Lecture 2: G0, G1 and S phase Flashcards
How is CDC2 involved?
CDC2 is required for both G1/S and G2/M transitions.
• CDC2 is positively regulated by cdc25 and cycB. It is negatively regulated by Wee1.
• CLNs are CDC2 regulators at G1/S in yeast. Their activity is dependent on nutrient availability.
What is the restriction point?
The R point is a point in G1 of the cell cycle when the cell commits to the cell cycle.
• It was found that mammalian cells would arrest in G1 if deprived of GFs and nutrients.
• Cells in G2, M or S would complete the cell cycle then arrest.
• It was proposed that cancer cells lose this regulation.
Which cyclin is involved in G1?
Cyclin D is involved.
• It binds to Cdk4/6.
• Cyclin D accumulates gradually.
• It is inhibited by INK4 family CkIs P15, P16, P18 and P19.
Which cyclin is involved in G1/S?
Cyclin E is involved in G1/S.
• Cyclin E binds to Cdk2.
• CycE-Cdk2 promote the transcription of G2/M genes.
• They target cyclin D for degradation by SCF (a ubiquitin ligase).
How was cyclin E discovered?
Mutant complementation experiments.
1) Culture CLN mutant yeast, express CLN3 under control of a GAL inducible promoter.
2) Plate onto + GAL media with plasmids of a human cDNA library.
3) Replica plate onto -GAL and study cultures hat grow.
4) Plasmids found that cyclin E was rescuing Cdk function. It could bind to Cdk subunits and phosphorylate histones in vitro.
How can extracellular signals be used to stop passage through R?
Extracellular signals may be required to prevent passage through R.
• The TGF-β pathway can be used.
• TGF-β signals bind to receptors and activate a signalling cascade.
• This activates P factors such as P21 and P27 which acts CKIs for Cdk4-cyc D.
How do mitogenic signalling and Cdk4/6 work?
Mitogens are proteins which signal for a cell to continue through the restriction point. Examples of mitogens can be EGF.
1) Mitogens bind to receptor tyrosine kinases. RTKs dimerise and autophosophorylate.
2) In early G1, the RTK activates the MAPK pathway. RTK binds SH2-domain proteins which bind Sos.
3) Sos converts Ras to its active form. Active Ras activates the MAPK cascade. Ras activates Raf, which phosphorylates and activates MEK.
4) MEK phosphorylates and activates MAPK.
5) MAPK then moves to the nucleus and activates TFs to upregulate cyclin D production. Cyclin D slowly accumulates.
6) Cdk4-cycD move to the nucleus through p21/p27.
7) In late G1, the RTK activates the PI3K pathway. This causes the phosphorylation of p21 and p27, ending the repression of G1 and G1/S cyclins and Cdks.
8) RTK recruits PI3K, leading to PIP2 being phosphorylated to PIP3.
9) PIP3 recruits Akt, which phosphorylates p27 (preventing nuclear import). p27 is phosphorylated on T187 and T286. This is a recognition site for SCF. When T187 is mutated, p27 is not degraded. Ladders show that ubiquitination is occurring.
10) p21 is also phosphorylated (decreasing stability). G1/S and S phase cyclins can accumulate and encourage S phase transition.
11) p21 and p27 inhibition promotes the production of cyclin E. Cyclin E rises sharply as it phosphorylates p21 and p27 as part of a positive feedback loop.
12) Cyclin D concentration slowly decreases. It is phosphorylated by Cdk2-E which marks it for degradation by SCF and the 26S proteasome.
How does retinoblastoma protein work?
Rb is a tumour suppressor gene that is also known as the guardian of the restriction point.
• A loss of function mutation with Rb leads to defective control of cell proliferation. R point is dysregulated and tumours can form.
• HPV can cause cancer by its E7 protein which binds to Rb.
• In its active form it binds to E2F transcription factors and therefore suppresses them. It is unphosphorylated during G0.
• It is hypophosphorylated by Cdk4/6 and cyclin D during G1. This leads to low E2F activation.
• It is activated by hyperphosphorylation, leading to the release of E2F and the production of S cyclins. It remains in this state throughout the rest of the cell cycle.
What is p53 and how is it involved?
p53 is an intracellular protein which can signal the arrest of the cell cycle at R.
• p53 can trigger cell cycle arrest, apoptosis, DNA repair or block oncogenesis depending on the signals it receives.
• p53 integrates many different signals such as hypoxia, lack of nucleotides, UV radiation, ionising radiation and oncogene signalling.
• Cell stress causes p53 dependent induction of cell cycle inhibitors.
• p53 normally exists as homotetramer. This makes it very vulnerable to mutation (mutant is dominant).
• It is mutated with a high frequency in cancer.
