Signalling Mechanisms of growth and division Flashcards
When are cells in the quiescent phase
Most adult cells are not constantly dividing
In the absence of growth signals they enter G0 e.g. liver hepatocytes
What is c-Myc
Key role in cell cycle entry and G1
Transcription factor that stimulates the expression of cell cycle genes. Once a growth factor binds to a cell, levels of Myc go up.
What are the key components of signalling pathways
Regulation of enzyme activity by protein phosphorylation (kinases)
Adapter proteins
Regulation by GTP-binding proteins
Describe growth factor stimulation of signalling pathways in the first 15 minutes
(first 15 mins)
1. Mitogenic growth factor binds to a receptor protein tyrosine kinase (RPTK)
2. Phosphorylation of RPTK recruits adaptor and signalling proteins
3. Activation of Ras and docking site formation
4. Ras binding stimulates and mediates early-response gene expression from the
MAPK/ERK cascade, eg c-Myc expression
What happens in the second phase (hour) of growth factor stimulation of signalling pathways
Stimulation of expression of delayed-response genes, leading to the activation of the cell-cycle control system
What does tyrosine phosphorylation do
Provides docking sites for adaptor proteins e.g. Grb2 (growth-factor receptor binding protein 2)
Describe the importance of protein function in protein-protien interactions
Proteins are modular and contain domains
Some domains are important in molecular recognition (have no enzymatic function, simply bring other proteins together)
Give examples of mitogenic growth factors
growth signals from other cells, e.g. Hepatocyte Growth Factor released after liver damage
What is the effect of a monomer substrate on the receptor tyrosine kinases
Receptors exist in monomers
The ligand is a dimer
Therefore it brings two monomers of tyrosine kinase receptors together
Describe what occurs in the FG signalling pathway when adapter proteins bind to the RPTKs
- Binding of Grb2 recruits inactive Ras protein (associated with a GDP molecule) to the cytosolic surface of the plasma membrane
- Ras-activating proteins are then able to activate Ras.
- Use of exchange factor Sos, which exchanges a phosphate from a GTP molecule associated with the ras-activating protein for the GDP molecule associated with Ras
- The Ras complex with GTP is now active
What are the potential drug targets for adapter proteins
Grb2 binding and Ras activation
Describe what occurs in the GF signalling pathway after Ras has been activated
G-proteins act as a molecular switch to turn Ras off by GTP hydrolysis (GTP associated with Ras) using GTPase activating proteins (GAP)
Describe the structure of Grb2 and what its regions bind to
SH3 - SH2 - SH3
SH3 - proline-rich regions (constitutive)
SH2 - phosphorylated tyrosine (inducible, specific sequence context)
Describe the oncogenic activation of c-Myc
Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation
Mutations that increase the amount of active GTP-loaded Ras (either prevent GAP binding, or prevent GTP hydrolysis)
Which protein kinase cascade is activated by Ras
Extracellular signal-regulated kinase (ERK) cascade
Generically - Mitogen-activated protein kinase (MAPK) cascades
What is the cascade associated with Kinase I (generic and specific)
MAPKKK
Raf
What is the cascade associated with Kinase II (generic and specific)
MAPKK
MEK
What is the cascade associated with Kinase III (generic and specific)
MAPK
ERK
What is B-Raf
Oncogene
Mutationally activated in melanomas
What is the role of protein kinases
Stimulates changes in cell proteins and genes expression (e.g. c-Myc) to promote division
Describe Ras and c-Myc’s role in tumour formation
Mutated Ras and c-Myc act as oncogenes, leading to uncontrolled cell division and tumour proliferation
Which molecules control the cell cycle
Activated protein kinases, known as cyclin-dependent kinases (Cdks) which are present in proliferating cells throughout the cell cycle
What regulates the activity of cyclin-dependent kinases
Regulated by their interaction with cyclins, and their phosphorylation
What are cyclins
Cyclins are proteins which activate Cdks
They are transiently expressed at specific points in the cell cycle, and this level of expression is highly regulated
They are synthesized, but then degraded
What does cyclins form to trigger events in the cell cycle
They form cyclin-Cdk complexes with different complexes
What forms the mitosis promoting factor (MPF)
Mitotic cyclin (B) and Cdk1
Describe the activation of the MPF complex
Activating phosphorylation by CAK (cdk activating kinase)
Removal of inactivating phosphorylation by inhibitory kinase Wee1
Balance of Wee1 and CAK determines activation
When does removal of Wee1 occur and what does this drive
Occurs at the end of interphase using CDC25 phosphatase
Drives progression into mitosis
Which cyclins and Cdks are required during G1
Cyclin E and Cdk2
Which cyclins and Cdks are required during the S phase
Cyclin A and Cdk2
Give an example of how the cell cycle can be controlled by altering cyclins (via c-Myc)
c-Myc targets Cyclin D. Cyclin D is required for re-entry into the cell cycle from G0.
Cyclin D activates cdk4 and cdk6, which then stimulate cyclin E production (which is required for progression from through G1 into S phase)
What gives direction and timing to the cell cycle
Cdks are sequentially activated by cyclins and then stimulate synthesis of genes required for the next phase
Explain how cyclin dependant kinases affect cell cycle progression
Phosphorylation of proteins (on serine or threonine)
Give an example of how cdks affect cell cycle progression using MPF
MPF targets nuclear lamins; the phosphorylation of which cases breakdown of nuclear envelope (key step in prophase)
Give an example of how cdks affect cell cycle progression using Cdk4/6-cyclin D
Targets retinoblastoma protein (pRB); the phosphorylation of
which inactivates the protein.
This releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase
What is the role of Cdk inhibitors
Inhibits Cdks to prevent progression through the cell cycle, which is important in ensuring the integrity of the cell cycle is maintained
Must be degraded to allow cell cycle progression
Give 2 examples of Cdk Inhibitors
INK4 - inhibit Cdk4/6 therefore arresting G1 at the restriction checkpoint (coded by p16 gene)
CIP/KIP - all Cdks, especially in S phase (KIP coded by p27)
Give examples of how CDK inhibitors can be involved in cancer
Cancers can be caused by:
Loss of INK4 inhibition
Aver-production of cdk4/cyclin D
Loss of retinoblastoma protein (highly prevalent in lung cancer)
Give examples of oncogenes
EGFR/HER2 Ras Cyclin D1 B-Raf c-Myc
Give examples of tumour suppressors
Rb
p27KIP1
What does Cdk4/6-Cyclin D target
Retinoblastoma protein (pRB); the phosphorylation of which inactivates the protein. This releases E2F transcription factor, driving gene transcription of Cyclin E and allowing progression of the cell cycle from G1 > S phase
