Signalling Mechanisms of Growth and Division Flashcards
What is c-Myc, and briefly just say its function without going into detail?
- It is a transcription factor that increases following growth factor stimulation from G0 into G1 and then falls again at S phase. It is a transcription factor for genes involved in the cell cycle, so it enables cell cycle progression
- It is also an oncogene - often overexpressed in cancer
Outline the stepwise signalling pathway process that occurs when mitogenic growth factor binds a receptor to cause cellular growth and proliferation, stopping at the proteins involved in the cell cycle (not mentioning what these are and what they do in this question yet)
Note: use Grb2, Ras and c-Myc as your example molecules for some of the appropriate steps in the pathway
This is a huge question, large pathway
- Mitogenic growth factor (often dimeric) binds a monomeric receptor
- This receptor is essentially dimerised by this growth factor
- This brings certain protein kinase domains nearby within the receptor
- This results in cross-phosphorylation of various amino acid residues including serine, threonine and tyrosine by their kinases, using ATP to transfer phosphates to the amino acids
- This phosphorylation results in conformational change in the receptors which allows the binding of adapter proteins such as Grb2
- Grb2 has both SH2 and SH3 domains which selective for phosphorylated tyrosine and for proline-rich regions on other proteins. This allows it to anchor onto the receptor and also hereby influence downstream signalling
- Grb2 through its SH3 domain binds Sos protein which is an exchange factor which allows exchange of GDP for GTP
- GTP is now available for Ras to bind and become activated
- Ras, from this GTP-Ras complex can now interact with RAF
- RAF results in further downstream signalling via kinase cascades, in this case the extracellular signal regulated kinase (ERK) cascade (a type of MAPK - mitogen activated protein kinase cascade)
- This eventually phosphorylates gene regulatory proteins, which in turn phosphorylate c-Myc
- c-Myc is a transcription factor for genes for proteins involved in the cell cycle
Describe the signalling and interactions involved in the Ras-GTP sort of ERK pathway (a type of MAPK pathway). You must also go into how it has its effect on cell cycle proteins to ultimately impact cell growth and proliferation but need not detail these cell cycle proteins or their functions
This includes what stimulates or brings about everything and then also anything that plays an inhibitory role
- Sos protein is an exchange factor that exchanges GDP for GTP (note there are no phosphorylation / dephosphorylation bits at this point yet!)
- The GTP is carried on a GTP-binding protein
- The Ras binds the GTP and is activated
- Note that GTP binding protein has intrinsic hydrolytic activity against the GTP to inactivate it by converting it into GTP
- The activated Ras on the Ras-GTP (+GTP binding protein) complex can now interact with RAF to trigger further downstream signalling as part of the ERK pathway … eventually resulting in phosphorylation of gene regulatory proteins and thence of c-Myc for example or other transcription factors which will influence the expression of cell cycle proteins and thus impact cellular proliferation and growth (increase them)
- GAP - GTPase Activating Proteins can also hydrolyse the GTP into GDP to turn off or inactivate Ras to silence this ERK pathway
- Ras also has some intrinsic hydrolytic activity to hydrolyse GTP and thus inactivate itself, its an autoregulatory mechanism
Describe the mutations of Ras that can result in oncogenic activation and the pathophysiology of these cause cancer, then name these mutant forms
- V12Ras: Glycine in position 12 of Ras is changed into VALINE. Thus the side chain goes from being a simple hydrogen (from glycine) to a hydrophobic side chain (from valine). This prevents GAPs (GTPase activating proteins) from binding Ras, thus meaning that Ras is constantly binding GTP and activated, so the ERK pathway is hyperactive, resulting in XS cell growth and proliferation - cancer
- L61Ras: Glutamine in position 61 is converted to LEUCINE. The side chain goes from an amide to a hydrophobic side chain. This impairs the intrinsic hydrolytic activity of Ras, therefore there is more GTP for Ras to bind and be activated, resulting in a hyperactive ERK pathway and thus XS cell growth and proliferation - cancer
Cyclin dependent kinases are ….. ….. kinases, not ….. kinases
Cyclin dependent kinases are serine threonine kinases, not tyrosine kinases
What are 3 things that regulate the activity of CDKs (cyclin dependent kinases)?
- Cyclins
- Phosphorylation
- CKI (cyclin dependent kinase inhibitors)
How is cyclin activity regulated?
