The Cell Cycle

Jonathon PinesGurdon Institute

j.pines@gurdon.cam.ac.uk

http://www.gurdon.cam.ac.uk/~pineslab/New_Web_Site/Site/Lectures.html

Outline

How do we know there is a cell cycle?

How is the cell cycle controlled?

What goes wrong in cancer?

What does a cell need to do to proliferate?

Chromosome Separation

DNA replication

M

S

Oscillator?

Product - Substrate?

How do we know there is a cell cycle?

+

+

+

=

=

=

G1 or S or G2 MM

G1

S

S S

G2 G2 delay

Rao & Johnson, 1970, Nature 225, 159-164; Johnson & Rao, 1970, Nature 226, 717-722 After Murray and Hunt, 1993

M phase and S phase are different cellular states

Problems to be solved:Alternation and Completion

Chromosome Separation

DNA replication

M

S

Murray and Hunt, 1993The Cell Cycle: an introduction.

Morgan, D.O., 2007The Cell Cycle: Principles of Control

M

S

• Three converging lines of evidence: Yeast genetics

Xenopus meiosis

Translational control in sea urchin eggs

‘S phase’Cyclin-CDK

‘M phase’Cyclin-CDK

Cyclin-CDK complexes drive the cell cycle

How can you isolate cell cycle regulators?

Schizosaccharomyces pombe (fission yeast): Position in cell cycle related to length of cell

cdc2

Wee1, mik1

cdc13 (Cyclin B)

cdc25

Mitosis

After Murray and Hunt, 1993.

Interphase

Screen for genes that accelerate or slow down the cell cycle

cdc2 is a conserved protein kinase required at 2 points in cell cycle and with a wee allele

MPF: a potent trigger for mitosisXenopus laevis: arrested in G2 of meiosis I, egg arrested in metaphase of meiosis II

Factor in egg cytoplasm forces oocyte to enter M phase - M phase promoting factor (MPF)

Self amplifies and does not require protein synthesis: pre-MPF in oocyte

Universal property of M phase cells

Masui and Markert, 1971, J. Exp. Zool 177, 129. After Murray and Hunt, 1993.

Inject cytoplasmProgesterone

Cyclins: Coincidence or cause?

Sea urchins: large eggs arrested in G1 of first mitotic cell cycle

Fertilisation causes large increase in translation

One protein only translated after fertilisation and destroyed at each mitosis: Cyclin

Evans et al. 1993. Cell 33, 389

M MS S

Cyclin

MPF

MPF eventually shown to consist of cyclin and Cdc2

(Dorée and Hunt, 2002, J Cell Sci, 115, 2461-4)

Cyclin-CDK complexes define the state of the cell cycle

START

DNA Synthesis InitiationDNA Synthesis

Mitosis Initiation

G1

M

G2

S

cdc13-cdc2

cig2-cdc2

Hayles et al., 1994, Cell 78, 813 and Broek et al., 1991, Nature 349 , 388.

Degrade cdc13 or cdc2

Re-initiate DNA synthesis

The cell cycle as alternation of CDK activity

Low CDK activity: Assemble and fire origins of replication

High CDK activity: Repress origins of replication

Build mitotic apparatus

Norton and Diffley, 2000, Molecular Cell, 5, 85-95

S. pombe paradigm - the Threshold hypothesisStern and Nurse, 1996, Trends Genet. 12, p345-350

Testing the Cdk threshold hypothesis

Coudreuse & Nurse, 2010 Nature 468, 1074-1079

Analogue Sensitive Cdc2 Analogue Sensitive Cdc2 KinaseKinaseCyclin B (Cdc13)Cyclin B (Cdc13)

Cyclin-cdc2 fusion drives cell cycle in absence other cyclins

Control amount of kinase activity through analogue sensitive Cdc2

Low concentrations of inhibitor block mitosis

High concentrations block DNA replication

Add different inhibitor concentrations to drive cell cycle with non-degradable Cdc13

Block G2 cells and release into low concentrations of inhibitor - Cells re-replicate

