Signalling Mechanisms in Growth and Division Flashcards
What transcription factor is stimulated by growth factor signalling and is vital to starting the cell cycle?
c-Myc
Describe what happens to tyrosine kinase receptors when growth factors bind to them.
TK receptors are usually present on membranes as monomers
Most GFs are dimers, so on binding, they bring TK receptors close together
Allows TK receptors to cross-phosphorylate (using gamma phosphate from ATP to phosphorylate tyrosine residues in proteins)
Phosphorylated domains on TK receptors act as docking sites for adaptor proteins
Give an example of an anti-cancer drug that targets tyrosine kinase receptors.
Herceptin
Inhibits HER2 TK receptor (important in many tumours e.g. breast)
Prevents ligand binding + subsequent signalling
Name an important adaptor protein.
Grb2
Describe the structure of Grb2.
Modular
SH2 domain: binds to docking sites
2 SH3 domains: bind to proline-rich regions of proteins
Describe how receptor protein tyrosine kinases can signal to Ras.
Grb2 is constituently bound to an exchange factor: Sos
When TK receptors become active + docking sites become available, Grb2 binds to the docking site
This brings Sos close enough to the membrane + Ras, to allow it to exchange the GDP on Ras for GTP
GTP bound Ras is active
What must the Ras protein be bound to for it to become activated?
Plasma membrane
Interference with membrane binding of Ras can make a good anti-cancer drug
How is Ras turned on and off?
On: Exchange factors e.g. Sos exchange GDP on Ras to GTP
Ras has intrinsic GTP hydrolysis capability: GTPase activity is stimulated by GTPase-activating proteins (GAPs)
Broadly speaking, how might Ras signalling be different in cancer?
Ras could be permanently switched on (GTP bound form), thus it constantly signals cell division
Describe two mutations that lead to an increase in the amount of active Ras.
V21Ras: glycine replaced by valine. Prevents GAP binding Ras, thus prevents inactivation of Ras.
L61Ras: glutamine replaced by leucine. Prevents GTP hydrolysis so Ras remains in the active, GTP bound form.
What cascade does Ras activate?
ERK cascade (Extracellular signal-regulated kinase cascade)
What is the family that the ERK cascade belongs to called?
MAPK cascade (Mitogen-activated protein kinase cascade)
What are the three kinases involved in the ERK cascade?
Raf (MAPKKK)
MEK (MAPKK)
ERK (MAPK)
What does the last kinase in the cascade phosphorylate?
Gene regulatory proteins (transcription factors e.g. c-Myc), which go on to regulate the expression of genes
Also phosphorylates other proteins + change their activity
What are c-Myc and Ras classed as? What would mutation in either result in?
Oncogenes
Mutation causes Cancer
What type of kinase are cyclin-dependent kinases (Cdks)?
Serine-threonine kinases
What conditions do Cdks require to become activated?
Binding to cyclin
Phosphorylation (activating phosphorylation + removal of inhibitory phosphorylation)
(+ degradation of Cdk inhibitors)
What does the mitosis-promoting factor (MPF) consist of?
Cdk1 + cyclin B
What are the requirements, in terms of phosphorylation, for MPF to become active?
Activating phosphorylation by CAK (Cdk activating kinase)
Removal of the inhibitory phosphorylation (that was placed by Wee1) by Cdc25 phosphatase
What activates MPF at the end of interphase?
Removal of the inhibitory phosphorylation by Cdc25 phosphatase
Describe the positive feedback loop that is formed by MPF activation.
Removal of the inhibitory phosphorylation by Cdc25 produces active MPF, which then phosphorylates Cdc25 + increases its activity meaning that more MPF can be activated
How does MPF put mitosis on hold before progressing to the next stage?
At end of metaphase, it phosphorylates key substrates + inhibits their action (thus putting mitosis on hold)
Signal from fully attached kinetochores causes cyclin B degradation, Cdk1 is inactivated + the substrates become dephosphorylated + hence active.
Which Cdk/cyclin is required for G1/S phase?
Cdk2-cyclin E
Which Cdk/cyclin is required for S phase?
Cdk2-cyclin A
How can the same Cdk be used for two different stages?
Cyclin binding alters the substrate specificity of Cdk
Also, substrate availability changes throughout cell cycle
What is one of the most important transcription targets of c-Myc? What does this do?
Cyclin D
Stimulates transcription of other genes
What is the first Cdk/cyclin complex that is formed when a cell goes from G0 to G1?
Cdk4/6-cyclin D
The Cdk4/6-cyclin D complex then stimulates the expression of the next cyclin in the cell cycle. What properties does this system give to the cell cycle?
Gives cell cycle direction + timing (because Cdk-cyclin complexes must reach a certain concentration before they can trigger the next stage)
Give an example of a phosphorylation target of MPF that allows the cell cycle to progress.
Phosphorylation of nuclear lamins allows breakdown of the nuclear envelope
What is start kinase and what is one of its most important targets?
Start kinase = Cdk2-cyclin E
Retinoblastoma protein
Describe the role of retinoblastoma in the quiescent G0 state.
Retinoblastoma is unphosphorylated
It binds to + sequesters a group of transcription factors: E2F
What effect does Cdk4/6-cyclin D have on retinoblastoma?
It multiply phosphorylates retinoblastoma
On phosphorylation it loses its affinity for E2F + releases E2F
E2F transcription factors regulate gene expression + promote progression of the cell cycle
What is one of the main targets of E2F?
Cyclin E (the next cyclin in the cell cycle)
What type of gene is retinoblastoma?
Tumour suppressor gene (acts a brake on the cell cycle)
State some important genes that are regulated by E2F.
Proto-oncogenes: c-Myc, n-Myc
Cell cycle: E2F-1,2,3, pRb, cyclin A, cyclin E, CDK4, CDK2
DNA synthesis: thymidine kinase, thymidine synthetase, dihydrofolate reductase, DNA polymerase
The initial release of E2F allows transcription of cyclin E leading to the formation of Cdk2-cyclin E. What effect does this complex have on retinoblastoma?
Cdk2-cyclin E further phosphorylates retinoblastoma so more E2F is released + conc. of E2F increases
What is the significance of the increasing concentration of E2F?
E2F can now bind to targets with a lower affinity (e.g. cyclin A gene promoter isn’t activated until the E2F concentration is high enough)
What are the 2 families of Cdk inhibitors?
INK4
CIP/KIP
During which phase do each of the families act and how do they inhibit Cdk?
INK4: G1 phase: displaces cyclin D from Cdk4/6-cyclin D complex
CIP/KIP: S phase: binds to Cdk/cyclin complexes + inhibits them
These inhibitors need to be degraded at various stages for the cell cycle to progress
State 5 common and important oncogenes.
EGFR/HER2: mutationally activated or over-expressed in many breast cancers
Ras: mutationally activated in many cancers
Cyclin D1: overexpressed in 50% of breast cancers
B-Raf: mutationally activated in melanomas
c-Myc: overexpressed in many tumours
State 2 important tumour suppressor genes.
Rb: inactivated in many cancers
p27KIP1: under-expression correlates with poor prognosis in many malignancies
Describe the presence of Cyclins in the cell cycle
Transiently expressed at specific points
Regulated at level of expression
Synthesised then degraded quickly
