5: Signalling & Mechanisms in Growth/Division Flashcards
How is entry into cell cycle controlled?
c-Myc is an oncogene overexpressed in tumours
Its a transcription factor that stimulates expression of cell cycle genes
GFs increase Myc levels
What are the key components of signalling pathways?
- Kinases - regulation of enzyme activity
- Adapter Proteins
- GTP-binding proteins (G-proteins)
Describe the GF stimulation of signalling pathways
- Mitogenic growth factor
- RPTK
- Small G-Protein (Ras)
- Kinase cascade
- Immediate early genes (e.g. c-Myc which is a TF) control expression of other genes
What are adaptor proteins?
Proteins that recognise and bind to specific phosphorylated tyrosines
What GFs are overexpressed in breast cancers and how do we treat it?
EGFR/HER2
Use Anti-HER2 antibody (Herceptin) to block binding of ligand to RPTK
Describe the structure of Grb2
SH2/SH3 regions
SH2 recognises phosphorylated tyrosines
SH3 is proline-rich region, docking site for other proteins
What does Grb2 do?
Recruits Exchange Factor Sos, which is a Ras activating protein
How is Ras activated?
Exchange Factor (like Sos) exchanges GDP on Ras to GTP
What is the name of proteins that inactivate G proteins like Ras?
GTPase Activating Proteins (GAP)
What are common oncogenic mutations of Ras?
V12Ras (glycine -> valine at position 12) prevents GAP binding
L61Ras (glutamine -> Leucine at position 61) prevents GTP hydrolysis
What happens when Ras is activated?
Ras activates ERK cascade (Extracellular signal Regulated Kinase)
What kinases are involved in the ERK cascade?
- Raf
- MEK
- ERK
Give an example of a type of cancer originating from the ERK cascade and state its cause
Melanoma caused by activation of the oncogene B-Raf
What are the two main effects of ERK cascade?
Altered gene expression
Altered protein activity
How do growth factors/ERK cascade affect cell cycle?
Expression of proteins that go inside the nucleus and upregulate Myc, which regulates cell cycle and proliferation. Myc is also an oncogene
What proteins primarily regulate the cell cycle?
Cdks (Cyclin-dependent Kinases)
Present in proliferating cells throughout the cell cycle
CYCLICAL activity regulated by cyclins and phosphorylation
What are cyclins?
Proteins that bind to and activate Cdks triggering different events in the cell cycle
Transiently expressed at specific points of cell cycle (i.e. not there all the time - up/downregulated)
What are cyclins?
Proteins that bind to and activate Cdks triggering different events in the cell cycle
They also alter substrate specificity in Cdks
Transiently expressed - synthesised and quickly degraded at specific points of cell cycle, hence cyclical
Give 2 examples of Cdk/Cyclin complexes and the proteins that they can phosphorylate
Cdk1 + cyclin B - phosphorylates Nuclear Lamins causing breakdown of nuclear envelope
Cdk2 + cyclin E - phosphorylates Retinoblastoma proteins (tumour suppressor)
Describe how Cdk activation is regulated
When cyclin binds to Cdk it is still inactive
2 phosphorylations occur: activating phosphorylation (by Cdk-activating Kinase) AND inactivating phosphorylation (Inhibitory Kinase)
Dephosphorylation of inactivating phosphate by Cdc25 phosphatase activates Cdk1 at the end of interphase (before mitosis starts)
There are also Cdk inhibitors (CKIs)
How do Cdks regulate mitosis?
- Cdk1/cycB is active during first stages of mitosis
- It phosphorylates key substrates and puts mitosis on hold
- At the mitotic checkpoint (before anaphase) signals from fully attached kinetochores causes degradation of cyclin B
- This inactivates Cdk1 and dephosphorylates the key substrates
- Mitosis continues
What Cdk/cyc complexes are required in the different stages of the cell cycle?
Mitosis: M-Cdk (Cdk1/cycB)
G1 phase: G1-Cdk (Cdk4/6/cycD)
G1/S phase: G1/S-Cdk (Cdk2/cycE)
S phase: S-Cdk (Cdk2 cycA)
How can GF stimulation of signalling pathways promote G0 to G1 transition?
GF activates immediate early gene transcription factors such as c-jun, c-Fos and c-Myc
c-Myc stimulates transcription of cyclin D
Cyclin D activates Cdk4/6 (G1-Cdk) which stimulates production of cyclin E
Cyclin E activates Cdk2 (G1/S-Cdk)
State why regulated expression of Cdks/cyclins is important in the cell cycle
Cdks become sequentially active and stimulate synthesis of genes/proteins required for the next phase
This gives DIRECTION and TIMING to the cycle
How do Retinoblastoma cells regulate the cell cycle?
In G0, activated Rb protein binds to E2F transcription factor and inactivates it (holds it), preventing expression of cyclins like cyclin E
Cdk4/6/cycD PHOSPHORYLATES the Rb protein at multiple sites and INACTIVATES IT, releasing the E2F and resulting in expression of cyclin E which is required for progression of cell cycle
Therefore Rb is a TUMOUR SUPPRESSOR
Summarise the sequence of interactions that occur between Cdk, cyclin and Rb proteins throughout the cell cycle
c-Myc makes cyclin D
Cyclin D activates Cdk4/6 which phosphorylates Rb
Rb releases E2F which makes Cyclin E
Cyclin E activates Cdk2 which phosphorylates Rb more
Rb -> E2F -> Cyclin A
Cyclin A -> Cdk2 -> phosphorylates Rb more -> E2F -> Cyclin B -> Cdk1 -> Mitosis
What are the two families of Cdk inhibitors and how do they work?
INK4 (G1 phase CKIs): Inhibit Cdk4/6
CIP/KIP (S phase CKIs): Inhibit ALL Cdks
CKIs MUST be degraded for cell cycle progression
What is an example of a CKI tumour suppressor?
p27KIP1
