Growth Autonomy 1 Flashcards
What is passage through the cell cycle phases co-ordinated by?
(1 mark)
Cyclins and CDKs
What are cyclins and what do they do?
(2 marks)
- Protein that binds to and activates cdks (v specific binding)
- Concentration dependent on transcription of its gene and is regulated at degradation of protein level
What are Cdks and what do they do?
(3 marks)
- Serine/ threonine kinases - phosphorylate target proteins on either serine or threonine
- Cellualr concentration doesn’t vary
- Regulatory mechanisms include:
- association with cyclins
- association with cdk inhibtors e.g. p21, p27
Which cyclin is the molecular link between growth factors and cell proliferation?
Cyclin D
What happens in teh G1 and G2 phase of the cell cycle?
(1 mark)
Cells prep themselves for division
What happens during the G0 phase in the cell cycle?
(1 mark)
- Most cells outside the cell cycle
- GF send signalling to triggered cell to go into cell cycle
What does cycle D bind to in the cell cycle and what does this do?
Binds to cdk4/6 and pushes cell past restriction point- stablitity reliant on GF signalling
What are growth factors?
(4 marks)
- Peptides that interact with cognate receptors mainly receptors tyrosine kinases
- Mitogens that drive mitosis and cell differentiation
What is progression through G1 checkpoint facilitated by?
(1 mark)
Co-operation of different Ras induced signal transduction
Outline the EGF signallling pathway.
8 marks
- Many signal transduction pathways set off from growth receptors
- EGFR dimerises activates the kinase in the intracellular region which will phosphorylate its target tyrosine which is a tyrosine in the opposite receptor
- Phosphorylated tyrosines are docking sites for signalling molecules to lock onto and start relaying signal
- Adaptor protein GRB2 recognises the phosphorylated tyrosine binds onto it so it changes it shape reveals a binding site for SOS so binds onto GRB2 reevals a binding site allowing it to bind onto Ras
- RAS active swapping molecule of GDP for GTP, can set off two pathways. Promotes activation of Raf which activates MEK and then MAPK
- RAS can also bind pi3k and convert pip2 into pip3 facilitating action of AKT
- Pathways cooperate together block one of these pathways block cell proliferation
What is Raf?
2 marks
- Kinase and activated Raf carries teh signal away from teh membrane and phoshporylates MAPKK and MEK
- Exists as inactive monomer in cytoplasm, binds active Ras and subsequently dimerises
What is ERK activated by?
1 mark
Its a key MAPK activated by growth factors - MAPKs affect activity of trasncription factors via phoshporylation
What is the most prevalent kinase cascade?
2 marks
MAP kinase cascade 3 - major families of MAPKs
First kinase is MAPKKK, MAPKK and third is MAPK
What are the MAPK pathways?
1 mark
MAPK, JNK, p38 - normally trigger apoptosis
Which Raf isoform is commonly mutated in cancer?
B-Raf
What happens in teh Raf/MEK/ERK pathway when ras is active?
2 marks
- Active Ras binds to Raf
- Raf needs to form dimer with another raf molecule, kinase has to be phosphorylated to activate it
What does Raf go on to activate in the RAF/MEK/ERK pathway?
2 marks
Raf goes on to activate MEK, MEK then phoshporylayes and activates ERK, then ERK can go off into nucleus and affect teh molecules driving the cell cycle
What is the AP-1 transcription factor?
3 marks
- Dimer composed of Fos and Jun protein
- Binds to TPA/CRE response elements in target gene e.g. cyclin d
- Binds to DNA regulates expression of genes involved in growth, differentiation adn cell death
How does ERK induce AP-1 levels and activity by?
2 marks
- Phosphorylating and activating other transcription factors that regulate Fos and Jun gene expression
- Phosphorylation of Fos in AP-1 dimer which increases its DNA binding ability - AP-1 activity increases so does continuation of transcriptional regulation
How does Ras effect cell-cycle components by Raf/MEK/ERK signalling?
(4 marks)
- Induces expression of cyclin D - by activation of AP-1 transcription factors
- Increases levels of cdk inhibitor p21
- Promotes assembly of cyclin d - cdk4 complexes by interactions with p21
- Reduces level of cdk inhibitor p27
- With complex of cdk4 and p21, it helps cyclin d bind to complex thus reducing cdk inhibtor p27
What effects does Ras have on cell cycle components?
