5. Signalling mechanisms in growth and division Flashcards
What is c-Myc?
• (Proto-)oncogene + transcription factor
• As soon as growth factor binds to cells, c-Myc is expressed
• Over-expressed in many tumours
- stimulation to express cell cycle genes
Outline the expression of c-Myc throughout the cell cycle
- Low conc. in G0
- If you trigger cell division, rapid and dramatic rise in Myc
- Peaks then goes down going into the S phase
- Plateaus at intermediate level in the S phase
- Stays at that level into G1
What binding protein does a growth factor act via when it binds to a (tyrosine kinase type) receptor and what does this trigger?
- GTP-binding protein (Ras)
- This triggers a kinase cascade (fast)
- Activation of genes required for the progression through the cell cycle (slow - requires transcription + translation)
At what point of the signalling pathway is c-Myc triggered?
Early in the kinase cascade
What are mitogenic growth factors?
Growth factors that induce mitosis in cells
What is herceptin, what does it do and why is it important in tumours?
- Anti-HER2 antibody
- Blocks the early stage of growth stimulation
- Important as HER2 can be over-expressed in a number of tumours e.g. breast cancer
What are one of the important adaptor molecules that are recruited to activated tyrosine kinase receptors?
Grb2 - important for bringing Ras onto the signalling unit
Which part of ATP do tyrosine kinases use to phosphorylate tyrosine residues in proteins?
Gamma phosphate of ATP
What do the phosphorylated domains on the tyrosine kinase receptors act as?
Docking sites for adaptor proteins
Describe function of the domains of adapter proteins
- Functional/structural units that are copied in many proteins
- Mixed and matched to give the protein different properties
- Important in molecular recognition
- No enzymatic function - only bring proteins together so enzymes and substrates meet
How many domains does Grb2 have and what are they?
- 3 domains
- SH2 - binds to the phosphorylated tyrosines of the receptor
- (2x) SH3 - binds to the proline-rich regions of other protiens
What is Ras?
- GTP-binding protein (+ pro-oncogene)
* Can either be on (GTP-bound) or off (GDP-bound)
Is the switching on of Ras phosphorylation?
- No
* Merely the exchange of GDP for GTP (catalysed by Sos)
How can Ras turn itself off?
Can hydrolyse GTP do GDP itself (intrinsic GTP hydrolysis capability)
How can Ras be turned on and off externally?
- On - Exchange factors e.g. Sos
- Off - GTPase activating proteins (GAPs)
Almost always control the cycle
Are the GTP binding proteins kinases?
No
What happens to Ras in cancer?
- Mutated to always be in the GTP bound form
* Leads to uncontrolled division
Which domain is Grb2 bound to the RPTK via?
SH2 domain
Which domain is Grb2 bound to Sos via?
SH3 domains
Outline how the activation of RPTK leads to the activation of Ras?
- RPTK activated - phosphorylated
- Grb2, with Sos attached, binds to the phosphorylated tyrosine domains
- Sos is then close enough to the membrane to activate Ras (GDP => GTP)
- Activated Ras can signal downstream
Where does the Ras protein need to be bound to work?
Plasma membrane (interference with this can make a good anti-cancer therapy)
What does V12Ras describe, with reference to mutations?
- Glycine => valine (position 12 of Ras)
- Simple hydrogen => hydrophobic side chain
- Prevents GAPs from binding to Ras
- Ras can’t be turned off easily and constantly gives growth stimulatory signals
What does L61Ras describe, with reference to mutations?
- Glutamine => leucine (position 61 of Ras)
- Single base change
- Amide => hydrophobic side chain
- Prevents intrinsic GTPase hydrolysis
- Ras is constantly turned on and giving growth stimulatory signals
What is the specific kinase cascade for growth stimulatory signalling and the generic name for the cascades?
- Growth stimulatory signalling: extracellular-signal-regulated kinase (ERK) cascade
- Generic: mitogen-activated protein kinase (MAPK) cascade
Which oncogene can be blocked to temporarily prevent melanomas from growing?
B-Raf
What are cyclin-dependent kinases?
- Family of kinases
- Serine-threonine kinases (not tyrosine kinases)
- In the cell throughout the cell cycle
- Not active until they bind to cyclin
- Also controlled by phosphorylation (extra level of control)
Describe how cyclins work
• Transiently expressed during the cell cycle (specific points of the cell cycle)
• Expressed during mitosis
• Cyclins dock onto the cyclin-dependent kinases => activate them
• Activated CDKs phosphorylate proteins
• Degrade once they activate the CDKs and it has fulfilled its role
- low during interphase, then rise again into mitosis
What is the M-phase promoting factor?
