The Cell Cycle Jonathon Pines Gurdon Institute j

The Cell Cycle Jonathon Pines Gurdon Institute j. pines@gurdon. cam. ac. uk http: //www. gurdon. cam. ac. uk/~pineslab/New_Web _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? G 1 or S or G 2 M = + G 1 S + + M S G 2 = S G 2 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 cdc 2 is a conserved protein kinase required at 2 points in cell cycle and with a wee allele Wee 1, mik 1 cdc 25 cdc 2 Interphase cdc 13 (Cyclin B) Mitosis After Murray and Hunt, 1993.

MPF: a potent trigger for mitosis Xenopus laevis: arrested in G 2 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 G 1 of first mitotic cell cycle Fertilisation causes large increase in translation One protein only translated after fertilisation and destroyed at each mitosis: Cyclin MPF S M Evans et al. 1993. Cell 33, 389 MPF eventually shown to consist of cyclin and Cdc 2 (Dorée and Hunt, 2002, J Cell Sci, 115, 2461 -4)

Cyclin-CDK complexes define the state of the cell cycle Mitosis Initiation cdc 13 -cdc 2 M Degrade cdc 13 or cdc 2 G 1 S DNA Synthesis START Re-initiate DNA synthesis DNA Synthesis Initiation cig 2 -cdc 2 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, p 345 -350 Norton and Diffley, 2000, Molecular Cell, 5, 85 -95

Testing the Cdk threshold hypothesis Cyclin B (Cdc 13) Analogue Sensitive Cdc 2 Kinase Cyclin-cdc 2 fusion drives cell cycle in absence other cyclins Control amount of kinase activity through analogue sensitive Cdc 2 Low concentrations of inhibitor block mitosis High concentrations block DNA replication Add different inhibitor concentrations to drive cell cycle with non-degradable Cdc 13 Block G 2 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 B 1 -CDK 1 Cyclin A-CDK 1/2 G 0 M G 2 G 1 START or R point Cyclin D-CDK 4/6 Completion of DNA Synthesis S Initiation of DNA Synthesis Cyclin A-CDK 2 Cyclin E-CDK 2

Waves of cyclin-CDK kinase activity during the human cell cycle G 1 S G 2 M G 1 Kinase activity Cyclin B-CDK 1 Cyclin A-CDK 2 Cyclin E-CDK 2 M Growth Factors Cdc 25 A time

But Cdk 2 is Not Essential for Mitosis Ortega et al. , 2003, Nature Genetics, 35, p 25 -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 • • • Cdk 2 - spermatogenesis, oogenesis Cdk 4 - pancreatic beta cells, pituitary, mammary epithelium Cdk 4 or Cdk 6 - 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 B 1 -CDK 1 Cyclin A-CDK 1/2 G 0 M Polo Aurora G 2 G 1 Cyclin D-CDK 4/6 Completion of DNA Synthesis S Initiation of DNA Synthesis Cyclin A-CDK 1 & 2 Cyclin E-CDK 2 S

Coordination between mitotic kinases Kinase Domain • PBD Polo box binds to S-p. T or S-p. S - Elia et al. , 2003, Science 299, p 1228 • • Often generated by Cdk (S-T-P) Thus Cdk phosphorylation generates Plk substrate Eg: Cdk 1 phosphorylates INCENP to recruit Plk 1 to kinetochores - Goto et al. , 2006, Nature Cell Biology 8, p 180

Regulating a CDK Cyclin Proteolysis (Ubiquitin) Cyclin Binding Thr 14 & Tyr 15 Phosphorylation (wee 1/mik 1) Thr 14 & Tyr 15 dephosphorylation (Cdc 25) T-loop Thr dephosphorylation (p 24 KAP ) T-loop Thr phosphorylation (CAK) Inhibitor binding Inhibitor removal T 14 Y 15 Deactivate CDK Activate T 160 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

Wee 1 - Generating a Robust Switch • Fission yeast wee 1 and mik 1 prevent premature mitosis • mik 1 stabilised by unreplicated DNA • Wee 1 in animal cells blocks mitosis in interphase Ultrasensitive response to inhibition by Cdk 1 - bistable SP SP SP Sites not well conserved Not inhibitory SP SP state Kinase Domain 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 Sic 1 Cln 2 -Cdc 28 Clb 5 -Cdc 28 Cdc 34 - SCF p 40 Sic 1 Cdc 28 -Clb 2 M Sic 1 p 40 Degradation Cdc 28 -Clb 5/6 G 1 S Phase Cdc 4

Sic 1 Inactivation has Parallels with Wee 1 Inactivation Sic 1 is processively phosphorylated by Cln 2 and Clb 5 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, p 1670

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 = Skp 1 + Cullin + F-box protein APC/C = Anaphase Promoting Complex/Cyclosome

Ubiquitination E 1 ATP ADP + Pi Ubq UBC Ubiquitin Ligase E 2 E 3 Ubq Su b st r at e Ubq DESTRUCTION 2 6 S Prot eoso me Ubq

