The Cell Cycle
Jonathon Pines
Gurdon Institute
j.pines@gurdon.cam.ac.uk
http://www.gurdon.cam.ac.uk/~pineslab/New_We
b_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
M
Product - Substrate?
Oscillator?
S
DNA replication
How do we know there is a cell cycle?
G1 or S or G2
M
=
+
G1
S
+
+
M
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
M
Murray and Hunt, 1993
The Cell Cycle: an
introduction.
Morgan, D.O., 2007
The Cell Cycle:
Principles of Control
S
DNA replication
Cyclin-CDK complexes drive the cell cycle
• Three converging lines of evidence:
Yeast genetics
Xenopus meiosis
Translational control in sea urchin eggs
M
‘M phase’
Cyclin-CDK
‘S phase’
Cyclin-CDK
S
How can you isolate cell cycle
regulators?
Schizosaccharomyces pombe (fission yeast): Position in cell cycle related to length of cell
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
Wee1, mik1
cdc25
cdc2
Interphase
cdc13 (Cyclin B)
Mitosis
After Murray and Hunt, 1993.
MPF: a potent trigger for mitosis
Xenopus 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
Progesterone
Inject cytoplasm
Masui and Markert, 1971, J. Exp. Zool 177, 129.
After Murray and Hunt, 1993.
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
Cyclin
MPF
S
M
S
M
Evans et al. 1993. Cell 33, 389
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
Mitosis Initiation
cdc13-cdc2
M
Degrade cdc13 or cdc2
G2
G1
S
DNA Synthesis
START
Re-initiate DNA synthesis
DNA Synthesis Initiation
cig2-cdc2
Hayles et al., 1994, Cell 78, 813 and Broek et al., 1991, Nature 349 , 388.
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
S. pombe paradigm - the Threshold hypothesis
Stern and Nurse, 1996, Trends Genet. 12, p345-350
Norton and Diffley, 2000, Molecular Cell, 5, 85-95
Testing the Cdk threshold hypothesis
Cyclin B (Cdc13)
Analogue Sensitive Cdc2 Kinase
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
Coudreuse & Nurse, 2010 Nature 468, 1074-1079
The Cell Cycle: Alternation
Metaphas
e
Initiation of Mitosis
Cyclin B1-CDK1
Cyclin A-CDK1/2
G0
M
G2
G1
START or R point
Cyclin D-CDK4/6
Completion of DNA Synthesis
S
Initiation of DNA Synthesis
DNA Synthesis
Cyclin A-CDK2
Cyclin E-CDK2
Waves of cyclin-CDK kinase activity during the human cell cycle
G1
S
G2
M
G1
Kinase activity
Cyclin B-CDK1
Cyclin A-CDK2
Cyclin E-CDK2
Cyclin D-CDK4/6
time
M
Growth Factors
Cdc25
A
But Cdk2 is Not Essential for Mitosis
Ortega et al., 2003, Nature Genetics, 35, p25-31
Cyclins E and A are partially redundant
Cyclin E is only essential for endo-replication
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
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
Why are there multiple Cyclin-Cdks?
• Partial Redundancy?
S. cerevisiae paradigm
• Tissue specific roles
The Cell Cycle: Alternation
Metaphas
e
Initiation of Mitosis
Cyclin B1-CDK1
Cyclin A-CDK1/2
G0
M
Polo
Aurora
G2
G1
Cyclin D-CDK4/6
Completion of DNA Synthesis
S
Initiation of DNA Synthesis
DNA Synthesis
Cyclin A-CDK1 & 2
Cyclin E-CDK2
S
Coordination between mitotic kinases
Kinase Domain
•
PBD
PBD
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
Regulating a CDK
Cyclin Proteolysis (Ubiquitin)
Cyclin Binding
Thr14 & Tyr15 Phosphorylation (wee1/mik1)
Thr14 & Tyr15 dephosphorylation (Cdc25)
T-loop Thr dephosphorylation (p24 KAP )
T-loop Thr phosphorylation (CAK)
Inhibitor binding
Inhibitor removal
T14 Y15
Deactivate
CDK
Activate
T160
Morgan 1995, Nature 374, 131.
Cyclin-CDK inactivation: the paradigm
N-term
P
P
ATP
P
Cyclin
Box
KINASE
Cyclin
Fold 2
C-term
Cks
OFF
ON
Wee1 - Generating a Robust Switch
•
Fission yeast wee1 and mik1 prevent premature mitosis
•
mik1 stabilised by unreplicated DNA
•Wee1 in animal cells blocks mitosis in interphase
Ultrasensitive response to inhibition by Cdk1 - bistable
state
SP SP SP
SP SP
Kinase Domain
Sites not
well conserved
Not inhibitory
Conserved
inhibitory sites
Kim & Ferrel, 2007, Cell 128, 1133-1145
Cyclin-CDK inactivation: the Paradigm 2
Cki
N-term
ATP
P
Cyclin
Box
KINASE
Cyclin
Fold 2
C-term
Cks
OFF
ON
CKIs inhibit both the CDK and the cyclin
Russo et al, 1996
Nature 382, 325.
