11.1 Cyclins and cyclin-dependent kinases #
The study of Cyclins and cyclin-dependent kinases provides fundamental principles governing cellular architecture, molecular interactions, and physiological regulation.
Caspase Dependent Apoptosis Pathways In Myofibroblasts
11.1.1 The discovery and the principle #
The cell cycle is a biochemical oscillator, not a chain of unrelated events. Three lines of work converged. Hartwell isolated yeast mutants that arrested at particular points (cdc mutants). Nurse found that one of those genes, cdc2, encodes a protein kinase required at Start and again at mitosis, and that the human equivalent will replace it. Hunt saw proteins in sea-urchin eggs that accumulate and then disappear each cycle, and named them cyclins. The kinase is constant in amount. The cyclin that activates it comes and goes. Together they are a cyclin-dependent kinase (CDK).
The principle: different cyclins, synthesised at different times, direct the same or related CDKs to different substrates. Phosphorylation of those substrates is the next phase of the cycle. Destruction of the cyclin ends that phase and permits the next.
11.1.2 The main pairs in animal cells #
| Time | Cyclin–CDK | What the activity causes |
|---|---|---|
| G1 | Cyclin D with CDK4 or CDK6 | Phosphorylates Rb protein, partly releasing the transcription factor E2F. Responds to growth factors. This is the link from the outside world to the cycle |
| G1/S | Cyclin E with CDK2 | Completes Rb phosphorylation, starts S-phase transcription, and helps fire replication origins |
| S | Cyclin A with CDK2 | Keeps S phase going and prevents re-assembly of pre-replication complexes |
| G2/M | Cyclin A, then cyclin B, with CDK1 | Cyclin B–CDK1 is the classical maturation-promoting factor (MPF). It phosphorylates lamins (envelope breakdown), condensin, and microtubule regulators, and drives entry into mitosis |
Plant Cell Structure Frp
Yeast uses fewer CDKs (one CDK in fission yeast) and a different cyclin nomenclature. The logic is identical, which is why a human CDK gene can rescue a yeast mutant.
11.1.3 Switching CDK activity #
Four controls stack, and an exam answer that mentions only cyclin synthesis is incomplete.
1. Cyclin synthesis and destruction. Mitotic cyclins are ubiquitinated by the anaphase-promoting complex (APC/C) and destroyed by the proteasome. G1 cyclins respond to signalling.
2. Activating phosphorylation. CDK-activating kinase phosphorylates a threonine on the CDK. Without it the cyclin-bound CDK is still weak.
3. Inhibitory phosphorylation. Wee1 kinase phosphorylates CDK1 on two residues and holds MPF inactive while the cell finishes G2. The phosphatase Cdc25 removes those phosphates and releases the block. Cdc25 is itself activated by the CDK it activates, which makes entry into mitosis switch-like once it begins.
4. CKI proteins. Stoichiometric inhibitors bind cyclin–CDK complexes. p21 and p27 are the ones to know. p21 is a direct transcriptional target of p53 and is how DNA damage stops the cycle in G1.
11.2 Once per cycle: replication licensing #
A replication origin that fired twice in one S phase would duplicate some genes and not others. The cell prevents this by separating the loading of the helicase from its activation.
- In late mitosis and G1, when CDK activity is low, origins are licensed. ORC marks the origin. Cdc6 and Cdt1 load the MCM helicase, forming the pre-replication complex. The helicase is loaded inactive.
- In S phase, cyclin–CDK and a second kinase (DDK) start the loaded helicases. The same CDK activity prevents new loading, by phosphorylating Cdc6 and ORC and by allowing geminin to inhibit Cdt1.
- Only after CDK activity collapses at the end of mitosis, and geminin is degraded, can origins be licensed again.
Loading and firing are mutually exclusive. That is the mechanism behind “DNA replicates once per cycle.”
