Cell Cycle Flashcards
What are the phases of the cell cycle?
G0, G1, S, G2, M
What happens in G1 phase?
Cell growth and preparation for DNA synthesis
What happens in S phase?
DNA synthesis
What happens in G2 phase?
Cell growth and preparation for mitosis
What happens in M phase?
Mitosis
What are cyclins and CDKs and how do they work together?
- Cyclin = protein expression oscillates over cell cycle
- Cyclin dependent kinase = stable expression
- Cyclin proteins bind to cyclin dependent kinases
- Each have low activity individually, but when bound CDK will phosphorylate
What cyclin-Cdk combos are present in G1?
Cdk4-cyclin D and Cdk6-cyclinD
What cyclin-Cdk combo is present in S and G2?
Cdk2-cyclin A
What cyclin-Cdk combo helps with the G1/S transition?
Cdk2-cyclin E
What cyclin-Cdk combo is present in G2 and M?
Cdk1-cyclin B
How is CDK/Cyclin activity regulated?
Availability of corresponding proteins:
- regulated expression of cyclin genes
- regulated degradation of cyclin genes
Activity of complex depends on:
- CKI activity
- Inhibitory/activating phosphorylations of Cdk/cyclin
Describe the temporal expression of cyclin and Cdk genes.
Cdk = constant
Cyclin = oscillates across different phases of the cell cycle
What is the pathway for the cyclic degradation of cyclins?
For M-cyclin, APC/C (an E3 ubiquitin ligase) controls proteolysis. Other cyclins have other E3 ligases (F-box proteins):
- Activation of APC/C:
- Cdc20 activating subunit binds inactive APC/C - Ubiquitylation of cyclin:
- E1, E2, and APC/C ubiquitylate the cyclin
- polyubiquitin chain is added to K residue - Degradation in proteasome
What determines cyclin stability?
Cyclin ubiquitylation
How are inhibitory and activating phosphorylation involved in cell cycle progression?
- Cyclin and Cdk bind, forming an inactive complex
- Protein complexes add inhibitory and activating phosphates
- Inhibitory phosphate trumps activating - Activating protein phosphatase removes the inhibitory phosphatase
- Active cyclin-Cdk complex
What are the 4 mechanisms of Cdk activity regulation?
- Availability of cyclins
- Activating phosphorylation
- Inhibitory phosphorylation
- Association with Cdk Inhibitors (CKIs)
How does cyclin activation progress through the cell cycle?
S: Cyclin A-Cdk2
G2: Cyclin A-Cdk1, cyclin B-Cdk1
M:
G1: Cyclin D-Cdk4, cyclin D-Cdk6, cyclin E-Cdk2
What is a CKI?
Cyclin inhibitor protein: inhibits the active cyclin-Cdk complex (ex: p27)
How (temporally and mechanistically) is CKI proteolysis regulated?
Cyclically regulated through the ubiquitylation pathway
- CKI phosphorylated by a kinase
- Active SCF (F-box protein) complex (an E3 ubiquitin ligase) controls proteolysis: ubiquitylation of CKI by E1-E3.
- Degradation in proteasome
What Cdk-cyclin complexes are inhibited by the INK4 family of CKIs?
G1 phase: Cdk2,6-cyclinD
What Cdk-cyclin complexes are inhibited by the CIP/KIP family of CKIs?
G1 phase: Cdk2-cyclin E
S phase: Cdk2-cyclin A
G2 phase: Cdk2-cyclin A
M phase: Cdk1-cyclin B
Where in the cell cycle are the checkpoints? What is necessary to pass the checkpoint?
G1 checkpoint (major checkpoint) requires:
- cell size
- nutrient availability
- growth factors present
- no DNA damage
S phase: check for DNA damage
G2 checkpoint requires:
- cell size
- successful chromosome replication (no DNA damage or unreplicated DNA)
Metaphase checkpoint requires:
- all chromosomes attached to mitotic spindle
What are the downstream mechanisms by which cyclin-Cdk activity determine cell cycle position?
- Change gene expression and activate cell cycle stage specific transcription
- Modify activity of existing proteins, exerting direct control over cell cycle specific processes
What signals regulate exit from G0 and entry into the cell cycle?
