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The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006. Outline of Class 1. History and fundamentals 7. Discussion of papers 2. MPF and the discovery of CDKs 5.

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Presentation on theme: "The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006. Outline of Class 1. History and fundamentals 7. Discussion of papers 2. MPF and the discovery of CDKs 5."— Presentation transcript:

1 The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006

2 Outline of Class 1. History and fundamentals 7. Discussion of papers 2. MPF and the discovery of CDKs 5. DNA replication control 3. CDK regulation of mitotic entry and exit 4. Regulation of G1-S 6. Cell cycle checkpoints

3 Key points 1. Essential components of the cell cycle and what they do 2. Logic of cell cycle circuitry and switches 3. Experimental approaches Many topics we have no time to cover, or will cover in little detail: chromosome dynamics, cytokinesis, centriole replication, cancer cell cycles, developmental/alternative cell cycles, checkpoints, DNA replication mechanisms, meiotic cell cycle, growth control....

4 What is a Cell Cycle? 1850’s Schultze cytosol (protoplasm) and nucleus defined as separate entities in animal and plant cells 1830’s Schleiden & Schwann organisms are made of cells 1850’s Remak and Virchow omnis cellula e cellula --all cells come from other cells 1880’s Fleming and Strasburger mitosis (chromosomes look like threads) 1950’s Stages of cell cycle are defined DNA replication as a discrete event (S phase) 1960’s Continuous and discontinuous events RNA synthesis, protein synthesis, cell growth--continuous DNA synthesis, Mitosis--discontinuous Process by which cells replicate themselves

5 Cell Cycle Fundamentals 1 M G1 S G2 Mitosis Interphase 1 2 G1MG2S Relative Amount time Protein RNA DNA Mass/Size S phase and Mitosis are defined by processes G1 and G2 (gap phases) are defined by timing G1 (and G2?) can be split into more meaningful sub-stages by molecular and physiological criteria: Restriction Point in mammalian tissue culture cells defined by serum sensitivity Restriction Point

6 Cell Cycle Fundamentals 2 Mitosis is subdivided into different stages

7 Cell Cycle Fundamentals 3 The cell cycle can be broken into subcycles whose relationships change in different cell types and under different circumstances Timing in a typical somatic cell: 1. DNA replicationS phase 4. Cell Division (cytokinesis) Mitosis 3. Nuclear Division (karyokinesis)Mitosis 5. Cell GrowthThroughout 2. Centrosome duplication (microtubule organizing center) S phase In a typical somatic cell 1-5 are all regulated and coupled

8 Cell Cycle Fundamentals 4 Non-canonical cell cycles are found throughout nature and play a critical role in cell and developmental biology 1. DNA replication 3. Nuclear Division (karyokinesis) 4. Cell Division (cytokinesis) 5. Cell Growth 2. Centrosome duplication (microtubule organizing center) Somatic cell 1-5 regulated Embryonic cell cycle: 1,2,3,4 (division with no growth) Meiotic cell cycle 2,3,4: (division with no DNA replication) Megakaryocytes, slime molds: 1,2,3,5 (replication and nuclear division) Liver cells, fly salivary glands, many plant tissues: 1,2,5 (endoreplication=S phase with no mitosis) Oocyte formation: 5 (growth with no division) Some green algae: 5 and 1,2,3,4 separated (Chlamydomonas) or 1,2,3,5 and 4 separated (Scenedesmus) Ciliated epithelial cells, some protozoans: 2 (centriole amplification)

9 Cell Cycle Fundamentals 5 Cell Cycle States are Regulated I. Johnson and Rao (1975) Cell fusion experiments

10 Cell Cycle Fundamentals 5 Cell Cycle States are Regulated: nucleocytoplasmic ratio controls replication II. (Sudbery and Grant, 1976) Physarum (slime mold) Starved plasmodium no growth, no DNA replication UV irradiate ~50% of nuclei inactivated Remaining nuclei replicate and divide until nucleo-cytoplasmic ratio is restored

11 Cell Cycle Fundamentals 6 Models for Cell Cycle Analysis Xenopus oocytes/extracts Simple biochemical system budding yeast (Saccharomyces cerevisiae) Powerful genetics, G1-S regulation fission yeast (Schizosaccharomyces pombe) Powerful genetics, G2-M regulation mammalian tissue culture cells e.g. HeLa cells, NIH3T3 Closest model for human cells, regulation is more complex Details vary between organisms, general principles are similar

