Signalling Mechanisms of Growth and Division Flashcards
what is c-Myc and what does it do?
a transcription factor that stimulates expression of cell cycle genes
what are c-Myc levels in the quiescent phase and cell division phase?
Concentration of Myc is low in the quiescent phase and then in triggered cell division, Myc rapidly rises
what are the 3 components of the signalling pathway?
- Regulation of enzyme activity by protein phosphorylation – kinases.
- Adapter proteins.
- Regulation of GTP-binding proteins.
how is the signalling pathway stimulated at first?
by growth factor which triggers a kinase cascade
what is the main effect of the kinase cascade?
the activation of immediate early genes (which c-Myc affects) required for the progression the cell cycle (transcription and translation)
what does the phosphorylated receptor protein tyrosine kinase (RPTK) recruit?
the adaptor molecule
e.g. Grb2
and signalling proteins
what is the action of the dimeric Growth Factor (GF)?
draws together two tyrosine kinase receptors which then cross-phosphorylate using ATP
where do adaptor proteins bind?
to the phosphorylated domains which act as docking sites
e.g Grb2
example of substance that can block the binding of the dimeric growth factor?
Herceptin (anti-Her2 antibody) blocks growth early
what gives adaptor proteins different properties?
Different domains are mixed and matched to give the protein different properties. Important in molecular recognition
do adaptor proteins have enzymatic functions?
molecules have no enzymatic function; they simply bring other proteins together.
what are type of interactions that Grb2 make?
o SH2 – binds to phosphorylated tyrosine of receptor.
o SH3 – bind to proline rich regions of other proteins.
SH2 is important here
what is the role of adaptor protein in relation to the RTK?
brings in the Ras (a GTP binding protein)
What sort of molecule is Ras?
a GTP binding protein which is switched on when attached to GTP and switched off when there is no GTP (GDP instead)
it is not a kinase
in what conditions is Ras active?
has to be bound to the plasma membrane
then must have a GTP bound
what enables GTP to bind to Ras?
exchange factors e.g. Sos
what switches off Ras?
GTPase Activating Proteins (GAPs) that hydrolyse the GTP to GDP
what role does Ras play in cancer?
Ras is usually in the switched off position
but mutations mean it could have GTP bound all the time leading to constant cell cycle signals being made
act as oncogene
where does the Ras-activating protein e.g Sos bind?
to the adaptor protein at SH3, at all times
what binds to SH2 and SH3?
adaptor protein is bound to the RTK via SH2
the Ras-activating protein (exchange factors like Sos) is ALWAYS bound to Grb2 via SH3
what is the effect of a Ras mutation?
Ras can be oncogenically activated by mutations that increase amount of active GTP-loaded Ras.
examples of Ras mutations
V12Ras – glycine in position 12 –> valine
o Prevents GAP binding so prevents inactivation. (GAP ensure GTP to GDP on Ras)
L61Ras – glutamine in position 61 –> leucine.
o Prevents GTP hydrolysis.
what is the role of activated (GTP-bound) Ras?
initiate the ERK (a MAPK cascade form) cascade so the kinases can activate another
phosphorylate Raf
what is the kinase cascade called
Extracellular Signal-Regulated Kinase (ERK) cascade.
same as Mitogen-Activated Protein Kinase (MAPK) cascade
what are the 3 kinases specific to ERK cascade?
o Raf – MAPKKK.
o MEK – MAPKK.
o ERK – MAPK.
what is the role of protein kinases?
changes in cell proteins (activity) and gene expression to promote division via phosphorylation
what does the ERK kinase in the ERK cascade do?
this is the last kinase in the cascade which phosphorylates proteins to change their activity e.g the transcription factors so they can regulate gene expression
an example of a gene that will be expressed is c-Myc as a result of the cascade
what are key oncogenes that can be mutated in human cancers/tumours?
Myc and Ras
what proteins regulate the cell cycle (CC) ?
cyclin-dependent kinases (Cdks)
these are not tyrosine kinases
when are Cdks present?