The Cell Cycle: Alternation

START or R pointG1

M

G2

S

G0

Cyclin D-CDK4/6

Cyclin E-CDK2Cyclin A-CDK2

Cyclin A-CDK1/2Cyclin B1-CDK1

Metaphase

Initiation of DNA Synthesis

Completion of DNA Synthesis

DNA Synthesis

Initiation of Mitosis

Waves of cyclin-CDK kinase activity during the human cell cycle

G1 S G2 M G1

M A

Cdc25

Cyclin B-CDK1

Cyclin E-CDK2

Cyclin A-CDK2

Growth Factors

Kin

ase

acti

vity

time

Cyclins E and A are partially redundant

Geng et al., 2003 Cell 114, 431-443

Cyclin A is only essential in early embryos & stem cells

Kalaszczynska et al., 2009 Cell 138, 352-365

Cyclin E is only essential for endo-replication

But Cdk2 is Not Essential for Mitosis

Ortega et al., 2003, Nature Genetics, 35, p25-31

Most mitotic cycles only require one Cdk

Santamaria et al., 2007, Nature 448, 811-815

• Looks like dividing animal cells are just like fission yeast

• But for cells to differentiate and form particular tissues they need specialised Cdks

• Cdk2 - spermatogenesis, oogenesis

• Cdk4 - pancreatic beta cells, pituitary, mammary epithelium

• Cdk4 or Cdk6 - haematopoesis, cell size

• Partial Redundancy? S. cerevisiae paradigm

• Tissue specific roles

Why are there multiple Cyclin-Cdks?

The Cell Cycle: Alternation

START or R point

G1

M

G2

S

G0

Cyclin D-CDK4/6

Cyclin E-CDK2Cyclin A-CDK1 & 2

Cyclin A-CDK1/2Cyclin B1-CDK1

Metaphase

Initiation of DNA Synthesis

Completion of DNA Synthesis

DNA Synthesis

Initiation of Mitosis

PoloAurora

• Polo box binds to S-pT or S-pS

- Elia et al., 2003, Science 299, p1228

• Often generated by Cdk (S-T-P)

• Thus Cdk phosphorylation generates Plk substrate

Eg: Cdk1 phosphorylates INCENP to recruit Plk1 to kinetochores

- Goto et al., 2006, Nature Cell Biology 8, p180

Coordination between mitotic kinases

Kinase DomainKinase Domain PBDPBD PBDPBD

Regulating a CDK

CDK

Cyclin Proteolysis (Ubiquitin) Cyclin Binding

Thr14 & Tyr15 Phosphorylation (wee1/mik1) Thr14 & Tyr15 dephosphorylation (Cdc25)

T-loop Thr dephosphorylation (p24KAP) T-loop Thr phosphorylation (CAK)

Inhibitor binding Inhibitor removal

Deactivate Activate

T14 Y15

T160Morgan 1995, Nature 374, 131.

Cyclin-CDK inactivation: the paradigm

N-term

C-term

KINASE

CyclinBox

CyclinFold 2

Cks

ATP

P

P

P

ONOFF

• Fission yeast wee1 and mik1 prevent premature mitosis

• mik1 stabilised by unreplicated DNA

Wee1 - Generating a Robust Switch

•Wee1 in animal cells blocks mitosis in interphase

Ultrasensitive response to inhibition by Cdk1 -

bistable stateSP SP SP SP SPKinase DomainKinase Domain

Kim & Ferrel, 2007, Cell 128, 1133-1145

SP SP SP SP SPKinase DomainKinase Domain

Conservedinhibitory sites

Sites notwell conserved

Not inhibitory

Cyclin-CDK inactivation: the Paradigm 2

N-term

C-term

KINASE

CyclinBox

CyclinFold 2

Cks

Cki

ATPP

ONOFF

CKIs inhibit both the CDK and the cyclin

Russo et al, 1996Nature 382, 325.

p40Sic1

Cdc28-Clb5/6

S PhaseG1

p40Sic1

M

Cdc28-Clb2

Cln2-Cdc28

Degradation

Cdc34 - SCFCdc4

Yeast CKI Paradigm: Cell Cycle Co-ordination - Sic1

Clb5-Cdc28

Sic1 Inactivation has Parallels with Wee1 Inactivation

Kõivomägi et al., 2011, Nature 480, 128–131

Sic1 is processively phosphorylated by Cln2 and Clb5

Clb5 phosphorylation generates a positive feedback loop

Generating a Robust Switch also requires Regulating Antagonistic Phosphatases

How to turn off a specific complex?