Schematic of RING E 3 ligases COP 9/Signalosome UBQ F-Box/ BTB/ SOCS Box Substrat e E 2 RING DOC 1 Nedd 8 Cullin Mammalian cells: F-box proteins 69 F box proteins - bind to Cul 1 BTB domain: proteins Fbw 1 - bind to Cul. Fbw 2 3 Fbw>200 = WD 40 = b. Trcp 1, = b. Trcp 2, Fbw 7 = Cdc 4 SOCSrich boxdomain: proteins. Fbl 1 - bind to Cul 2/5 Fbl >50 = leucine = Skp 2 DDB 1 proteins - bind to Cul 4 Fbx>15 = other domains

Nedd 8 closes the gap between E 2 and substrate Nedd 8 Duda et al. , 2008 Cell 134, 995 -1006

Interplay between the SCF and the APC/C • • SCFSkp 2 is degraded by the APC/C Allows p 27 (Cdk inhibitor) to accumulate in G 1 phase Bashir et al. , 2004, Nature 428, 190 -193

Ubiquitination: Mitosis Destruction box proteins UBC (UBC 10/UBC 5 + Ube 2 S) E 2 Cdc 20 E 2 E 3 Cyclin B Securins P APC/C Ubq APC 7 APC 3 APC 10 APC 6 APC 11 Apc 6 APC 4 APC 2 Su b st rat e Apc 3 Ubq APC 1 Ubq Pines, J. 2011. Nature Reviews Molecular and Cell Biology 12, 427 -438 APC 8 Apc 8 APC 5

Co-ordinating Mitosis by Proteolysis Antephase Pro-metaphase NEBD Metaphase Chromosome Chromatid Attachment Separation Anaphase Cytokinesis Telophase Spindle Disassembly Spindle checkpoint Cyclin A Securin Cyclin B 1 Cdc 20 Plk 1 Aurora A Ubc. H 10 Pines, J. 2006, Trends Cell Biol. , 16, 55 -63

APC 10 and Cdc 20 form the Destruction Box receptor EM reconstructions Da Fonseca et al. , 2011, Nature 470, 274 -278

APC/C specificity: two co-activators Cdc 20 Cdh 1 Essential for Mitosis Required for correct G 1 phase & to degrade Aurora kinases Regulated by spindle checkpoint proteolysis by Cdh 1 Proliferating cells phosphorylation (by CDKs) Rca 1/Emi 1 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 G 2 Checkpoints Ds breaks UV Morphogenesis Checkpoint Prophase Checkpoint Spindle Checkpoint M S

Checkpoints Think about the biology – DNA damage: budding yeast arrest in mitosis fission yeast and animal cells in G 2 What should be the phosphorylation state of Cdk 1? What should be the targets of the checkpoint?

Checkpoints • Budding yeast do use Cdk 1 Y 18 phosphorylation • Part of the morphogenesis checkpoint to prevent budding in inappropriate conditions • Major regulation is on the stability of Swe 1 p • Degradation of Swe 1 accompanied by relocalisation to bud neck via binding to Hsl 7 p Mc. Millan et al. , 2002, Mol. Biol. Cell. , 13, p 3560 -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 D 1 binds to notch promoter - Bienvenu et al. , 2010 Nature 463, 374 D-type Cyclins are basically there to turn on E-types Rescue Cyclin D 1 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 E 2 F-family

CKIs are important Regulators of G 1 Phase Cyclin-Cdks INK 4 family inhibit only D-Cdk 4/6 CIP/Kip (p 21/p 27/p 57) inhibit E & A Ubiquitin-mediated proteolysis is important to regulate Ckis and to maintain quiescence Wirth et al. , 2004 Genes Dev 18, 88 -98 SCFSkp 2 is degraded by the APC/C - allows p 27 to accumulate in G 1 phase

Balancing proliferation & differentiation Number of progenitor divisions will determine tissue or organ size E. g. : Neural cortex in Cdh 1 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. : PI 3 Kinase pathway, activates AKT & promotes survival and growth Activated/amplified RTKs & AKT PTEN is a commonly deleted tumour suppressor

PI 3 K PATHWAY Engelman 2009 Nature Reviews Cancer

Hit the gas Chromosome rearrangements drive proliferation signals Frequent for the D-type Cyclins (D 1 & D 2) Parathyroid adenoma

Hit the gas • Mantle cell lymphoma: Cyclin D 1 next to Ig. H or Cyclin D 2 next to Ig. K fused to Cyclin D 2

Cut the Brakes Rb & the pocket proteins p 16 p 21 & p 27 - Skp 2 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: Cdc 4 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 Cdh 1 - Garcia-Higuera et al. , 2008, Nat Cell Biol 10, 802 -811

Loosen the Steering Genomic Instability DNA damage repair pathways ATM p 53 or Chk 2 - Li-Fraumeni syndrome

Loosen the Steering Genomic Instability Chromosome segregation Very few genetic diseases because essential Mosaic variegated aneuploidy - Bub. R 1 - Hanks et al. , 2004 Nat Genet 36, 1159 -1161

The Spindle Assembly Checkpoint Requires Mad 1, Mad 2, Bub 1, Mad 3/Bub. R 1, Bub 3, Mps 1, kinases STOP GO CDC 20 + 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