Cell Cycle Regulation: CDKs, Checkpoints, Rb/E2F, and Replication Licensing

What happens in G1?

Cell growth and synthesis of RNAs/proteins needed for DNA synthesis; ends at START/restriction point.

START / restriction point

Late G1 point after which the cell is committed to DNA synthesis (about 2-3 h before S phase in mammals).

What happens in S phase?

DNA synthesis and centrosome duplication.

What happens in G2?

DNA repair if needed, lipid/protein synthesis, mitochondrial fission, Golgi duplication, chromosome condensation begins.

Interphase

G1 + S + G2.

G0

Cells exit the cycle (e.g. post-mitotic differentiated cells); some can re-enter, many never do.

Five things cell cycle checkpoints monitor

Growth, DNA damage, DNA replication, kinetochore attachment to the spindle, spindle positioning.

Mitogen vs anti-mitogen

Mitogens (growth factors, e.g. EGF) push cells through G1; anti-mitogens (e.g. TGF-beta) block entry.

The "R-factor" idea

A labile factor accumulates in G1; past a threshold the cell is committed. It is cyclin D (with cyclin E feedback) in mammals; Cln3 in budding yeast.

Three biochemical reactions behind all CDK regulation

1) Phosphorylation/dephosphorylation 2) Molecular recognition/binding (cyclins, CKIs) 3) Ubiquitin-mediated destruction.

Why are CDKs inactive on their own?

They need a bound cyclin; CDK levels stay roughly constant, cyclin levels change.

What does cyclin binding do to a CDK?

Repositions the activation loop and catalytic residues, and helps set substrate specificity.

How is cyclin accumulation controlled?

Transcription of cyclin genes.

How is cyclin disappearance controlled?

Ubiquitin-mediated proteasomal degradation (E3 ligases: SCF and APC/C).

Cyclin D-CDK4/6

G1 / cell cycle entry; levels respond to mitogens.

Cyclin E-CDK2

G1/S transition; commits the cell.

Cyclin A-CDK2

S phase (cyclin A also binds CDK1 at the start of mitosis).

Cyclin B-CDK1

Mitosis; also called MPF.

What is CAK and is it rate-limiting?

CDK-activating kinase; phosphorylates activating Thr160 in the A-loop. Constitutively active, so not rate-limiting.

Wee1

Kinase that puts inhibitory phosphates on Thr14/Tyr15 of CDK1 (in the ATP-binding G-loop).

Cdc25

Phosphatase that removes the inhibitory Thr14/Tyr15 phosphates and activates CDK1.

Why does Thr14/Tyr15 phosphorylation inhibit CDK1?

It adds negative charge in the G-loop that interferes with ATP binding.

INK4 family (e.g. p15)

CKIs that bind CDK4/6 monomers and block cyclin D binding.

Cip/Kip family (p21, p27, p57)

CKIs that bind cyclin-CDK complexes.

p21 is induced by what?

p53 after DNA damage.

What does p27 do?

Blocks G1-to-S when growth factors/nutrients are limited; p27 knockout mice are larger with organ hyperplasia.

Who discovered cyclins and how?

Tim Hunt, in sea urchin eggs: a protein cycled in abundance and was destroyed before cell division (cyclin B).

What is MPF made of?

CDK1 (~32 kDa) + cyclin B (~45 kDa).

Mitogen to cyclin D pathway (EGF example)

EGF -> EGFR -> Ras -> Raf -> MEK -> MAPK -> fos/jun -> Myc -> cyclin D transcription.

How does TGF-beta cause G1 arrest?

Promotes transcription of CKI p15INK4b, causing G1 arrest or entry into G0.

Role of Rb in early G1

Binds E2F, keeping it inactive; also recruits chromatin modifiers that condense chromatin and repress E2F target genes.

What do E2F target genes encode?

DNA synthesis proteins and other cyclins (cyclin E, cyclin A), CDK2, and E2F itself.

Rb/E2F pathway steps

Cyclin D-CDK4/6 phosphorylates Rb -> Rb releases E2F -> E2F makes cyclin E/A -> cyclin E-CDK2 phosphorylates Rb more (positive feedback).

When is Rb dephosphorylated?

After mitosis as CDK activity falls (Rb stays phosphorylated through S, G2, M).

Retinoblastoma

Childhood eye cancer caused by loss of Rb; Rb is inactivated in many cancers.

