4. Signalling Mechanisms in Growth and Division Flashcards
Outline how c-Myk triggers cell division
Most adult cells are NOT constantly dividing
- In the absence of growth signals they go into G0 (quiescent phase)
- (hepatocytes are in quiescent phase much of the time because they don’t divide very regularly)
Oncogene c-‐Myc (when in italics -> GENE)
- 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)
Explain how ligands which activate tyrosine kinase receptors signal through the small G protein, Ras, to activate the extracellular signal-regulated kinase (ERK) cascade.
Growth Factor Stimulation by Signalling Pathways
- The growth factor arrives and it binds to a receptor (usually tyrosine kinase type receptors)
- 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
Example of a mitogenic growth factor = hepatocyte growth factor
Signalling by Peptide Growth Factors
- The receptors normally sit on the plasma membrane as monomers but most growth factors are dimeric
- When the dimeric growth factor binds to two receptor tyrosine kinase molecules, it brings them closer together
- When the receptors are close together, the tyrosine kinase domain is able to cross-‐phosphorylate the partner receptor (Trans-autophosphorylation)
- Tyrosine kinases use the gamma phosphate of ATP to phosphorylate tyrosine residues in proteins
- The phosphorylated domains on the tyrosine kinase receptors act as docking sites for adaptor proteins (which are recruited to the activated tyrosine kinase receptors)
- The adaptor proteins contribute to downstream signalling
This is one of the first places that you can interfere with growth factor signalling:
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
- The anti-‐Her2 antibody can be used to block the early stage of growth stimulation
- One of the important adaptor molecules that is recruited is called Grb2
Discuss adaptor proteins
Adaptor Proteins
- Adaptor proteins are often modular – different domains are mixed and matched to give the protein different properties.
- Important in molecular recognition.
- Adaptor molecules have no enzymatic function; they simply bring other proteins together.
- Grb2 – has only 2 types of protein-protein interactions:
- SH2 – binds to phosphorylated tyrosine of receptor. (SH2+3 aminoacid sequence)
- SH3 – bind to proline rich regions of other proteins.
(image) On the right you have the activated receptor with all the adaptor molecules bound to it -‐ the phosphorylated tyrosines act as docking sites
GTP Binding proteins (Ras)
- Grb is bound to the RPTK via its SH2 domain and it binds to a protein called Sos through its SH3 domains
- Sos is an exchange factor (swaps out GDP to GTP) for Ras (a signalling molecule that sits in the membrane of the cell)
- NOTE: Grb2 is always bound to Sos
- 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
- Grb2 brings Ras onto the RTK.
- Ras is a GTP-binding protein (powerful molecular switch) that is either ON (GTP-bound) or OFF.
- Exchange factors (e.g. Sos) turn it ON.
- GTPase activating proteins turn it OFF.
- Most Ras in a cell is usually in the OFF position. In cancer, the Ras protein may be mutated and may be constantly in the GTP-bound state à signals.
- The GTP binding proteins are not kinases.
Outline Ras mutations
Ras can be oncogenically activated by mutations that increase amount of active GTP-loaded Ras.
V12Ras
- glycine in position 12 -> valine due to mutation
- The side chain goes from being a simple hydrogen (in glycine) to a hydrophobic side chain (valine)
- Prevents GAP binding so prevents Ras inactivation
L61Ras
- glutamine in position 61 -> leucine
- This is a single base change in the genome
- The side chain goes from being an amide to a hydrophobic side chain
- This mutation inhibits the intrinsic GTPase activity of the Ras protein -> Prevents GTP hydrolysis.
- Ras ends up constantly being in the GTP bounds (on) state and therefore giving growth stimulatory signals
Ras activates a protein kinase cascade
Ras – Protein Kinase Cascade:
- Ras activates ERK cascade (a form of a MAPK cascade).
- Extracellular Signal-Regulated Kinase (ERK) cascade.
- Mitogen-Activated Protein Kinase (MAPK) cascade.
The Ras then directly initiates the kinase cascade where each kinase activates another kinase.
Explain how the ERK cascade pathway regulates gene expression and leads to progression through G1 of the cell cycle.