12 Xenopus oocytes/extracts Properties of Xenopus system: Cell free extracts can cycle between S phase and Mitosis Extracts can be manipulated to effect a mitotic arrest: chelate calcium > stabilize cytostatic factor (CSF) Feedback controls/checkpoints are missing* -extracts can cycle without nuclei G1 regulation is absent

13 Discovery of MPF (maturation promoting factor) Yoshio Masui and co-workers Serial injections can be repeated indefinitely until original source of MPF is diluted away MPF activity fluctuates and is present in both oocytes and fertilized embryos Tim Hunt and co-workers Cyclin synthesis and abrubt degradation mirrors rise and fall of MPF activity

14 Biochemical nature of MPF I Lohka and Maller Stabilize MPF in vitro from Xenopus extracts and fractionate: MPF has two components 32 kd and 45 kd; MPF posseses kinase activity 45 kd protein = cyclin B Kinase activity only present when both subunits are present Several groups: Murray and Kirschner Take cell free extract and RNase treat to destroy all endogenous mRNAs Inactivate RNase with inhibitor and add no RNA or cyclin mRNA activate extract to induce interphase and add sperm nuclei

15 Biochemical nature of MPF II Murray and Kirschner ∆13 equiv. to wt cyclin B ∆90=nondegradable cyclin missing destruction box Cyclin synthesis drives activation of MPF Cyclin destruction is required to inactivate MPF and drive cells into interphase MPF is required for cyclin destruction Cyclin destruction is NOT required to initiate anaphase Oscillator behavior of cell cycle is explained: cyclin MPF

16 Yeast Genetics and Unification of the Cell Cycle Main control point is G1-S boundaryMain control point is G2/M boundary

17 cdc mutants are critical for identifying cell cycle components Hartwell and colleagues, Nurse and colleagues budding yeast fission yeast mitotic cdc mutant cdc28 (budding yeast) has two arrest points pre-S phase, and pre-M cdc2 (fission yeast) shows arrest at G2/M, but also has dominant alleles that give a wee phenotype phenotypes suggest that these two cdcs have a critical role in cell cycle regulation G1-S blocked cdc mutant

18 Unifying observations for the cell cycle field budding yeast CDC28 = fission yeast cdc2 = Xenopus MPF 32kd subunit aka CDK1 budding yeast daf1/whi1 = a cyclin (later renamed Cln3) fission yeast cdc13 = a B-type cyclin homolog Key enzyme for cell cycle regulation is now defined as a cyclin dependent kinase complex (CDK) composed of a catalytic kinase subunit and a cyclin that activates the kinase 2001 Nobel Prize in Physiology or Medicine given to Tim Hunt, Lee Hartwell, and Paul Nurse for their pioneering work in cell cycle regulation Since then things got more complicated: Multiple CDKs, Multiple cyclins and Interacting proteins discovered All eukaryotes use the same set of proteins for cell cycle regulation with some species specific variation

19 Understanding the somatic cell cycle Somatic cells and yeasts have a G1 period with low CDK activity Somatic cells and yeasts have feedback controls that gate each transition to ensure proper completion of previous events Somatic cells and yeasts have multiple cyclins or CDKs that control progression through G1, S phase and mitosis

20 Nomenclature How to deal with all these gene names Protein yeasts budding fission animalsplants G0/G1 CDKs Cdc28 cdc2 CDK3 (G0), CDK4, CDK6 CDKA S CDKs Cdc28 cdc2 CDK2, CDK1CKDA G2/M CDKs Cdc28 cdc2 CDK1CDKA, CDKB G0/G1 cyclins Cln1, Cln2, Cln3 puc1 Cyclin D, Cyclin C (G0)D cyclins S cyclins Clb,5,6 cig2 Cyclin E, Cyclin AA cyclins G2/M cyclins Clb 1,2,3,4 cdc13 Cyclin BB cyclins

21 CDK Regulation How is CDK activity controlled during the cell cycle? Activation by cyclin binding Activation by phosphorylation (CAK) Inactivation by cyclin destruction (Anaphase Promoting Complex/Cyclosome aka APC/C) Inactivation by phosphorylation (Wee1) Inactivation by inhibitory binding proteins (KIP/CIP/WAF/KRP and INK) Activation by dephosphorylation (CDC25, Cdi1) Activation by destruction of inhibitor (Skp1-Cullin-F box complex aka SCF) Substrate specificity (CDK-cyclin combinations) Subcellular localization CDK abundance usually not regulated

22 CDK regulation I Cyclin concentration or CDK activity time Why is CDK kinase activity non-linear with respect to cyclin concentration?

