Cancer 5: Signalling mechanisms of growth and division Flashcards
What causes cell to go into the G0 phase?
Absence of growth factors.
What is c-Myc?
This is an oncogene - overexpressed in tumours. It is a transcription factor - stimulates the expression of cell cycle genes
What are the key components of signalling pathways?
- Regulation of enzyme activity by protein phosphorylation (kinases)
- Adapter proteins
- Regulation by GTP-binding proteins
Describe the growth factor stimulation signalling pathway?
1) Mitogenic growth factor (Hepatocyte growth factor released after liver damage)
2) Receptor protein tyrosine kinase
3) Small G (GTP-binding) protein (Ras)
4) Kinase cascade
5) Immediate early genes (c-jun, c-Fos, c-Myc) - control the expression of other genes.
See slides
Describe what happens a peptide GF binds to a tyrosine kinase protein receptor
In the presence of a ligand the receptors form dimers. They are then activated by phosphorylation.
When phosphorylated it recruits adaptor and signalling proteins –> Bind to phosphorylated tyrosines.
Herceptin (antibody) is used to treat HER2 positive metastatic breast cancer. In many breast cancers the EGFR/HER2 is mutationally activated or over expressed. Herceptin blocks the binding of GFs.
Describe adaptor proteins?
- Tyrosine phosphorylation provides docking sites for adapter proteins
- Protein-protein interactions: protein binding – They bring proteins together so they can signal to each other.
Proteins are modular and contain domains, i.e. functional and structural units that are copied in many proteins. (Kinase domain)
Some domains are important in molecular recognition – have no enzymatic function of their own, simply bring other proteins together
What are GTP binding (G) proteins?
G proteins such as Ras bind to GTP to become activated. They are inactivated with the hydrolysis of GTP to GDP with GAP (GTPAse Activating Proteins).
The GDP bound to ras is exchanged with an exchange factor such as Sos.
What is the Grb2 protein?
Grb2 (adaptor protein) - contains 3 domains. SH3-SH2-SH3.
SH3 - proline rich (another docking site for other proteins)
SH2 - recognises phosphorylated tyrosines
Main function is to bind to Ras
Describe the function of Sos?
Sos is an exchange factor which is constitutively active (always) bound to Grb2 via the SH3 domains.
Exchanges GDP with GTP activating Ras proteins bound to the plasma membrane - essentially the RAS activating protein.
What are some mutations in RAS leading it to be oncogenically activated?
V12RAS - consitutatively active (prevents GAP binding - prevents inactivation)
L61RAS - constituatively active (Prevents GTP hydrolysis)
Both forms of mutations effectively ensure that RAS remains active.
See slides
What does the activated RAS do?
Activates a protein kinase cascade - by docking
This kinase will activate the protein kinase by phosphorlyating kinase I –> KII –> KIII
These kinases downstream of the EGFR and Ras are called: Extracellular signal-regulated kinase (ERK) cascade = (Generically - Mitogen-activated protein kinase (MAPK) cascades)
Describe the ERK cascade
Kinase I = Raf
Kinase II = MEK
Kinase III = ERK
The ERK modifies Myc and Ras (involved in cell proliferation) This means that both of the genes can be oncogenes if mutated or expressed in high amounts.
What is b-Raf?
Oncogene - mutationally activated in melanomas
What are cyclin-dependent kinases (Cdks)
Control cell cycle - phosphorylate proteins (serine or threonine) to drive cell cycle progression.
Present in proliferating cells throughout cell cycle
Activity is regulated by:
- Interactions with cyclins
- Phosphorylation
What are the cyclins?
Transiently expressed at specific points in the cell cycle
Regulated at level of expression
Synthesised, then degraded - highest during the mitosis phase of the cell cycle.
Different pairs of cyclins and Cdks control different parts of the cell cycle.
What do activated Cdks do?
They phosphorylate proteins (on Serine or Threonine) to drive cell cycle progression
Cdk1 binds to cyclin B (mitotic cyclin) –> phosphorylates nuclear laminins (causes breakdown of nuclear envelope)
Cdk2 binds to cyclin E (G1 cyclin).
Retinoblastoma protein (pRb) promotes G1 progression –> causes production of cyclin E.
Tumour suppressor - inactivated in many cancers
How are Cdks regulatated?
Regulated by phosphorlyation. Just binding of the cyclin to the Cdks will not activate it.
Requires activating phosphorylation (Cdk - activating kinase = CAK)
AND
removal of inactivating phosphorylation (Wee 1 inhibitory kinase)
1) Cyclin binds to Cdk
2) The cyclin Cdk complex is phosphorylated by CAK and Wee1.
3) It is still inactive so dephosporylation by Cdc25 (phosphatase) activates it at the end of interphase.
What causes cyclin B to be degraded?
Signals from fully attached kinetochores causes cyclin B to be degraded
- Cdk1 inactivated
- key substrates dephosphorylated
- mitosis progresses
What cyclins and Cdks are required at different stages at the cell cycle?
- Cyclins activate Cdks but also alter substrate specificity
- substrate accessibility changes through cell cycle
Mitosis - Cdk1 and cyclin B
G1–>S - Cdk2 and cyclin E
S–>G2 - Cdk2 and cyclin A
What do the genes c-jun, c-Fos and c-Myc do?
They stimulate the transcription of cyclins. Therefore GF are directly related to the regulation of the cell cycle promoting growth.
How does Rb protein regulate gene expression?
The retinoblastoma protein binds to and holds the inactive E2F transcription factor.
In the proliferating cells Cdk4/6 and cyclin D phosphorylate the Rb protein, inactivating it.
This causes the release of the active E2F transcription factor which transcribes cyclin E
pRb acts a brake on the cell cycle. The Cdks phosphorylate at multiple sites & progessively inactivate pRb. pRb is a tumour suppressor.
See slides
See slides for the progressive inactivation of pRb
See slides
What are CKIs?
Cdk inhibitors - These are often found mutated in tumours.
Two CKI families:
INK4 family - G1 phase CKIs: Inhibit Cdk4/6 by displacing cyclin D
CIP/KIP family - S phase CKIs: Inhibit all Cdks by binding to the Cdk/cyclin complex.
p27^KIP1 tumour suppressor - reduced expression correlates with poor prognosis in many malignancies.
In normal cell cycle the CKIs have to be degraded at their appropriate step.
