Lecture 2 - G1 Control and Restriction Point Flashcards
What is confluence?
When the amount of cells is kept constant by equaling cell division and cell death
What happens in normal contact inhibition in a petri dish?
Cells divide until there is no space in the monolayer and then reach confluence
What do cancer cells do to do with contact inhibition in a petri dish?
Do not stop dividing and go above the monolayer
Where is the restriction point?
Just before S phase
What is the restriction point also called?
Rb switch
What are cyclin dependent kinases
Heterodimeric protein complex of cyclin and cyclin-dependent protein kinase (Cdk).
Kinases that phosphorylate a selected set of substrate proteins
How are cyclin dependent kinases specific?
- Sequence recognition
- Temporal availability (synthesised and destroyed in a cyclic way)
- Spatial location (in the nucleus)
Which cyclins are involved in which parts of the cell cycle?
M - cyclin B
G1 - cyclin D
R - cyclin E
S - cyclin A
Cyclin levels vary, what happens to Cdk levels?
Kept fairly constant
What does CDK1 (CDC2) complex with?
Cyclins A and B
What does cyclin D complex with?
CDK4/6
What does CDK2 complex with?
Cyclins E and A
What happens when you knock out a CDK?
Another CDK will take over
What happens when cyclin binds to CDK?
Cyclin pulls the activation loop away from the active site of CDK and exposes the ATP
What is CAK and what does it do?
CDK activating kinase.
Required for full activation of CDK
How do some CDKi (CDK inhibitors) work?
Block ATP binding at the active site
What CDKi’s can inhibit E,A and B?
p57
p27
p21
What CDKi’s are needed to inhibit D in G1 phase and what is the family called?
INK4 family p16 p15 p18 p19
How is expression of the INK4 family usually regulated and give an example
Growth factor signalling
Antigrowth signals instigated by TGF B up-regulate p15 resulting in inhibition of cyclin D/Cdk4
3 ways pathways can feed into and regulate cyclin D
Activity (CDKi)
Complex formation
Accumulation
What activates the expression of p15 and p21?
Dimerisation of Smad3 and Smad4 after 3 is phosphorylated by TGF B receptor kinase
What else does SMAD3 do when phosphorylated?
Dimerises with E2F and inhibits Myc - an oncogene
What does Myc do when activated?
Inhibits p15 and p21
How many cancers is Rb disrupted in?
Probably all
How have children with retinoblastoma usually got it?
Usually have inherited one faulty copy and random loss of the second leads to tumour formation (recessive)
What happens to the Rb protein as it goes through the restriction point?
Hypophosphorylated (1) pre restriction point
Hyperphosphorylated post restriction point
How does the hyperphosphorylation of Rb activate txn?
Removes it from E2F (family of transcription factors)
What does the activation of Rb lead to?
Txn of genes required for next cell cycle phase including DNA replication factors
What regulates the hypophosphorylation of Rb?
Cyclin D-Cdk4/6
What regulates the hyperphosphorylation of Rb?
Cyclin E-Cdk2
Why is Rb hypophosphorylated first and what does this mean?
Allows slight txn which includes cyclin E.
Cyclin E is under the control of E2F so unrestricting it leads to massive txn (positive feedback loop)
What does E-Cdk2 also phosphorylate and what does this mean?
CDKi p27
Passage through R is irreversible
How can cancer mutate to stop the effect of p27?
In some breast cancers it may not become localised to the nucleus effectively
Give an example of how DNA tumour viruses render Rb ineffective?
HPV encodes a protein E7 which sits in the pocket of Rb
What does Cdk2 activity have an effect on in yeast?
Low Cdk2 activity - supports replication complex assembly in G1
Rising Cdk2 activity - prevents further assembly (to stop multiple repetitions) AND activates complexes
How do mammalian cells ensure DNA is only replicated once?
Cyclin E supports replication complex assembly
Cyclin A activates replication complexes and inactivates assembly proteins
Describe a pre-replication complex
Origin recognition complex (ORC) binds origin.
Cdc6 cooperates with others to load helicase (Mcm complex)
Presence of helicase defines activatable replication origin
What does cyclin A-Cdk2 do in late G1 and early S phase?
Late G1 - Phosphorylates and inactivates Cdc6 and Mcm so they cannot be reused
Early S - phosphorylates DNA polymerases and promotes assembly
What is the nuclear matrix?
The structural framework the protein complexes are mounted on
What is Ciz1?
Protein that attaches DNA replication complexes to the nuclear matrix
What binds to the Ciz1 protein?
Cyclin A and E
What happens on Ciz1 when cyclin A levels increase and why?
Cyclin A pushes off E - this coordinates them to function in the right order
