Cancer 5: signalling mechanism of growth and division Flashcards
T/f most adult cells are constantly dividing
Most adult cells are not constantly dividing.In the absence of growth signals they go into the G0, or quiescent, phase (e.g. liver hepatocytes)
What is c-Myc
An oncogene….- transcription factor - stimulates the expression of cell cycle genes
When might c-Myc increase
In response to presence of growth factor
What are the key components of signalling pathways
Regulation of enzyme activity by protein phosphorylation (kinases)
Adapter proteins
Regulation by GTP-binding proteins
What is mitogenic growth factor
(i.e. growth signals from other cells, e.g. Hepatocyte Growth Factor released after liver damage)
Activate the kinase cascade
Outline the stages following growth factor binding
Receptor protein tyrosine kinase
Small G (GTP-binding) protein (Ras)
Kinase cascade
Immediate early genes
(c-Jun, c-Fos, c-Myc - transcription factors) – control the expression of other genes
What happens in response to phosphorlation of tyrosine on growth factor receptors
Binding of adaptor proteins
What can happen in HER2-positive preast cancer
HER2/EGFR (both ErbB receptor family, oncogenes) activated or overexpressed in breast cancers
What drugs can be used in HER2-positive breast cancer
Anti-HER2 Ab
Herceptin
Name an important adaptor protien
Grb2
What is a protein domain
functional and structural units that are copied in many proteins
What type of domains do adaptor proteins contain
domains are important in molecular recognition – have no enzymatic function of their own, simply bring other proteins together
What domains do Grb2 contain and which is this important
2 x SH3… to proline rich regions
SH2… to phosphoylated tyrosines (inducible, specific sequence context), I.E. THE PHOSPHORYLATED TYROSINES IN THE RECEPTOR
What is Src homology regions
The domains on Grb2 where first found on the src oncogenes
How can RAS be activated and switched off
Exchange factor (i.e. SOS) can cause activation of RAS by exchange of GTP for GDP
GTPase Activated Proteins will remove phosphate to switch off
NOT KINASE
What will happen in response to GF activation
Activated receptor tyrosine kinase…
binding of Grb2, Sos binds (it’s always bound Grb2 via SH3 domains)… this activates RAS by binding GTP to RAS
What must RAS do to be actiated
Ras must bind to the plasma membrane to become activated
Outline how RAS could be constitutively actived
V12RAS…. glycine 12 –> Valine
(prevents GAP binding, prevents inactivation)
L61 RAS glutamine 61–> Leucine prevents GTP hydrolysis
How does Ras activate protein kinase cascade
Specifically - Extracellular signal-regulated kinase (ERK) cascade
(Generically - Mitogen-activated protein kinase (MAPK) cascades)
Outline the specific ERK casade
(active Ras protein –>) Raf, MEK, ERK
MAPKKK, MAPKK, MAPK
What is B-Raf
B-Raf is an oncogene - mutationally activated in melanomas
What can happen in response to the signalling cascade
Changes in protein activty/changes in expression
What genes could ERK avticate
c-Myc, cell
proliferation
Give two examples of oncogenes fromt he ERK cascade
Myc and Ras are “oncogenes
What is cell cycle control based on
cyclically activated protein kinases….. called
Cyclin-dependent kinases (Cdks)
When are Cdks present
Present in proliferating cells throughout
cell cycle
How are Cdks regulated
Interaction with cyclins
Phosphorylation (activating and inhibitory)
When are cyclins present
Transiently expressed at specific
points in the cell cycle
How are cyclins regulated
Regulated at level of expression
Synthesised, then degraded
What do activated Cdks do
They phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression
Serine-threonine kinases
What does cdk1 bound with cyclin B do
Cyclin B is a mitotic cyclin
Nuclear lamins (causes breakdown of nuclear envelope)
What does Cdk2 bound to cyclin E do
Cyclin E is a G1 cyclin, so this is causing the cell to begin
Phosphorylates retinoblastoma protein (pRb)
Tumour suppressor - inactivated in many cancers
Cyclin E/CDK2 phosphorylates retinoblastoma protein (Rb) to promote G1 progression. Hyper-phosphorylated Rb will no longer interact with E2F transcriptional factor, thus release it to promote expression of genes that drive cells to S phase through G1 phase
T/f binding of cyclin alone will activate the cdk and why
F….
Requires activating phosphorylation
AND
removal of inactivating phosphorylation
What is the 3 ways of controlling activation of cdk
Clycin, phsophorylation and removal of inactivating phosphorylation
What proteins give activating phosphrylation to Cdk
CAK
What proteins might give inhibitory phosphrylation to Cdk
Wee1
What is the function of Cdc25 and when does it work
Phosphatase (this is how the Cdk1 is activated, following cyclin binding and addition of activating phosphate by CAK)
Removes the wee1 kinase (dephosphorylation activates Cdk1 at the end of interphase), so that nuclear membrane can breakdown )
How do checkpoint signals from kinetochores influence Cdks
Signal from fully attached kinetochores
causes cyclin B to be degraded:
Cdk1 inactivated
key substrates dephosphorylated
mitosis progresses
What is the function of Cdk1/cycB, when is it active
So when Cdk1/cycB becomes active, it moves the cell from G2 to M phase. However, whilst Cdk1/cycB is activated, the cell cannot move beyond metaphase of the M phase
It keeps mitosis ‘on hold’ until all the key substrates for metaphase have been activated
When does Cdk1/cyclin B become inactive
Once all the key substrates have been phosphorylated …
How is Cdk1/cyclin B inactivated
Signal from fully attached kinetochores
causes cyclin B to be degraded
SO THIS IS THE BASIS OF THE METAPHASE CHECKPOINT!