Present in proliferating cells throughout the cell cycle.
how do Cdks regulate the CC?
interaction with cyclins & phosphorylation
when and how are Cdks activated? how does deactivation occur?
activation by phosphorylation when bound to cyclin by: Cdk activating kinase (CAK)
activation by removal of deactivating phosphorylation:
- the deactivating phosphorylation = Wee1 phosphate
- remover of this =Cdc25, a phosphatase
after Cdk has been activated, the cyclin detaches and it degraded
at the end of interphase (ready to enter mitosis)
when are cyclins expressed?
at specific points of the CC, regulated at the level of expression
Cdks don’t really change
how do Cdk-cyclin carry out their overall function in the CC?
there are multiple Cdk-cyclin complexes (all different) that trigger the different events of the CC
Cdk-cyclin complex that induced mitosis
Mitosis promoting factor (namely Cdk-1 +cyclin B) induces mitosis
cyclin B is “mitotic cyclin B”
what is the key process that regulates Cdk?
phosphorylation and dephosphorylation
by Cdk activating kinase (CAK)
what causes the final activation step of Cdk1-cyclin B complex?
in mitosis:
phosphatase Cdc-25 removes the inhibitor that prevents the activation of the complex despite being bound together
allows progression from interphase to mitoses
at what stage does Cdc25 (a phosphatase) remove the Wee1 phosphate?
end of interphase when Cdk1 should be activated to go into mitosis
what effect does the activated Cdk1-CycB complex (aka Mitosis Promoting Factor) have on the Wee1-removing Cdc25?
phosphorylates Cdc25 to increase activity and drives a +ve feedback.
it can now remove the inhibitory phosphate
what is the mitotic promoting factor doing at end of metaphase?
puts mitosis on hold while key substrates for the mitotic process are phosphorylated
what does MPF do once kinetochores have attached to the sister chromatids?
releases a signal to degrade its cyclin B
what happens when cyclin B is degraded?
MPF degraded
Cdk1 becomes deactivated
substrates are dephosphorylated so mitosis can proceed
what is the Cdk-cyclin complex allowing G1 to S?
Cdk2+ cyclin E
what is the Cdk-cyclin complex in S?
Cdk2+ cyclin E
Cdk2+ cyclin A
what phase is Cdk2-CycE involved in?
G1 into S
what phase is Cdk2- CycA involved in?
S
why is the cyclin different but the Cdk is the same when the CC goes from G1 to S?
same Cdk is bound but with a different cyclin so the cyclin changes the substrate specificity (to phosphorylate different substrates).
what does Growth Factor stimulation of the signally pathways promote?
the transition from G0 to G1
what is the pathway of GF to the formation of CC relating Cdk-cyc complexes?
GF binds to the RPTK –> cascades via Ras –> transcription factor phosphorylation–> expression of c-Myc–> stimulate transcription of cyclin D.
what Cdk does cyclin D bind to?
Cdk4 and Cdk6
what is the effect of cyclin D activating Cdk4/6?
the synthesis of Cyclin E (used in G1) therefore the activation of Cdk4/6 starts off the CC
what is the order of Cdk-Cyc complexes created starting from the effects of c-Myc?
GF–> c-Myc expression
1) Cdk4/6+CycD (G1 to S)
2) Cdk2+CycE (G1 to S where is falls)
3) Cdk2+CycA (G1 to S, falls at mitosis)
4) Cdk1+CycB (MPF, peaks at mitosis)
DEAB cyclins
46221 Cdks
how is Cdk-cyc expression a regulated process?
Different cyclins and different Cdks are required at different stages of the cell cycle.
Each Cdk produced is involved in stimulating synthesis of the next Cdk by stimulating synthesis of genes required.
Gives direction and timing to cycle
which amino acids do Cdks target when they phosphorylate?
example of protein that is phosphorylated?
Serine or Threonine
the phosphorylation of proteins drives the CC
phosphorylated retinoblastoma protein (pRb) has been phosphorylated by Cdk2-CycE at these positions
what type of protein is the retinoblastoma protein?
