1st February Flashcards
Within the cell cycle, in which phase is the restriction point located?
G1
During which phase is the cell responsive to extracellular growth factors?
G1
Which Cdk-cyclin complexes control passage through the restriction point?
Cdk4/6 - Cyclin D
Why are the growth phases of the cell cycle important in adult cells?
Give the cell time to respond to both internal and external factors.
Prevents the cell going into mitosis with DNA damage
Ensures the cell has enough mass to create 2 new cells
Outline the process of pre-replicative complex formation
- During early G1 2 large helicases bind to the origin of replication in their inactive form by Cdc6, Cdt and ORC
- In S-phase the Cdk2 activates the pre-RC by phosphorylating specific initiator proteins. DDK also phosphorylates specific helicase subunits.
- The helicases unwinde the DNA, replication starts and the pre-RC leaves the DNA
- Mitosis occurs
- Repeat
When is ORC bound to the origin of replication?
Throughout the cell cycle
What prevents the pre-RC reassembling on the DNA after replication?
APC/C is inactivated in late G1 helping prevent further pre-RC assembly later in the cycle
The 1st stage of pre-RC assembly can only occur without Cdks present
What prevents the pre-RC ‘firing’ before S phase?
It requires Cdk2 activity therefore the presence of cyclin E is required which only occurs during S phase
How does Cdk2 prevent the assembly of the pre-RC?
It phosphorylates Cdc6 triggering it for destruction
What is the function of Rb?
It suppresses cyclin E function through the recruitment of HDAC
Outline the mechanism of activating cyclin E expression
OFF - E2F1 is bound to the promoter, to which Rb binds. This recruits a HDAC which deacetylates the promoter –> heterochromatin meaning that the gene can not be transcribed
ON - Cdk4/6 phosphorylates Rb meaning that HDAC can no longer bind. This means HAT can come and acetylate the DNA –> expression of cyclin E. Once cyclin E has been produced it creates a positive feedback loop as the Cdk2-cyclin E complex further phosphorylates Rb
How is cyclin E expression under the control of GFs?
Cyclin D is required for activation of Cdk4/6 which is required for the phosphorylation of Rb to relieve the inhibition on the cyclin E gene.
Cyclin D is under the direct control of GFs
Name 3 examples of proteins in the Rb family
Rb
P107
P130
Name 2 CKIs acting on Cdk2-cyclin E
p27
p21
Is p21 subject to internal or external cues?
Internal cues such as cell stress and DNA damage
Is p27 subject to internal or external cues?
External cues such as growth signals and anti-mitogenic factors
Can p21 and p27 bind to CDK4/6 - Cyclin D complexes?
Yes however they can not inhibit it, causing CDK4/6-Cyclin D to sequester the CKIs
Outline the mechanism behind regulation of p27 stability
p27 is phosphorylated by p27
Phosphorylated p27 is ubiquitinated by SCF E3 Ub ligase
It is the polyubiquitinated and sent to the 26s proteasome for degradation
How is p27 stability increased during G1?
During G1 APC/C degrades SCF preventing p27 degradation which prevents the premature activation of Cdk2
Which cdk does the INK4 family inhibit?
Cdk4
List the members of the INK4 family
p14, p16, p18 and p19
How do INK4 family proteins inhibit CDK4?
They bind to the CDK preventing cyclin binding
How are p15 levels affected by TGF-beta signalling?
They are increased
Give examples of some of the ways in which G1/S control is lost in cancer
Inherited Rb mutations Inherited p16 mutations Acquired Rb/p16 mutations Epigenetic silencing of p16 through promoter methylation Reduced expression of p27 Cyclin D OE
What form of cancer do inherited Rb mutations lead to?
Retinoblastoma
What form of cancer do inherited p16 mutations lead to?
Familial melanomas
List some examples of targeted approaches of treatment aimed at the restriction point
anti-HER2 receptor MoAbs anti-HER1, HER2, HER4 Tyrosine kinase inhibitors RAS farnesyltransferase inhibitors RAF inhibitors MEK inhibitors mTOR inhibitors
Are mice lacking Cdk2 viable?
Yes
What is the only essential substrate of Cdk4/6?
Rb
What is observed when mice have all 3 cyclin D genes KO?
Die at mid/late stage gestation due to severe anaemia indicating that hematopoeitic cells are cyclin D dependent
Organs develop normally indicating that other cells are not cyclin D dependent
How does Akt aid CDK activation
It relieves 2 constraints:
Akt –I GSK3beta preventing it from phosphorylating and destabilising cyclin D
Akt –I FOXO TFs preventing them from producing p27Kip1 and P21Cip1