Generating a Robust Switch - Target a Specific Phosphatase Complex with an Inhibitor

Mochida et al., 2010 Science 330, p1670

States of the Cell Cycle are generated by Proteolysis

Different complement of proteins present in

different cell cycle states

The Cell Cycle is Co-ordinated by Ubiquitin-dependent Proteolysis

Effectively an interplay between the SCF and the APC/C

SCF = Skp1 + Cullin + F-box protein

APC/C = Anaphase Promoting Complex/Cyclosome

Ubiquitination

E1

Ubq

ATP

ADP + PiE1 E2

UBC

E2 E3

Substrate Ubq

Ubq

Ubq

E3

Ubq

26 S Proteosome

DESTRUCTION

Ubiquitin Ligase

Schematic of RING E3 ligases

Cullin

RING

Nedd8DOC1

E2

UBQ COP9/Signalosome

SOCS SOCS BoxBox

F-F-Box/Box/BTB/BTB/ SubstraSubstra

tete

Mammalian cells:69 F box proteins - bind to Cul 1>200 BTB proteins - bind to Cul 3>50 SOCS box proteins - bind to Cul2/5>15 DDB1 proteins - bind to Cul 4

F-box proteins

Fbw = WD40 domain: Fbw1 = bTrcp1, Fbw2 = bTrcp2, Fbw7 = Cdc4Fbl = leucine rich domain: Fbl1 = Skp2Fbx = other domains

Nedd8 closes the gap between E2 and substrate

Duda et al., 2008 Cell 134, 995-1006

Nedd8

• SCFSkp2 is degraded by the APC/C

• Allows p27 (Cdk inhibitor) to accumulate in

G1 phase

Interplay between the SCF and the APC/C

Bashir et al., 2004, Nature 428, 190-193

Ubiquitination: Mitosis

E2

UBC (UBC10/UBC5 + Ube2S)

E2

Ubq

Ubq

Ubq

Ubq

Substrate

E3

E3 PP

Cyclin B

Securins

Destruction box proteins

Cdc20

Pines, J. 2011. Nature Reviews Molecular and Cell Biology 12, 427-438

APC/C

Apc8Apc6

Apc3APC11

APC2

APC1

APC4 APC5

APC8APC6

APC7

APC3APC10

Co-ordinating Mitosis by Proteolysis

Antephase Pro-metaphase Metaphase Anaphase Telophase

Chromosome Attachment

ChromatidSeparation

Spindle Disassembly

Cyclin A

Cyclin B1

Securin

Spindle checkpoint

Plk1 Aurora ACdc20UbcH10

CytokinesisNEBD

Pines, J. 2006, Trends Cell Biol., 16, 55-63

APC10 and Cdc20 form the Destruction Box receptor

Da Fonseca et al., 2011, Nature 470, 274-278

EM reconstructions

APC/C specificity: two co-activators

Yu, H. 2007, Mol Cell., 27, 3-16; Garci-Higuera et al., 2008, Nature Cell Biology, 10, 802-811Floyd et al., 2009 Curr Biol., 18, 1649-1658

Cdc20 Cdh1

Essential for MitosisRequired for correct G1 phase & to degrade Aurora kinases

Regulated by spindle checkpoint Regulated by phosphorylation (by CDKs)

Rca1/Emi1proteolysis by Cdh1

Proliferating cells Somatic & Differentiated cells (brain & trophectoderm)

Completion: Timing and Checkpoints

Chromosome Separation

DNA replication

M

S

Completion: Timing

Budding yeast mitosis (at least under laboratory conditions)

Embryonic cell cycles

Xenopus (all you need is cyclin B)

Drosophila

Problem: High error rate Inflexible

Completion: Checkpoints

“The events of the cell cycle .. are ordered into dependent pathways in which the initiation of late events depends on the completion of early events.

First defined by Weinert and Hartwell, 1989, Science 246, 629.

Control mechanisms enforcing dependency in the cell cycle are here called checkpoints.”

Some dependencies can be relieved by mutation ….suggesting that the dependency is due to a control mechanism and not an intrinsic feature of the events themselves.

Checkpoints:

S phase checkpoint

G2 CheckpointsDs breaksUV

Morphogenesis Checkpoint

Prophase Checkpoint

Spindle Checkpoint

S

M

Checkpoints

Think about the biology

– DNA damage: budding yeast arrest in mitosis

fission yeast and animal cells in G2

What should be the phosphorylation state of Cdk1?

What should be the targets of the checkpoint?

Checkpoints

• Budding yeast do use Cdk1 Y18 phosphorylation

• Part of the morphogenesis checkpoint to prevent budding in inappropriate conditions

• Major regulation is on the stability of Swe1p

• Degradation of Swe1 accompanied by relocalisation to bud neck via binding to Hsl7p

McMillan et al., 2002, Mol. Biol. Cell., 13, p3560-75

Summary

The cell cycle is driven by alternation of high and low Cdk activity

Robust switches are driven by ultrasensitivity and phosphatase

feedback loops

Alternation in Cyclin-Cdk activity is underpinned by proteolysis

In somatic cells checkpoints ensure that the switch is not thrown

until previous stage is complete (embryos often rely on timing)