23 CDK activation and inactivation by feedback loops Cdc2-cyclin B Cell cycle target proteins wee1 Inhibitory kinase cdc25 Activating phosphatase APC/C Ubiquitin ligase for cyclin CAK (CDK Activating Kinase) CKI/KIP ICK/KRP

24 Structural basis for CDK activation by cyclins and phosphorylation I Inactive monomer ATP misoriented, substrate binding occluded by T-loop, PSTAIRE helix mispositioned Cyclin bound ATP properly oriented via interaction with repositioned T loop and PSTAIRE helix. Substrate binding cleft suboptimal. Tyr14 site in roof of ATP binding cleft is available for Wee1phosphorylation (not shown)

25 Structural basis for CDK activation by cyclins and phosphorylation II CDK Thr160 +cyclin T loop flattened. Phospho T160 forms stabilizing interactions that optimize binding site With substrate peptide SPXK-containing peptide fits into pocket and interacts with T loop, including Phospho T160.

26 Phosphorylation/Dephosphorylation of CDKs cdc2 CAK (CDK activating kinase) --largely unregulated Wee1 Major negative regulator Cdc25 Major positive regulator Balance between Cdc25 and Wee1 activities regulates mitotic entry

27 Postive Feedback Loop for CDK Activation inactiveactive inactive Loop leads to explosive auto-activation of CDK once its activity rises above a certain threshold What are the substrates CDK1-cyclin B that lead to mitotic entry and progression? Cdc25, Histone H1, lamins, cyclin B and many more

28 Mitotic Exit is Regulated APC/C I APC/C is a E3 specificity factor for ubiquitin ligase pathway Cdc20 Hct1 core complex activator and specificity factor or + RXXL destruction (D) box- containing substrates Cdc20, Hct1 (Cdh1) B cyclin Pds1 RXXL + or KEN-box substrates Hct1 only Cdc20 Targeting by APC/C leads to rapid degradation by the 20S proteasome examples: KEN Cin8

29 Mitotic Exit is Regulated APC/C II B Cyclins and Pds1(Securin) are key substrates of APC/C: Nondegradable cyclin blocks MPF destruction but does not block anaphase APC/C has at least one more target whose destruction promotes anaphase Pds1/Securin destruction releases a protease, separase, that degrades cohesisns and allows sister chromatids to separate

30 Budding yeast mitotic exit Decreased CDK activity and Separase release activate FEAR (Cdc14 early anaphased release) Cdc14 activates a second pathway called MEN (mitotic exit network) that initiates cytokinesis Cdc14 dephosphorylates and activates cdh1 subunit of APC and Sic1 (a CDK inhibitory protein) to establish a stable G1 state with low CDK activity Cdc14 is a protein phosphatase that plays a central role in exiting mitosis

31 Overview of APC activation and mitotic exit CDK1-Cyclin B activates APC-Cdc20 directly or indirectly through phosphorylation Regulation of APC/C by CDK1-Cyclin B generates a negative feedback to drive mitotic exit A time lag between APC-Cdc20 activity and other essential mitotic events is essential Decreased CDK activity allows activation of Cdc14 mitotic exit pathway, Cdh1/Hct1 and establishment of a stable G1 state

32 G1 and G1-S regulation G1 is characterized by low CDK activity and high APC-Cdh1 activity What triggers initiation of S phase and cell cycle re-entry?