Yeast equivalent of Rb/E2F

Whi5 (repressor) phosphorylated by Cln3-CDK, then Cln1/2 phosphorylate more Whi5 (same feedback logic).

Two ways cyclin E-CDK2 promotes S phase

1) Promotes S-cyclin accumulation (more transcription + inhibits Cdh1) 2) Triggers degradation of Sic1/p27.

Cdh1

APC/C co-activator active from late mitosis through G1; degrades S and M cyclins.

How do G1/S CDKs inactivate Cdh1?

They phosphorylate Cdh1 so it dissociates from APC/C, letting S/M cyclins accumulate.

Sic1 (yeast) / p27 (metazoans)

CKI that holds S-phase CDKs inactive in G1; degraded by SCF after CDK phosphorylation.

Why does Sic1 have six sub-optimal CDK phosphorylation sites?

All six must be phosphorylated before SCF binds well, so only high G1/S-CDK activity destroys it: switch-like, rapid S-phase entry (vs a slow dimmer-switch with one optimal site).

Why is Sic1/p27 degradation irreversible?

Proteolysis is irreversible, so S-phase entry cannot be reversed.

SCF: what is it and how does it pick substrates?

E3 ubiquitin ligase (Skp1, Cullin, F-box protein); the F-box protein recognizes phosphorylated substrates (substrate is phosphorylated).

SCF targets (list)

Sic1/p27 (G1/S) and Wee1 (via beta-TrCP, at G2/M).

APC/C: how is it regulated?

The APC/C itself is regulated (phosphorylation and co-activator binding); 13-14 subunits; inactive without Cdc20 or Cdh1.

APC/C-Cdc20 vs APC/C-Cdh1

Cdc20: metaphase-anaphase (securin, cyclins A/B). Cdh1: late anaphase through G1 (S and M cyclins).

SCF vs APC/C regulation (key difference)

SCF: the substrate is phosphorylated. APC/C: the ligase itself is phosphorylated/activated.

ORI licensing happens when?

G1 (low S-CDK activity).

ORI firing happens when?

S phase (high S-CDK + DDK).

Pre-RC (pre-replication complex)

ORC at origins + Cdc6/Cdt1 + loaded inactive MCM helicase; assembled in G1.

Steps of DNA replication initiation (5)

1) ORC recognizes origins 2) Cdc6/Cdt1 load MCM helicase (pre-RC) 3) recruit helicase cofactors 4) activate helicase 5) recruit DNA polymerase machinery.

Why can ORI licensing only happen in G1?

MCM helicase can only be loaded when non-phosphorylated, which requires low S-CDK activity.

Why can ORI firing only happen in S?

Helicase is activated only when phosphorylated by S-CDK and DDK (high activity).

How does the cell ensure DNA is replicated once per cycle?

Helicase loaded only at low CDK (G1) and activated only at high CDK (S); S-CDK also phosphorylates Cdc6/Cdt1 so no new loading occurs.

DDK

Dbf4-dependent kinase (Cdc7); second kinase needed with S-CDK to activate the helicase. (Slide spells it DKK.)

CMG helicase

Cdc45-MCM-GINS; the active replicative helicase.

Geminin (vertebrates)

Binds and inhibits Cdt1 from late S through mitosis; destroyed by APC/C at end of mitosis.

Cohesin subunits

Smc1, Smc3, Scc1, Scc3.

Cohesin in G1

Loaded (Scc2-Scc4) and unloaded (Pds5-Wapl) continually; not yet cohesive.

How does cohesin become cohesive?

During replication: Smc3 acetylation by CoAT plus sororin stabilization; rings entrap both sister DNAs.

Non-cohesive functions of cohesin in G1

Partitioning the genome into TADs and DNA looping to regulate transcription (cohesinopathies).

Cyclin B-CDK1 problem at G2/M

Cyclin B accumulates in S/G2 and binds CDK1 but stays inactive until Wee1 is overcome and Cdc25 is activated.

Role of 14-3-3 at G2/M

Binds phospho-Cdc25C and holds it inactive until Pin1 and Plk1 release it.

Cdc25B vs Cdc25C

B = starter phosphatase at the centrosome; C = workhorse in the cytoplasm.