Yeast CKI Paradigm: Cell Cycle Co-ordination Sic1
Cln2-Cdc28
Clb5-Cdc28
Cdc34 - SCF
p40
Sic1
Cdc28-Clb2
M
Sic1
p40
Degradation
Cdc28-Clb5/6
G1
S Phase
Cdc4
Sic1 Inactivation has Parallels with Wee1
Inactivation
Sic1 is processively phosphorylated by Cln2 and Clb5
Clb5 phosphorylation generates a positive feedback loop
Kõivomägi et al., 2011, Nature 480, 128–131
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 Ubiquitindependent 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
E1
ATP
ADP + Pi
Ubq
UBC
Ubiquitin Ligase
E2
E3
E2
E3
Ubq
Su b st r at e
Ubq
DESTRUCTION
2 6 S Prot eoso me
Ubq
Ubq
Schematic of RING E3 ligases
COP9/Signalosome
UBQ
F-Box/
BTB/
SOCS
Box
Substrat
e
E2
RING
DOC1
Nedd8
Cullin
Mammalian
cells:
F-box
proteins
69 F box proteins
- bind to Cul 1
BTB domain:
proteins Fbw1- bind
to CulFbw2
3
Fbw>200
= WD40
= bTrcp1,
= bTrcp2, Fbw7 = Cdc4
SOCSrich
boxdomain:
proteinsFbl1
- bind
to Cul2/5
Fbl >50
= leucine
= Skp2
DDB1
proteins
- bind to Cul 4
Fbx>15
= other
domains
Nedd8 closes the gap between E2 and
substrate
Nedd8
Duda et al., 2008 Cell 134, 995-1006
Interplay between the SCF and the APC/C
•
•
SCFSkp2 is degraded by the APC/C
Allows p27 (Cdk inhibitor) to accumulate in G1
phase
Bashir et al., 2004, Nature 428, 190-193
Ubiquitination: Mitosis
Destruction box proteins
UBC (UBC10/UBC5 + Ube2S)
E2
Cdc20
E2
E3
E3
Cyclin B
Securins
P
APC/C
Ubq
APC7
APC3
APC10
APC6
APC11
Apc6
APC4
APC2
Su b st rat e
Apc3
Ubq
APC1
Ubq
Ubq
Pines, J. 2011. Nature Reviews Molecular and Cell Biology 12, 427-438
APC8
Apc8
APC5
Co-ordinating Mitosis by Proteolysis
Antephase
Pro-metaphase
NEBD
Metaphase
Chromosome Chromatid
Attachment Separation
Anaphase
Telophase
Cytokinesis
Spindle
Disassembly
Spindle checkpoint
Cyclin A
Securin
Cyclin B1
Cdc20 Plk1
UbcH10
Aurora A
Pines, J. 2006, Trends Cell Biol., 16, 55-63
APC10 and Cdc20 form the Destruction Box receptor
EM reconstructions
Da Fonseca et al., 2011, Nature 470, 274-278
APC/C specificity: two co-activators
Cdc20
Cdh1
Essential for Mitosis
Required for correct G1 phase & to degrade Aurora
kinases
Regulated by
Regulated by
spindle checkpoint
proteolysis by Cdh1
Proliferating cells
phosphorylation (by CDKs)
Rca1/Emi1
Somatic & Differentiated cells
(brain & trophectoderm)
Yu, H. 2007, Mol Cell., 27, 3-16; Garci-Higuera et al., 2008, Nature Cell Biology, 10, 802-811
Floyd et al., 2009 Curr Biol., 18, 1649-1658
Completion: Timing and Checkpoints
Chromosome Separation
M
S
DNA replication
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
First defined by Weinert and Hartwell, 1989, Science 246, 629.
“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.
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.
Control mechanisms enforcing dependency in the cell cycle are here called
checkpoints.”
Checkpoints:
S phase checkpoint
M
G2 Checkpoints
Ds breaks
UV
Morphogenesis Checkpoint
Prophase Checkpoint
Spindle Checkpoint
S
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
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?
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 Point
Committment
D-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
Committment
Turning on genes required for S phase and
mitosis requires de-repressing the E2F-family
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
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?
Dysregulation
Hit the gas
Cut the brakes
Loosen the steering
Hit the gas
The 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 PATHWAY
Engelman 2009 Nature Reviews Cancer
Hit the gas
Chromosome rearrangements drive proliferation signals
Frequent for the D-type Cyclins (D1 & D2)
Parathyroid adenoma
Hit the gas
•Mantle
cell lymphoma: Cyclin D1 next to IgH or
Cyclin D2 next to IgK
IgK fused to Cyclin D2
Cut the Brakes
Rb & the pocket proteins
p16
p21 & p27 - Skp2
Senescence and telomerase
Loosen 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 Steering
Genomic 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- EckholmReed, 2004, J. Cell Biol 165, 789-800
Lack of Cdh1 - Garcia-Higuera et al., 2008, Nat
Cell Biol 10, 802-811
Loosen the Steering
Genomic Instability
DNA damage repair pathways
ATM
p53 or Chk2 - Li-Fraumeni syndrome
Loosen the Steering
Genomic 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
Requires Mad1, Mad2, Bub1, Mad3/BubR1, Bub3,
Mps1, kinases
STOP
GO
CDC20
CDC20 + APC/C
Checkpoint monitors kinetochore attachment
Loosen the Steering
The Spindle Assembly Checkpoint detects most
errors in chromosome attachment but not
merotely
Loosen the Steering
Genomic Instability: Tetraploidy as a permissive state
- Fujiwara et al., 2005, Nature 437, 10431047
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 Steering
Extra centrosomes increase lagging chromosomes and mis-segregation