Rb protein (acts as a “brake” on the cell cycle)
what effect does phosphorylation of a Rb protein like retinoblastoma protein (pRb) have on it?
phosphorylation of the Rb protein inactivates it
why do Rb proteins like pRb need to be phosphorylated to allow cell cycle progression?
Rb is a “tumour suppressor”. Phosphorylation deactivates it and therefore allows the progression of the CC
How does the Rb (like pRb) carry out its tumour suppressor function?
in a G0 cell, the Rb protein is in the dephosphorylated state (i.e activated Rb)
in this state, the Rb inactivates the E2F transcription factors
(which enables the expression of Cyclin E for the CC to progress) by binding to it
NB phosphorylation of Rb does NOT allow it to do its function
How does the Cdk prevent the Rb carrying out its tumour suppressor function? Why must this happen?
Cdk phosphorylates the Rb protein and therefore deactivates it.
This means is lets go of its E2F transcription factors that it had previously kept deactivated.
The E2F TF is now active and can allow the expression of Cyclin E and therefore CC progression
which Cdk-Cyc complex actually phosphorylates the Rb protein to allow the expression of Cyclin E by E2F TF?
Cdk4/6-CycD
what gene does EF2 TF allow to be expressed?
cyclin E for the progession of the CC
what TF do Rb proteins hold onto?
E2F
what type of gene expresses Rb proteins?
tumour suppressor gene
therefore reduced Rb –> tumour
name targets of EF2 TF?
myc proteins
cyclin E
Now that EF2 can allow Cyclin E expression, what effect does this have on Rb proteins downstream?
positive feedback for greater Rb deactivation:
Cyclin E can form Cdk2-CycE which phosphorylates further Rb proteins and causes the release of more E2F in a +ve feedback
what is the concentration of E2F by the time Cyclin A can be expressed?
high
what are the inhibitors that regulate Cdks?
Cdk-Is in 2 families:
o INK4 family
– e.g. p15INK4b
o CIP/KIP family
– e.g. p21CIP1/WAF1.
what phase does the INK family of Cdk-I affect and therefore what does it do?
G1
inhibits Cdk4/6-CycD by displacing cyclin D
what phase does the CIP/KIP family of Cdk-I affect and what does it do?
S
inhibits all Cdk-Cyc complexes by binding to them
what needs to be done to the Cdk-Is in order for the CC to progress?
they need to be degraded
what are the genes that are typically lost in cancers?
tumour suppressor genes:
o Rb – inactivated in many cancers.
o P27KIP1 – under expression correlates with poor prognosis.
however p53 being a TSG is often over expressed because a mutated p53 has a poor protein turnover
which genes are commonly over-expressed in cancers?
oncogenes:
o EGFR/HER2 – mutation activated in breast cancer
o Ras – mutation activated
o Cyclin D1 – overexpressed in 50% of breast cancer.
o B-Raf – mutation activated in melanomas
o cMyc – overexpressed in many tumours.
how is EGFR/Her2 over-expression treated?
– treated with Herceptin antibody (in HER2+ cancer).
Stop proliferative signalling
how is Ras over-expression treated?
treated with membrane attachment inhibitors
needs attachment to the membrane for activation
how is B-Raf over-expression treated?
treat with kinase inhibitors.
one of the kinases in the ERK cascade
what is the purpose of the ERK cascade?
stimulate changes in cell proteins and gene expression to promote division.
how does p27KIP actually inhibit the Cdks?
p27KIP complexes with the Cdk4-cyclinD
- but does not inhibit its action until the complex is phosphorylated by GSK3b
what is GSK3b?
what is it inhibited by?
phosphorylates the p27KIP-Cdk complex to inactive the Cdk
inhibited by Akt/PKB so cell cycle progression can occur
what is the function of SOS?
exchange factor swapping GDP on the GDP-loaded Ras for GTP to activate Ras