G2/M positive feedback loop 1

Active cyclin B-CDK1 phosphorylates Cdc25C; Pin1 and Plk1 modify it and release it from 14-3-3; active Cdc25C activates more cyclin B-CDK1.

G2/M positive feedback loop 2

Cyclin B-CDK1 phosphorylates Wee1; Plk1 adds a site for SCF-beta-TrCP; Wee1 is ubiquitinated and degraded, giving more CDK1 activity.

Why is G2/M abrupt and irreversible?

Two positive feedback loops: activate the activator (Cdc25C) and destroy the inhibitor (Wee1).

Pin1

Proline isomerase; changes conformation of phospho-Ser/Thr-Pro on Cdc25C.

Plk1

Polo-like kinase 1; phosphorylates Cdc25C (activates) and Wee1 (targets for destruction); spindle formation.

What does nuclear cyclin B-CDK1 do?

Phosphorylates nuclear lamins (nuclear envelope breakdown) and with Plk1/Aurora promotes chromosome condensation and spindle formation.

Nuclear lamins

Intermediate filaments (A, B, C) of the nuclear lamina; depolymerize when phosphorylated by CDK1.

Centrosome duplication: role of Plk4

With G1/S CDKs, triggers each centriole to make a daughter procentriole during S.

Centrosome duplication: role of Nek2

Cleaves the linker connecting the two original centrioles.

Centrosome maturation kinases

M-CDKs, Plk1, Aurora A accumulate pericentriolar material (PCM).

Three types of spindle microtubules

Kinetochore MTs, polar (interpolar) MTs, astral MTs.

Kinetochore MTs

Attach to kinetochores; separate sister chromatids (anaphase A).

Polar MTs

Overlap and slide to separate the poles.

Astral MTs

Contact the cell cortex to orient the spindle.

Prophase pole separation

Kinesin-5 (bipolar) walks toward (+) ends of antiparallel MTs, sliding them apart.

Three prometaphase problems

1) Attach kinetochores to MT plus ends 2) Attach sister kinetochores to opposite poles (bi-orientation) 3) Align chromosomes at the metaphase plate.

Why does capture persist?

Kinetochore capture reduces MT catastrophe frequency, so the attachment lasts.

Dynein in prometaphase

Moves chromosome (attached to MT side/end) toward the pole ((-) end).

Congression

Aligning chromosomes on the metaphase plate via coordinated motors and MT growth/shrinkage regulators.

Kinesin-4 in congression

(+) end directed; pushes chromosomes toward the spindle center, away from poles.

Kinesin-7 in congression

Holds the kinetochore as the attached MT grows.

Kinesin-13 in congression/anaphase A

Depolymerizes MT ends; shortens kinetochore MTs.

What happens to dynein after congression?

Released from kinetochores and moves down kinetochore MTs to the poles.

Aurora B and the CPC

Chromosomal passenger complex at inner centromere; Aurora B phosphorylates Ndc80 when there is no tension, loosening attachments.

Ndc80

Outer kinetochore MT-binding complex.

How does tension tighten attachments?

Under tension Ndc80 stretches away from Aurora B; PP1 dephosphorylates it, giving tighter MT binding.

Spindle assembly checkpoint (SAC)

Blocks anaphase until all chromosomes are bi-oriented and under tension.

How does the SAC work?

Unattached kinetochores (Mps1 -> Knl1 -> Bub proteins -> Mad1-Mad2) generate the MCC that inhibits Cdc20, so APC/C-Cdc20 stays off.

Consequence of SAC failure

Nondisjunction and aneuploidy (cancer, Down syndrome via meiotic errors).

How is the SAC silenced?

PP1 dephosphorylates Knl1 and the MCC is disassembled.

Anaphase trigger chain

SAC satisfied -> APC/C-Cdc20 active -> securin degraded -> separase activated -> Scc1 of cohesin cleaved -> sisters separate.

Securin

Inhibitor that holds separase inactive.

Separase

Protease that cleaves the Scc1 cohesin subunit.

Anaphase A

Chromosomes move to poles; powered at least partly by kinesin-13 shortening MT ends.

Anaphase B

Poles separate; kinesin-5 sliding of polar MTs and dynein pulling on astral MTs at the cortex.

What mitotic exit events require CDK substrate dephosphorylation?

Anaphase spindle elongation, spindle disassembly, chromosome decondensation, nuclear envelope reformation.