- Growth signals (+ or -) from nearby cells
- TGFB signalling pathway
- Differentiaiton signals
- Cell-cell ECM contacts
- DNA damage
What are mitogens?
Growth factors
How do mitogens stimulate cell division?
Mitogens activate cyclins:
- Mitogen binds cell surface mitogen receptor
- Cytosolic signaling cascade involving Ras and MAP kinase
- Immediate activation of gene expression and delayed-response gene expression (via Myc regulatory protein)
- Increased synthesis of cyclin D and E2F
- Increased p27 degradation - Cyclins phosphorylate and inactivate Rb, activating E2F:
- active Rb keeps chromatin compact and represses transcription
- active G1-cdk complex phosphorylates and inactives Rb protein, freeing gene expression - E2F activates all S-phase genes:
- E2F, Cdk2, cyclinE, DNA replication factors, CyclinA
How do mitogen levels impact cyclins D and E, specifically?
Low mitogen: CKIs bind cyclins D and E
High mitogen:
- increased cyclin D expression
- cyclin D binds more CKIs
- freed cyclin E signals cell cycle progression
How does positive feedback control S phase initiation?
- Active E2F induces S-phase gene transcription, which increases E2F activity
- Active G1/S and S cyclins (E and A) increase E2F activity
- Active S-cdk increases E2F activity
What is replication licensing?
Limitation of the activation of the origins of replication to once per cell cycle to avoid re-replicating DNA
What is the mechanism of replication licensing?
G1:
- Cdc6 and Cdt1 proteins bind ORC, inhibiting helicase and thus replication
S:
- S-Cdk phosphorylates Cdc6, which releases from the pre-RC and is degraded
- Cdt1 is inhibited from geminin, releasing it from the pre-RC
- Preinitiation complex binds to the ORC and ORC is phosphorylated
- DNA replication
G2/M:
- ORC remains phosphorylated, preventing re-binding of Cdc6 and Cdt1
How does the G1/S checkpoint halt/restart the cell cycle to allow for DNA repair?
- DNA damage activates a kinase signaling cascade (ATM/ATR -> Chk1/Chk2)
- Kinases phosphorylate p53, releasing it from Mdm2 and activating it.
- Stable, active, phosphorylated p53 binds to regulatory region of p21 gene
- p21 (cyclin inhibitor) binds G1/Cdk (cyclinE/Cdk2) and S-Cdk (cyclinA)
- Cell cycle is inhibited during DNA repair
How is the cell cycle regulated during mitosis?
Replication forks suppress mitosis during DNA replication
What proteins mediate the G2/M DNA damage checkpoint?
ATM/ATR and DNA-PK -> p53, among others….
What is cellular senescence? What induces it?
Permanent cell cycle arrest to remove potentially damaged cells from the cell population.
Induced by DNA damage, mitochondrial dysfunction, aneuploidy, telomere attrition, oxidative stress
What is quiescence? What induces and reinforces it?
G0 = temporary removal of normal cells from the cell cycle
Induced by low mitogen and/or nutrients, contact inhibition
Reinforced by CKIs:
- INK4 binds cyclinD-Cdk4,6
- KIP1 inhibits cyclinE-Cdk2
- Cyclin D levels are reduced
- Rb is hypophosphorylated and bound to E2F
To escape: high levels of mitogen => increased cyclin D => outcompete INK and can bind p27-KIP, freeing cyclinE to resume the cell cycle
What proteins are involved in senescence?
Various upstream causes activate p53, p21, p16
These proteins inhibit cyclin-Cdks
Rb is kept active, maintainign senescence
What are telomeres?
ss ends of chromosomes that shorten with each replication due to the nature of replication (okazaki fragments)
What is the Hayflicks limit?
A limit to cellular replication caused by shortened telomeres => replicative senescence.
How can cells avoid telomere crisis?
If p53 is inactivated, normal p53 mediated senescence will not occur:
- Activation of salvage and NHEJ pathways -> breakage fusion cycles
- Telomerase is re-expressed, allowing for extension of telomeres