The Cell Cycle & Development

The Cell Cycle & Development

Cells must proliferate only in response to the correct cues

Proliferation, growth and differentiation must be coordinated to generate tissues and organs of the correct size and structure

To Cycle or Not?To Cycle or Not?The decision to proliferate or differentiate or become quiescent is made between mitosis and DNA replication

The Restriction point: the time when cells are committed to replicate their DNA and divide

The Restriction PointThe Restriction Point

CommittmentCommittmentD-type Cyclins are important signal transducers

D-type Cyclins are particularly important in specialised cell types

E.g.: Retina, Cyclin D1 binds to notch promoter - Bienvenu et al., 2010 Nature 463, 374

D-type Cyclins are basically there to turn on E-types

Rescue Cyclin D1 KO with a knock-in of Cyclin E - Geng et al., 1999 Cell 97. 767-777

CommittmentCommittmentTurning on genes required for S phase and

mitosis requires de-repressing the E2F-family

CKIs are important Regulators of G1 Phase Cyclin-Cdks

CKIs are important Regulators of G1 Phase Cyclin-Cdks

INK4 family inhibit only D-Cdk4/6

CIP/Kip (p21/p27/p57) inhibit E & A

Ubiquitin-mediated proteolysis is important to regulate Ckis and to maintain quiescence - Wirth et al., 2004 Genes Dev 18, 88-98

SCFSkp2 is degraded by the APC/C - allows p27 to accumulate in G1 phase

Balancing proliferation & differentiation

Balancing proliferation & differentiation

Number of progenitor divisions will determine tissue or organ size

E.g.: Neural cortex in Cdh1 heterozygotes

Microcephaly (centrosome proteins)

- Spindle orientation or cilia signalling?

DysregulationDysregulation

Hit the gas

Cut the brakes

Loosen the steering

Hit the gasHit the gasThe problem of ‘overexpression in cancer’

Some pathways are very frequently perturbed in cancer, e.g.:

PI3 Kinase pathway, activates AKT & promotes survival and growth

Activated/amplified RTKs & AKT

PTEN is a commonly deleted tumour suppressor

PI3K PATHWAYPI3K PATHWAY

Engelman 2009 Nature Reviews Cancer

Hit the gasHit the gasChromosome rearrangements drive proliferation signals

Frequent for the D-type Cyclins (D1 & D2)

Parathyroid adenoma

Hit the gasHit the gas•Mantle cell lymphoma: Cyclin D1 next to IgH or Cyclin D2 next to IgK

IgK fused to Cyclin D2

Cut the BrakesCut the Brakes

Rb & the pocket proteins

p16

p21 & p27 - Skp2

Senescence and telomerase

Loosen the SteeringLoosen the Steering

Genomic Instability

Cancer Genomes can undergo massive rearrangements - mechanism unclear but could be due to double stranded breaks in mitosis (IR or telomeres) - Stephens et al., 2011 Cell 144, 27-40

Loosen the SteeringLoosen the SteeringGenomic Instability

DNA replication - Beginning replication too early causes problems

E.g.: Too much Cyclin E, eg: Cdc4 mutation - Strhmaier et al., 2001, Nature 413, 316-322

Interferes with Pre-RC assembly- Eckholm-Reed, 2004, J. Cell Biol 165, 789-800

Lack of Cdh1 - Garcia-Higuera et al., 2008, Nat Cell Biol 10, 802-811

Loosen the SteeringLoosen the Steering

Genomic Instability

DNA damage repair pathways

ATM

p53 or Chk2 - Li-Fraumeni syndrome

Loosen the SteeringLoosen the SteeringGenomic Instability

Chromosome segregation

Very few genetic diseases because essential

Mosaic variegated aneuploidy - BubR1

- Hanks et al., 2004 Nat Genet 36, 1159-1161

The Spindle Assembly Checkpoint

STOP GO

Checkpoint monitors kinetochore attachment

CDC20 CDC20 + APC/C

Requires Mad1, Mad2, Bub1, Mad3/BubR1, Bub3,

Mps1, kinases

Loosen the SteeringLoosen the SteeringThe Spindle Assembly Checkpoint detects most errors in chromosome attachment but not merotely

Loosen the SteeringLoosen the SteeringGenomic Instability: Tetraploidy as a permissive state

- Fujiwara et al., 2005, Nature 437, 1043-1047

Tetraploidy could lead to aneuploidy because:i) Multipolar mitosis (Boveri’s centrosome theory)ii) Increase in improper kinetochore attachments

because too many chromsomes in the spindle

Loosen the SteeringLoosen the Steering

Extra centrosomes increase lagging chromosomes and mis-segregation