4 marks
- Prevents proteosome-mediated degradation of cyclin D - by inhibition of GSK3b
- Represses transcription of cdk inhibitors p21 and p27 - by p_hosphorylation and inactivation_ of FOXO4
- _Phosphorylates and inhibits the cdk inhibitor p2_1 - p21 exits nucleus when phosphorylated
What effects does Ras have on Akt signalling?
6 marks
- Cyclin d protein needs to stay stable and secure in cell
- Normally cyclin d phosphorylated by kinase gsk and targeted for degradation by proteosomal pathway
- AKT signalling phosphorylates the gdk, so cyclin d starts to accumulate in cell
- Akt can prevent trasncription of p21 and p27 gene so get balance right ot get just enough p21 to promote cyclin d formation
- Akt does it by inactivating TF aka as fox04 – so switches off all ^ genes (activated by foxo)
- Akt signalling preventing apopotosis occurring
- As well as stopping trans of p21 and p27 can directly phosphorylate protein and come out nucleus and cant act as transcription factors as not in the right place to do so.
What is MYC?
3 marks
Transcription factor
Short lived protein that promotes proliferation by regulating expression of specific target genes
Gene targets: N-Ras and p53
Needs Max – constitutively active family member to function – form heterodimers via basic helix-loop-helix leucine zipper domains and bind to E-box’s in target genes
How is the RB protein regulated by growth factors?
5 marks
- Rb bound to e2f and histone deactylases (HDAC) - binding to e2f blocks its transactivation domain so cant act with general TF
- E2f expression inhibited by HDAC
- Cyclin d binds to cdk4 and activates it and phosphorylates RB – phosphorylates it on b part of molecule allowing HDAC to move away relieving suppression on some genes
- So cyclin e can be transcribed and bind to cdk and activates it so cdk2 phosphorylates RB on different part of protein allowing it to come away from E2f and allow transcription of e2f sensitive genes which are involved in s phase
- Cell now pushed past g1 restruction point and irreversibly committed to cell cycle
What is teh role of RB?
Major point of control from G1 phase to S phase. Process regulated by serine/threonine phosphorylation
What are teh 2 steps of phoshporylation for RB?
3 marks
- Cyclin d cdk4 phosphorylate carboxyl-terminal residues of RB upon GF stimulation
- Cyclin e gene expressed after HDAC release.
- Cyclin e cdk3 phosphorylates additional AA residues of RB i.e. ser567 close to linker region causes conformational change of RB pocket domain and release of E2f and exp of its target genes i.e. cyclin A
What are the diffreent methods that can be explored for anti-cancer stratergies that target growth receptor signaling?
2 marks
- Monoclonal antibodies
- Small molecule inhibitors
What is the difference between the direct and indirect approach of mononclonal antibodies?
Indirect: maniuplation of immune cells
Direct: inducing cell death
What do monoclonal antibodies do?
- Bind to extracellular domain of receptor and stop ligand from binding
- Induce antibody dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP)
What happens after a monoclonal antibody is bound to the extracellular domain of receptor?
5 marks
- NK cells recognizes antibodies locks onto it and induces antibody dependent cellular excitoxicity (ADCC) or antibody dependent cellular phagocytosis (ADCP)
- mAbs can block binding of activating ligand or dimerization of a receptor therefore inhibiting signal transduction or inducing apoptotic signal
- Immune system kicking in so activate complement system and antibody cellular phagocytosis mediated by macrophages
- E.g. cetuximab – main drug used for colorectal cancer – targets EGFR - enhances internalization of receptor and degradation inside the cell
- Can tag monoclonal antibodes can tag them with toxins for targeted delivery
What are small molecule inhibitors and what do they do?
2 marks
Competitively bind to the ATP binding site in the tyrosine kinase
Prevent phosphorylation and subsequent signal transduction
What are the different types of small molecule inhibitors?
6 marks
- Type 1 – mimic hydrogen bonds presented by ATP – occupy adenine region in ATP binding site – most highly conserved region in kinases so quite difficult to make highly specific type 1 inhibitor
- Type 2 – occupy nearby hydrophobic pocket in an inactive kinase and allosterically affect kinase activity. More selective. Hydrophobic pockets show a bit more variability between different kinases so can be exploited to make more specific inhibitor
- Type 3 – covalently bind specific cysteines in target kinase e.g. the quinazolines. Specifically bind to cystines found in targeg kinases and quinazoline drugs act in this way and is specific for EGFR TRK
Why may multiple receptors need to be targeted in cancer therapy?
- As a result of redundancy in fucntionality in close family members or heterodimerization between family members of recepotrs e.g. erbB family - teh specific targeting molecule may have reduced efficacy
