPTKs
2. Contribute to downstream signalling
What does herceptin do?
Binds to the her-2 receptor tyrosine kinase ; important in breast cancer
Can be used to block the early stages of growth stimualtion
Name an important adapter molecule that is recruited?
Grb2
Adapter molecules are modular, meaning?
Various domains are mixed and matched to give the protein different properties
Domains used for molecular recognition
Adapter molecules have no enzymatic function. What do they do?
Bring other proteins together
What are the 2 protein-protein interactions of GRB-2?
SH2 - which binds to phosphorylated tyrosines of the receptor
SH3 - (2 copies) - binds to proline rich areas of other proteins
What type of protein is Res?
GTP binding protein
GTP binding proteins are powerful molecular switches (e.g. Ras)
When are they on and when are they off
GTP bound - ON
GTP hydrolysed/GDP bound - OFF
What exchange factor catalyses the exchange of GDP for GTP?
Sos (it is also a (proto/)oncogene
GTP binding proteins have intrinsic GTP hydrolysis ability. What does this mean?
GTP protein can turn itself off by hydrolysing GTP into GDP - so Ras can turn itself off
Hydrolysis of GTP to GDP can also be regulated by?
GTPase Activating Proteins (GAPs)
What are the 2 main factors controlling the cycling of GTP proteins?
- Exchange factors (e.g. Sos) that turn it on - GTP
2. GTPase Activating Proteins (GAPs) that turn it off - GDP
GTP binding proteins are not kinases, they simply switch signalling on or off. What happens in cancer with regard to Ras?
Ras = GTP binding protein
Ras mutated meaning that it is always in the GTP bound form
What is Sos and what does it activate?
Sos = Exchange factor
Activates Ras
Grb 2 (adapter protein) is bound to RPTK via which domain and to Sos (exchange factor) via which domain?
RPTK - SH2
Sos - SH3
(Grb 2 is always bound to Sos)
Explain the propogation of the signal
- RPTK activated via dimeric ligand (e.g. GF)
- RPTK phosphorylated
- Grb 2 with Sos attached binds to phosphorylated tyrosine residues
- Sos is then close enough to Ras to activate it
- 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
What is a prerequisite for Ras to work?
Ras must be bound to the plasma membrane
There are 2 oncological mutations that cause more Ras to switch to active GTP-loaded Ras. What are the 2 mutations
- 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 - 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)
What is the kinase cascade involved in growth stimulatory signalling called?
What generic family of cascades does it belong to?
Extracellular signal-regulated kinase (ERK) cascade specifically
Belongs to generic Mitogen-activated Protein Kinase (MAPK) cascades
What are the kinases involved in ERK cascade
Kinase 1 = Raf (MAPKKK)
Kinase 2 = MEK (MAPKK)
Kinase 3 = ERK (MAPK)
Must learn these
B-raf is the first kinase in the ERK cascade. It is an oncogene which is mutationally activated in?
Melanomas
What does the last kinase in the ERK cascade do? What are the knock on effects
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)
Myc and Ras are both?
Oncogenes
Cyclin Dependent Kinases (CDKs) are not tyrosine kinases. What are they
Serine-threonine kinases
Cyclins are present in proliferating cells throughout the cell cycle, but have 2 overarching control mechanisms, which are?
- Cdks only activated when they bind to cyclin
2. Cdks also controlled by phosphorylation
What do cyclins do?
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
How is Cdk activation regulated by phosphorylation
MPF = Mitosis promoting factor
- Cdk (1) binds to cyclin (B)
- CDK-cyclin complex is inactive
- Cdk activating kinase (CAK) puts an activating phosphorylation on CDK1
- Wee1 puts an inhibitory phosphorylation on CDK1.
- Inhibitory phosphate must be removed before CDK1 is activated
- CDC25 removes inhibitory phosphate
- Active MPF now present
What is the positive feedback that drives mitosis?
Activated MPF phosphorylates CDC25 - which means that more inhibitory phosphates are removed
In addition to activating Cdks, what else do cyclins do?
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
Myc is a TF - one of its jobs is to activate?
Cyclin D - cyclin D then goes onto activate Cdk 4/Cdk 6 which which stimulates synthesis of cyclin E
What do Cdks do?
Phosphorylate proteins in Serine or Threonine to drive cell cycle progression
What does Mitosis Promoting factor (MPF) do?
(Cdk 1 and mitotic cyclin/ cyclin B)
Phosphorylates nuclear lamins - causing breakdown of nuclear envelope
What is start kinase a complex of and what does it do?
CDK2 and G1 cyclin (/Cyclin E)
Most important protein it phosphorylates is Retinoblastoma protein (pRB)
pRB is a TSG. How does is act as a brake in the cell cycle
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
Which is one of the targets of E2F TF?
Cyclin E
E2F Tfs regulate proto-oncogenes including which proteins?
Myc proteins
How is there a positive signalling effect with E2F?
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
How is cyclin A activated?
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
What are the 2 families of Cdk inhibitors (CKIs) that regulate Cdks?
- INK4 family - inhibits Cdk4/6 by displacing cyclin D - so no functional complex can be formed - G1 phase CKI
- 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
Name 5 oncogenes commonly activated/overexpressed in cancers
- HER2/ EGFR
- Ras
- Cyclin D1
- B-Raf
- c-Myc
Name 2 TSGs inactivated/underexpressed in cancers
- Rb
2. p27KIP1