11.3 Checkpoints #
11.3.1 What a checkpoint is #
A checkpoint is a surveillance pathway that generates a wait signal when a precondition of the cycle is not met. It is not the event it guards. The spindle checkpoint is not the spindle. Remove the checkpoint and the cell does not wait; it proceeds and makes errors. That phenotype, proceeding despite damage, is how checkpoint genes were found.
11.3.2 DNA damage and replication stress #
Damage such as double-strand breaks is sensed by kinases, principally ATM, and stretches of single-stranded DNA at stalled forks are sensed by ATR. They activate Chk1 and Chk2, which have two fast effects and one slow one.
- They inhibit Cdc25, so CDK1 stays phosphorylated by Wee1 and the cell cannot enter mitosis with damaged or unreplicated DNA. This is the G2/M block.
- They stabilise p53, a transcription factor that is normally ubiquitinated by Mdm2 and kept scarce. Stable p53 induces p21, and p21 inhibits G1 and S CDKs. This is the G1 block, and it gives time for repair before DNA is copied.
- If damage is severe, p53 induces pro-apoptotic genes and the cell dies rather than pass on a broken genome (Chapter 13).
Loss of p53, by mutation or by viral proteins that bind it, removes this choice. The cell copies damaged DNA and survives genomic chaos. p53 is mutated in a large fraction of human cancers for this reason. It is a tumour suppressor: one wild-type copy is usually enough, and both copies must be lost (Knudson’s two-hit logic) before the checkpoint fails completely.
11.3.3 The spindle-assembly checkpoint #
The wait at metaphase is enforced by proteins at kinetochores that are not under tension or not occupied by microtubules. Mad2 and BubR1, recruited there, inhibit the co-activator Cdc20. Without Cdc20, the APC/C cannot ubiquitinate its mitotic substrates.
When every kinetochore is correctly attached, the inhibitor is silenced. APC/C–Cdc20 then ubiquitinates two crucial targets.
- Securin, the inhibitor of the protease separase. Free separase cleaves cohesin, and sister chromatids separate. This is the molecular start of anaphase.
- Cyclin B. Its destruction collapses CDK1 activity, which is required for exit from mitosis: envelope reassembly, decondensation, and cytokinesis. If cyclin B cannot be destroyed, the cell arrests in mitosis.
A second form of the APC/C, working with Cdh1, stays active into G1 and keeps CDK activity low so that origins can be licensed.
The checkpoint explains the pharmacology of spindle drugs. Taxol, vincristine, and related compounds either stabilise or depolymerise microtubules. Either way, kinetochores do not achieve stable bi-orientation, the checkpoint stays on, and cells arrest in mitosis and eventually die. Dividing tumour cells are hit harder than quiescent normal cells, which is the therapeutic window, imperfect because any rapidly dividing normal tissue (gut lining, bone marrow, hair follicles) is hit too.
11.4 Key terms, summary, and self-check #
Key terms. CDK; cyclin D; Rb; E2F; MPF; Wee1; Cdc25; p21; p53; licensing; MCM; geminin; ATM; ATR; spindle-assembly checkpoint; Mad2; APC/C; securin; separase.
Summary. Cyclins choose the substrates, and CDKs phosphorylate them. Wee1, Cdc25, and CKIs decide whether a cyclin–CDK pair is actually active. Origins are licensed only when CDK activity is low and fired only when it is high. DNA damage blocks CDKs through Cdc25 and p53/p21. Unattached kinetochores block the APC/C, and release of that block both separates sisters and ends mitosis.
Self-check.
1. Why does destroying cyclin B make the cell leave mitosis, rather than enter it?
2. A mutation prevents phosphorylation of CDK1 by Wee1. At which transition is control lost?
3. How does the cell ensure that an origin licensed in G1 does not get a second helicase loaded in the middle of S phase?
4. What is the direct substrate of separase, and what protects separase from acting early?
5. Why do microtubule drugs arrest cells at metaphase rather than at random points in the cycle?
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