- Regulated at the level of expression
- They are highly expressed during mitosis and low level of expression when cells are not dividing
- Cyclical activity
- Once they have fulfilled their activity, they are degraded
Briefly list which cyclins and CDKs act at different stages of the cell cycle
- G0 to G1: CDK4/6 and cyclin D
- G1 to S: CDK1 and cyclin E
- S: CDK2 and cyclin A
- M: CDK1 and cyclin B
Once the mitogenic growth factor binds its receptor, skipping past all the receptor activation then all the ERK pathway (all the intracellular signalling) up until the activation of c-Myc, describe what changes that activation of c-Myc or other transcription factors will result in, in terms of the downstream effects of the cycle with regards cell cycle proteins
- c-Myc is a transcription factor which stimulates the production of cyclin D
- Cyclin D activateds CDK4/6 to kickstart the cell cycle from G0 to G1
- Then there is sequential activation of the following cyclins by and thus also their partner CDKs at the other points in the cell cycle as the activated cyclins activate other cyclins involved in later parts of the cell cycle, like a domino effect
- Hence the whole pathway results in taking the cell through the cell cycle and thus increasing cellular growth and proliferation
Which cyclin and CDK is involved in kickstarting the cell cycle with the G0 to G1 transition and suggest one major target of this pathway and how it may lead to progression to the next stage of the cell cycle
- CDK4/6 and Cyclin D
- Major target is Rb
- At G0 (at rest) Rb protein sequesters E2F transcription factors
- These E2F transcription factors are for genes that code for cyclin E - the cyclin for the next phase of the cell cycle (from G1 to S). Therefore E2F allow the progression through the cycle through cyclin E
- So CDK4/6 phosphorylates the Rb protein and this causes conformational change which lowers its affinity for these E2F transcription factors so they are released
- Now these transcription factors are free to increase expression of cyclin E and progress the cell cycle
- CDK2 and cyclin-E forms a complex which further phosphorylates Rb to further release E2F and it can bind targets with lower affinity
- This lets it increase transcription of cyclin A - continuing the domino effect and so on
1) Which cyclin and CDK are involved in the G1 to S phase of the cell cycle?
2) Describe one of their major actions for sequential activation of the CDK and cyclin for the next phase of the cell cycle
1)
- CDK2 and cyclin E
- They have some function in enabling G1 - this wasn’t really detailed in the lecture but yeah
2)
- CDK2 and cyclin E, like CDK4/6 and cyclin D will phosphorylate Rb proteins which sequester E2F transcription factors which are transcription factors for genes coding cyclin E and also….
- The promoter for the gene for cyclin A is also only activated upon the concentration of E2F transcription factors reaching a certain level
- So this further phosphorylation by CDK2 and cyclin E allow progression to the next phase of the cell cycle by enabling cyclin A and CDK2
- Note that the same CDK2 has different actions based on the cyclin it is paired with
Which cyclin and CDK is involved in the S phase and describe the actions it has at this phase of the cell cycle?
- CDK2 and cyclin A
- They will trigger DNA replication machinery to enable S-phase
1) Which cyclin and CDK is involved in mitosis in the cell cycle - also what is another name for this complex?
2) Describe the ways the CDK is activated
3) What actions will it have in the cell cycle, give an example?
4) How is progression from mitosis enabled?
1)
- CDK1 and cyclin B
- Mitosis promoting factor (MPF)
2)
- Cyclin is one part of the activation
- CAK (CDK activating kinase) places an activating phosphorylation on CDK1
- Wee1 places an inhibitory phosphorylation on CDK1
- Cdc25 is a phosphatase which removes this inhibitory phosphate placed by Wee1
- CDK1 itself has a positive feedback effect. It phosphorylates Cdc25 to increase its activity which is as a phosphatase to remove the inhibiting phosphorylation placed on it by Wee1
3)
- It enables mitosis
- For example CDK1 will phosphorylate nuclear laminins which are part of the nuclear membrane to cause degradation of the nuclear envelope in mitosis
4)
- Towards the middle of mitosis, CDK1 and cyclin B (MPF) actually has a contradictory inhibiting effect on mitosis
- CDK1 puts a brake past metaphase by phosphorylating some proteins, i.e. it doesn’t allow progression past metaphase until signalled by a signal that is achieved once the kinetochores have been attached by the microtubule arrays from centrosomes (the mitotic spindle). This occurs via BUB-A and CENP-E on the kinetochores
- Once this signal is sent, this causes degradation of cyclin B and thus dismantles the MPF and thereby stops the inactivating phosphorylation that MPF has on proteins essential for steps past metaphase, allowing progression through the rest of mitosis
Describe how CKIs (cyclin dependent kinase inhibitors) can regulate the activity of CDKs and mention 2 families of CKIs and the CDKs / cyclin complexes they inhibit
INK4 FAMILY
- Inhibit G1 phase by inhibiting CDK4 / 6 by displacing cyclin D
CIP / KIP FAMILY
- Inhibit S phase in particular
- But inhibit ALL CDKs and cyclins
What is p27KIP1 and how is it implicated in cancer?
- It is a tumour suppressor gene which codes for the CIP / KIP family CKIs (cyclin dependent kinase inhibitors) which inhibit ALL CDKs and cyclins but are particularly in inhibiting S phase
- So inhibition of this TSG in cancer causes poor prognosis in tumour progression