The ERK Cascade
- Protein kinases stimulate changes in cell proteins and gene expression to promote division
- At the end of the cascade, the last kinase phosphorylates a number of proteins and changes their activity
- Among the proteins that are phosphorylated are gene regulatory proteins (transcription factors)
- Once phosphorylated, the transcription factors go on to regulate gene expression
- One of the most important genes that is turned on by this pathway is the c-‐ Myc gene
- So activating the growth factor pathway through the kinase cascade leads to the activation of a gene regulatory protein, which stimulates c-‐Myc production –> transctiption factor that leads to cyclin D production
- Myc and Ras are key molecules in stimulating growth -‐ they are commonly found to be mutated or over-‐expressed in many human tumours
- Myc and Ras are ONCOGENES
- Raf – MAPKKK.
- MEK – MAPKK.
- ERK – MAPK.
Summarise regulation of the cell cycle through oscillating amounts or activities of cyclins and their kinases.
Cell cycle control is based on CYCLICALLY activated protein kinases
Cyclin-dependent kinases (always present in cell):
(serine-‐threonine kinases - NOT tyrosine kinases)
- These Cdks are in the cell throughout the cell cycle but they are not activated until they bind to an activating protein called cyclin
- The Cdks are ALSO controlled by phosphorylation -‐ this is an extra level of control
Cyclins
(Cdks are activated by binding to cyclins)
- Cyclins are transiently expressed during the cell cycle
- Once they have activated the Cdks, the cyclins are degraded
- The cyclins are regulated at the level of expression
Different cyclin-Cdk complexes trigger different events in the cell cycle.
E.G. M-Phase-promoting factor initiates mitosis and S phase replication
Cdk1 + Mitotic Cyclin B: Mitosis Promoting Factor
- consists of Cdk1 and Mitotic cyclin (usually cyclin B)
Outline the phosphorylative regulation of cdks
Phosphorylation regulates Cdks
- Cdk1 binds to cyclinB which is inactive until phosphorylated (by 2 reactions)
- Cdk has to be activated at specific sites to become activated
This activation is performed by Cdk activating kinase (CAK)
Balancing this is an inhibitory kinase called Wee1
- CAK puts an activating phosphorylation onto Cdk1
- Wee1 puts an inhibitory phosphorylation onto Cdk1
- Even though Cdk1 is bound to cyclin, it needs the inhibitory phosphate to be taken off before it can function
- Cdc25 takes off the inhibitory phosphate that was put on by Wee1
- Then you get an active Mitotis Promoting Factor
Dephosphorylation activates Cdk1 at the end of interphase
- Dephosphorylation of the inhibitory site of Cdk1 by Cdc25 activates it towards the end of interphase
- Active MPF is able to phosphorylate Cdc25 to increase its activity
- This is a form of positive feedback that drives mitosis
- As soon as you get some active Cdk1 then you activate more Cdc25, which, in turn, leads to more dephoshorylation of the inhibitory site and this positive feedback pushes the cell through mitosis
Feedback Mechanism
- Dephosphorylation of the inhibitory Wee1 phosphate activates Cdk1 at the end of interphase.
- Active MPF (Mitosis Promoting factor) then phosphorylates Cdc25 to increase activity à drives a +ve feedback.
Cyclin B and regulation of mitosis and the cell cycle
Overview of Mitosis
- MPF, when active at the end of metaphase, phosphorylates a number of key substrates that are involved in the mitotic process
- This puts mitosis on hold when the substrates are phosphorylated
- They can’t progress to the next stage until a signal is sent saying that metaphase has been correctly achieved
- Once the kinetochores are correctly attached to the microtubule spindles, a signal is released that causes cyclin B to be degraded
- When cyclin B is degraded, Cdk1 becomes inactive and this means that the substrates (which were keeping mitosis on hold) are dephosphorylated so then mitosis can progress
Different cyclins and different Cdks are required at different stages of the cell cycle.
- G1/S – Cdk2, Cyclin E.
- S – Cdk2, Cyclin A.
- Note above how the same Cdk is bound but with a different cyclin so the cyclin changes the substrate specificity (to phosphorylate different substrates).
- Growth factor stimulation of signalling pathways promotes G0 to G1 transition.
- GF binds to the RPTK -> cascades via Ras à transcription factor phosphorylation -> expression of c-Myc -> stimulate transcription of cyclin D.