Cyclin B will not be degraded until the bubs proteins etc. have dissociated
What do cyclins to do Cdk
Cyclins activate Cdks but also alter substrate specificity
substrate accessibility changes through cell cycle
Outline overall how growth factor moves the cell out of G0 into G1
Production of ERK upregulates early gene transcrptipn factors e.g. c-Myc, c-Fos and c-Jun)
c-Myc transcription factor then stimlates transcription of other genes E.G. CYCLIN D!
What is cyclin D1….
Oncogene overexpressed in 50% of breast cancers
Which cdk must cyclin D bind to to move the cell out of G0?
Cdk 4/6
Cyclin D/Cdk4/6 stimulates expression of what
cyclin E, to give direction and timing to the cycle (i.e. it moves it into the next stage)
What is the effect of the regulated expression of cyclins/Cdks
Cdks become sequentially active and stimulate synthesis of genes required for next phase, e.g. cyclin D/Cdk4/6 stimulates expression of cyclin E – gives direction and timing to cycle
State the cyclin/Cdk complex at each stage
From G0 to G1 (Cdk4/6/cycD)
From G1 to S (G1/S-Cdk transition….. Cdk2/cyclin E)
During S (S-Cdk….. Cdk2/cyclin A)
Up to metphase of mitosis (M-Cdk….. Cdk1/cycB)
DEAB (dogs eat abused bitches)
How does cyclin D/Cdk4/6 cause cyclin E production
What is the importance of retinoblastoma protein
It phosphorylates retinoblastoma protein, inactivating it, allowing E2F transcription factor to transcribe cyclin E
pRb acts as a “brake” on the cell cycle in G0
Cdks phosphorylate (at multiple sites) &progressively inactivate pRb
Rb is a “tumour suppressor”
How does cyclin4/6-cyclinD affect Rb
So c-Myc has been activated due to ERK, and now cyclin D has been upregulated and complexed with cyclin…..
It phosphoylates the pRb, inativating it, releasing E2F, a transcrpttion factor, which then allows gene transcription of cyclin E…. the next cyclin required for the cycle!
Which genes are regulated by the transcprtion factor E2F
Protononcogenes (c-Myc, N-myc)
Cell cycle genes (retinoblastoma protein=pRb, E2F-1,2,3 cyclin A, cyclin E, CDK2, CDK4)
DNA synthesis (thymidine kinase etc.)
NOTE THAT IS IT NOT a TF to upregulate cyclin B (i.e. for the G2–>M phase… you can see on the slide that it is a transcription factor for the preceding cyclin E and A, but not B. It is also not a transcription factor for cyclin D. c-Myc, which is what binding of the mitogenic growth factor upregulated transcription of through the ERK cascade, causes upregulation of cyclin D and that’s really what sets this cycle off!
Outline the concept of the molecular clock
Just look at slide 32….
basically, with the formation of each complex, pRb is phosphorylated ever more, which allows ever more E2F to be liberated and transcribe cyclins in the next stage (E then A)
Then when pRb has been phosphorylated a lot, it can activate transcription factors (not E2F, as this does not activate cyclin B, look on slide 31, cyclin B not there ) which can lead to cyclinB/cdk1 expression
In addition to cyclins and phosphylation, how else can Cdks be regulated
Cdk Inhibitors (CKIs)
Outline the two CKI families
INK4 family:
CIP/KIP family:
p21CIP1/WAF1
p27KIP1
p57KIP2
What stage of the cell cycle do INK4 and CIP/KIP families of Cdk inhibitors inhibit
INK4- G1 phase CKIs
Inhibit Cdk4/6 by displacing cyclin D
CIP/KIP - S phase CKIs
Inhibit all Cdks by binding to the Cdk/cyclin complex
When is there degradation of INK4
These are Cdk inhibitors which inhibit Cdk4/6/cyclin D and are degraded in the G1 (to stop this from being prevented)
When is there degradation of CIP/KIP
These are Cdk inhibitors which inhibit all Cdks by binding to the Cdk/cyclin
And they inhibit the S phase so are degraded during S phase (Cdk2/cyclin A and Cdk2/cyclin E )
Outline how loss of Cdk inhibitors can affect cancer
Loss of a certain INK4 molecule is present in up to 80% of pancreatic cancer and ALL (the p16INK4a)
How can cyclin D be implicated in cancer
Overproduction of cyclin D involved in 50% of breast cancer and 90% of mantle cell lymphoma
Overproduction of Cdk4 involved in 40% of glioblastoma
How can retinoblastoma be involved in cancer
LOSS of Rb involved in 80% of small cell lung cancer
Thus, what type of genes are INK4, Cdk4, cyclin D and Rb
INK4, Rb tumour suppressor
Cdk4/cyclin D oncogenes
How many members of INK4, and how many of CIP/KIP
INK4= 4 members CIP/KIP= 3 members
T/F Ras is a G protein
This is true.
It binds GTP