- A type of cyclin-CDK complex (cyclin B/mitotic cyclin + CDK1)
- Triggers the mitotic machinery
What happens when S-cyclin binds to CDK?
- Induces protein synthesis
* Triggers DNA replication machinery
Give an example of the substrates that M-phase promoting factor phosphorylates and the effect this has
Nuclear lamins (proteins that form the nuclear membrane) => this breaks down the nuclear envelope
Describe how the cyclin-CDK complex is further activated in MPF?
- CDK activating kinase (CAK) => activating phosphorylation of CDK1
- Wee1 => inhibitory phosphorylation of CDK1 to balance this
- Cdc25 (phosphatase) then takes off the inhibitory phosphate put on by Wee1
- MPF is activated
- Active MPF can phosphorylate Cdc25 to increase its activity (positive feedback) => pushes cell through mitosis to metaphase
- Mitosis is then put on hold
When is a signal released to cause cyclin B to be degraded?
- When kinetochores are correctly attached to the microtubule spindles => CDK1 becomes inactive
- Mitosis can then progress from metaphase
Which cyclins and CDKs control G1/S and S?
- G1/S - cyclin E + CDK2
* S - cyclin A + CDK2
What does cyclin do to CDK to activate it?
Changes its substrate specificity and accessibility - so it phosphorylates different substrates depending on which cyclins are bound
Which cyclin does c-Myc stimulate the transcription of?
cyclin D
What does cyclin D do?
- Binds to CDK4 and CDK6
- Controls the entrance to the cell cycle
- Therefore (c-Myc and) cyclin D regulates the G0 => G1 transition
What does cyclin D/CDK4/CDK6 stimulate the expression of and what does this do?
- cyclin E
- Gives direction and timing to the cycle
- Gives timing as it takes time for the conc. of the cyclin to build up
What is the retinoblastoma protein and describe how it works?
- Tumour suppressor protein (acts as a brake in the cell cycle)
- Present throughout the cell cycle
- Unphosphorylated (active) in G0
- Binds to and sequesters (hides) E2F (transcription factor)
- E2F is held in the cytoplasm and everything is turned off
- CDK4/6-cyclin D kinase becomes active following c-Myc induction
- It phosphorylates the retinoblastoma protein (inactivation)
- RB loses affinity for E2F and releases it
- E2F can bind to promoter of genes involved in cell cycle progression (in nucleus) e.g. gene for cyclin E
What happens to the levels of retinoblastoma protein in tumours?
Reduced levels => E2F activity unregulated => cell cycle progression uncontrolled
What genes does E2F transcription factor control?
- Proto-oncogenese e.g. Myc proteins
- Genes involved in the S phase e.g. thymidine kinase
- cyclin E
Summarise the progression from c-Myc to cyclin E
- c-Myc turns on cyclin D
- cyclin D complexes with CDK4/6
- The complex phosphorylates retinoblastoma, deactivating it
- Inactive RB releases E2F
- E2F stimulates the production of cyclin E
What does cyclin E do?
- Forms complex with CDK2
- Complex further phosphorylates retinoblastoma protein
- Further increase in E2F - to the point it now binds to targets with lower affinity
- cyclin A starts to be transcribed instead
- cyclin A leads to the production of a transcription factor that causes cyclin B-CDK1 complex to be formed
- Mitosis induced
What are the 2 families of CDK kinase inhibitors (CKIs)?
- INK4
* CIP/KIP
What do CDK kinase inhibitors (CKIs) do?
- Proteins that dock onto the cyclin-CDK complex
* Render it inactive
When are INK4 inhibitors active and what do they do?
- Active in G1
* Inhibit CDK4/6 by displacing cyclin D
When are CIP/KIP inhibitors active and what do they do?
- Active in S phase
* Inhibit all the cyclin-CDK complexes by binding to them
What is p27KIP1 and what does reduced expression correlate with?
- Tumour suppressor in the CIP/KIP family of CKIs
* Reduced expression correlates with poor prognosis in many malignancies
Are CDK kinase inhibitors (CKIs) degraded?
Yes, they have to in order for the cell cycle to progress
Are cell surface tyrosine receptors oncogenes or lost tumour suppressor genes in cancer?
Oncogenes e.g. mutationally activated or over-expressed HER2
Is Ras mutationally activated or over-expressed in cancers?
Mutationally activated