Cyclin D activates Cdk4 and Cdk6 -> synthesis of Cyclin E.
This triggers the cell cycle.
Summarise the principle of the molecular timing process
RECAP: growth factor leads to the production of Myc (a transcription factor), which, in turn, stimulates the synthesis of cyclin D. Cyclin D leads to the production of an active Cdk4/6-‐cyclin D complex
- This Cdk-‐cyclin complex then stimulates the synthesis of the next cyclin and they then become sequentially active -‐ each cyclin is involved in stimulating the synthesis of the next cyclin
- This gives direction to the cell cycle
- It also gives timing because it takes time for the concentration of the cyclin to build up so the appropriate Cdk is activated
What do Cdks do?
MPF phosphorylates proteins involved in mitosis e.g. nuclear lamins (cause breakdown of the nuclear envelope)
Breakdown of the nuclear envelope is caused by phosphorylation of the nuclear lamins
Start kinase (from G1 to S) is a complex of Cdk2 and G1 cyclin (e.g. cyclin E) -‐ this phosphorylates substrates needed for that phase
The most important protein that is phosphorylated by start kinase is RETINOBLASTOMA
- Cyclin D and Cdk 4/6 phosphorylates RB -> Changes conformation and releases the RB protein -> Releases E2F goes into nucleus acts as a promotor and leads to cyclin E production
Regulation of Gene Expression by Retinoblastoma
pRb acts as a “brake” on the cell cycle.
Cdks phosphorylate & progressively inactivate pRb.
Rb is a “tumour suppressor”.
MoA:
- In G0, Rb protein is resting with E2F transcription factors in the cell cytoplasm.
- Cdk4/6-cyclin D kinase phosphorylates Rb and E2F is released and binds to gene promoters to drive transcription.
One target of E2F is the cyclin E, the cyclin required to continue cell cycle progression.
Rb acts as a brake in the cell cycle by holding onto the E2F – this makes it a TUMOUR SUPRESSOR GENE.
- Many tumours have reduced levels of Rb protein so can’t regulate E2F.
Genes Regulated by E2F Transcription Factors
E2F Transcription Factors
- E2F regulate proto-oncogenes including Myc proteins.
- Cyclin E is one of the first targets of E2F -> this pushes forward the cell cycle.
- Cyclin E -> binds to Cdk2 and “CyclinE-Cdk2 complex” phosphorylates Rb protein so another +ve feedback is initiated so more E2F is released by Rb.
Myc turns on cyclin D which complexes with Cdk4/6…
- Cyclins – D à E à A à B.
- Cdks – 4/6 à 2 à 2 à 1.
Ultimately leads to mitosis.
More and more pRb is phosphorylated to release more E2F (which is what initially creates cyclin E).
Cyclin A gene promoter is not activated until there is a HIGH concentration of E2F.
Summarise regulation of the cell cycle through inhibitor proteins.
Cdks are also regulated by inhibitors.
Two families of Cdk-Is exist:
- INK4 family – e.g. p15INK4b.
- G1-phase Cdk-I. Inhibits Cdk4/6 by displacing Cyclin D.
- CIP/KIP family – e.g. p21CIP1/WAF1.
- S-phase Cdk-I. Inhibits all Cdks by binding to the Cdk/Cyc complex.
Don’t need to remember the individual names but do remember the family names.
For the cell cycle to progress, these inhibitors need to be degraded.
Look to the left to see:
- INK4 family = G1 Cdk-I.
- CIP/KIP family = S Cdk-I.
Cell Cycle Regulators in Cancer
- Genes that are commonly lost in cancers are tumour suppressor genes.
- Rb – inactivated in many cancers.
- P27KIP1 – under expression correlates with poor prognosis.
Genes that are commonly over-expressed in cancers are oncogenes.
- EGFR/HER2 – mutation activated in breast cancer – treated with Herceptin antibody - really good at targeting things outside the cell (in HER2+ cancer).
- Ras – mutation activated – treated with membrane attachment inhibitors.
- Cyclin D1 – overexpressed in 50% of breast cancer.
- B-Raf – mutation activated in melanomas – treat with kinase inhibitors.
- cMyc – overexpressed in many tumours.
- p10, p53 - inhibition of the PIP3 pathway
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