Lecture 4: G2 to M Flashcards
How do cyclin concentrations change?
- There is a movement from cdk1/2-A to Cdk1-B and APC/C activation.
- In late S/G2, cyclin B concentrations accumulate in the low activity triple phosphate form. Cyclin B is rapidly activated by cdc25 upon the onset of M.
What did the ooctye experiments show?
Masui performed experiments in 1971 which proved the existence of a maturation promoting factor (MPF) and a cytostatic factor (CSF). They were later found with genetic and biochemical studies.
Maturation promoting factor
• Cytoplasmic activity in maturing eggs in metaphase can promote maturation of G2 arrested immature oocytes without hormonal stimulation.
• A small amount of cytoplasm was transferred. This implies that MPF works catalytically, like an enzyme.
Cytostatic factor
• Mature oocyte cytoplasm can stop the cell cycle and arrest embryonic cell divisions.
• CSF must inhibit an enzyme required for cell cycle progression.
What is MPF?
MPF is a mitotic kinase involved in stimulating the mitotic and meiotic phases of the cell cycle. MPF promotes entrance into M phase from the G2 phase by phosphorylating multiple proteins.
• It is highly conserved across evolution.
• In metazoans it consists of CDK1 and cyclin B.
• In S. pombe it is made up of CDC2 and CDC13.
• In S. cerevisiae it is CDC28 and CLB1/2.
• Wee1 phosphorylates CDC2 to give it lower activity. A phosphatase then removes these phosphate groups.
• Nurse showed that CDC25 counteracts Wee1 and gives it timely entry into mitosis.
What are egg extract systems?
Xenopus egg extraction systems can be used to isolate specific components of the cell cycle.
1) Induce oocytes to mature with progesterone.
2) Extract eggs.
3) Crush with low speed centrifugation at 10,000g. Use EGTA for calcium chelation.
4) This gives lipid, cytosol + membranes and nuclei organelles as fractions.
5) Another high-speed centrifugation at 200,000g gives cytosol and membranes.
6) Sperm can then be added to these extracts to follow the cell cycle state in vitro.
7) DNA is either decondensed as a ball (interphase) or condensed during mitosis.
8) Metaphase cytosol and calcium ions can be used to keep the egg extract cyclic.
How were egg extract systems used to isolate MPF components?
This system was used to great effect for isolating proteins involved in the cell cycle.
• MPF was purified into different sections.
• Each of these was tested for activity into G2 arrested immature oocytes.
• Western blotting confirmed that the 34kDa protein in MPF was an analogue of CDC2 in yeast.
• Cyclic B is the second subunit of MPF.
• There is a 1:1 stoichiometry.
This system was also used to show that cyclin synthesis is what drives the early embryonic cell cycle
• Cyclin B mRNA supported cycling of the extract.
• Cyclical synthesis and destruction of cyclin B protein.
• Both mitotic histone H1 kinase and MPF cycled in synchrony.
• RNase can be used to remove mRNAs.
• RNase inhibitors and cyclin mRNA can then be used to continue cycling.
How can is cyclin B activated?
Studies showed that cyclin B and CAKs aren’t enough. Cdc25 is required as well.
• Cdk1 phosphorylation was analysed in CDC25 mutants.
• The mutants were stalled in G2.
• In the mutants, it was found that tyrosine-15 of Cdk1 was phosphorylated in the predicted ATP binding site.
• CDC25 removes this extra phosphate group.
• Cyclin B accumulates in late S/G2.
• Activity spikes around G2/M.
• Wee1 adds the inactivating phosphates.
• CDC25 removes them.
• After Cdk1 is activated it inhibits the inhibitors (wee1 and myt1) and activates CDC25 with two phosphate groups.
How does CDC25 work?
CDC25 triggers Cdk activation upon mitotic entry.
• Genetic analysis has shown CDC25 opposed Wee1. The egg extract system with cyclin and CDC25 shows greater, earlier and more prolonged HI kinase activity.
• Cloning also shows that CDC25 has a dual specificity phosphatase domain. It can act on both phosphotyrosine and phosphoserine/threonine residues. Recombinant CDC25 can dephos Cdk on T14/Y15.
What is the importance of cellular phosphatases?
Protein phosphatases (PPPs) are proteins which remove phosphate groups.
• They are active in G2 and they prevent the accumulation of phosphorylated Cdk1 and cycB substrates.
• During mitosis, the phosphatases are not active. Phosphorylated substrates accumulate.
• There are 3 subfamilies of phosphatases: PP1, PP2A and PP5. The catalytic subunits are highly conserved. Specificity is achieved by recognising different substrates and binding with different subunits to form an holoenzymes.
What are the targets of cyclin B?
Cdk1-cyclin B have multiple targets.
• PP1.PP2A are inactivated by Cdk1-B in M phase. Cdk1-B directly inhibits Cdk1-B. It indirectly inhibits PP2A. It phosphorylates greatwall (positive feedback). This then phosphorylates ENSA which binds and sequesters PP2A.
• APC/C is phosphorylated, giving it a low activity. At G2/M it is inhibited by MCC (mitotic checkpoint complexes), however it can still degrade cyclin A. At anaphase it is bound by CDC20 and activated. It then degrades cyclin B and securing.
• CDH1 is phosphorylated. This stops it from binding to APC/C. APC/C must bind to CDC20 instead.
• PRC1 (protein regulator of cytokinesis 1) is also phosphorylated and inactivated. Spindle furrow formation is inactivated until after anaphase (when cyclin B levels have dropped enough).
How is the APC/C regulated from G1 to G2/M?
- In G1/S it is inhibited by Emi1.
- Emi1 is phosphorylated by Plk at the start of mitosis.
- CDH1 is phosphorylated and inhibited by Cdk1-B. APC/C therefore binds to CDC20 on the C box.
- APC/C-CDC20 is immediately inhibited. Cdk1-B is active from G2/M. The APC/C inhibitor must be present to stop early securing and cyclin B degradation.
- We can find the proteins responsible by mutagenizing yeast and allowing normal replication. Before the second replication a small amount of benomyl (mitotic poison is added). Mutants that die cannot arrest the cell cycle and they undergo mitotic failure.
- It was found that MAD1/2/3 genes and bub1/2/3 genes are responsible.
- MAD and bub form the APC/C inhibitor complex (MCC).
- MAD2c interacts with CDC20 and inhibits APC/C in vitor.
- MAD2c complexes with BUB1/3, BUBR1 and CDC20. This forms the MCC which blocks the APC/C binding site. Components were found with Co-IP experiments.
- This stops the APC/C degrading cyclin B and securing until chromosomes are aligned. It’s involved in the spindle checkpoint.
- Cyclin A can be degraded earlier than cyclin B and securing. The mechanism of this is unknown.
How is DNA damage checked in G2 before entering mitosis?
After S phase, the cell must check for DNA damage in G2 before entering M phase.
• DNA damage activates the ATM pathway.
• ATM phosphorylates Chk1 and Chk2.
• These in turn phosphorylate CDC25.
• CDC25 is degraded by SCF-BTRCP.
• This means that cdk-B activity decreases. G2 is elongated while DNA repair occurs.