33 G1 and G1-S regulation I G1 is a major control point for most cell types: Growth factors present and extracellular conditions favorable: S phase Differentiation factors present, unfavorable conditions: G0 (temporary or permanent withdrawal from cell cycle) G1 M What triggers entry to S phase, what mechanisms prevent it? In budding yeast and animal cells G1 CDK activity must reach a threshold value to trigger S phase APC-Cdh1 must be inactivated to allow S phase cyclin accumulation CDK inhibitory proteins must be destroyed or titrated away Cells must be growing and have reached a minimum size mammalian cells must not contact neighbors (contact inhibition)

34 G1 control points Budding yeast cells have a G1 control point termed Start Remove nutrients prior to Start--G1 arrest Remove nutrients after Start--cells complete S, G2 and M Star t Restriction point in animal cells occurs late in G1 Serum withdrawal before R point--cells arrest in G1 Serum withdrawal after R point-cells complete S, G2 and M Restriction point G1 S Pardee (1974) Serum Dependent Serum Independent

35 G1 and G1-S regulation II animal cells Growth factors in serum e.g. FGF,PDGF activation of RTK signaling transcription of D cyclins budding yeast nutrients (glucose, nitrogen etc.) increased protein translation rate Increased translation of Cln3 Similarities of Cln3 and D cyclins: messages and proteins are low abundance proteins are highly unstable length of G1 highly sensitive to dosage and expression levels control rate limiting step in G1-S transition neither are essential!

36 Triggering Start in budding yeast I G1 cyclins Cln1, Cln2, Cln3 In early G1: Sic1 and APC-Hct1/Cdh1 are dephos. and active. CDK activity is low Transcription factors SBF, MBF--activators of Cln1, Cln2 and other S phase genes Whi5--negative regulator of SBF, MBF SCF--Skp1-Cullin-Fbox--E3 ubiquitin ligase targets G1 substrates (Elledge and Harper 1996) Sic1--CDK inhibitor--disrupts CDK active site, prevents ATP binding F-box proteins are specificity factors in SCF, often require phosphorylation for binding target substrate Cullin F-box protein (adaptor) Skp1 Cdc4 is F box adaptor for Sic1

37 Triggering Start in budding yeast II SBF or MBF Cln1 Cln2 Clb5 Clb6 Whi5 Cln3-Cdc28 Cln1/2-Cdc28 Sic1Clb5/6-Cdc28Hct1/Cdh1SCF-Cdc4 As Cln3-Cdc28 activity builds: SBF/MBF become active Whi5 is phosphorylated and dissociates from SBF/MBF Cln1/2 and Clb5/6 are made SBF/MBF are further activated in a positive feed back loop Sic1 and Hct1 are inactivated in a negative feedback loop ?

38 Sic1 inactivation is key for S phase initation Triple mutant ∆cln1 cln2 cln3 is inviable ∆cln1 cln2 cln3 sic1 mutant--viability is rescued! Sic1 is key target of G1 cyclins Sic1 becomes multiply phosphorylated by CDKs during G1 followed by abrupt degradation Tyers and colleagues Cdc4 binding of Sic1 depends on 6+ phosphorylations Replacement with a high affinity Cdc4 binding site causes premature S phase initiation and genome instability

39 G1 control in mammalian cells G1 cyclins D1-D3-(like Cln3) G1-S cyclin E (like Cln1/2) CDK inhibitors p27 Kip1 (homologous to Sic1), INK family (no homolog in yeast) E2F complexes (transcription factors for S phase genes, Cyclin E (like SBF/MBF) RB/p107/p130--E2F repressors (like Whi5) CDK4, CDK6-specific for D cyclins CDK2-binds E and A cyclins SCF-Skp2--targets free cyclin E and p27 for degradation (like SCF-Cdc4)

40 Establishing functions of G1 CDKs and cyclins Can’t easily make cdc mutants with diploid mammalian cells overexpression (transfection) dominant negatives Strategies for genetic analysis: knockouts in ES cells, whole mice, or cell lines from KO mice siRNA-mediated knockdowns Harlow and colleagues Dominant negative CDK mutations However, CDK2 and CycE KO cells have only mild S phase entry defects! For many years CDK2/CycE thought to be a linchpin of G1-S regulation

41 Negative regulation of G1-S is critical for animal cells Unregulated cell division leads to defects in tissue morphogenesis, development and cancer Several G1 regulators are tumor suppressors or oncogenes-RB, INKs, D cyclins, CDK4 CDK inhibitors INK4 (CDKN2) family specific for CDK4/6-Cyclin D complexes p21, p27,p57 proteins inhibit all CDKs Two classes of negative regulators: RB-related proteins RB, p107, p130--bind to E2F complexes and repress S phase transcription Regulators show some functional overlap and tissue specificity e.g. RB is expressed in cycling cells, p107/p130 in quiescent cells, p27 is constitutively expressed,p21 is induced by checkpoint activation, p57 is expressed in neuronal cells, p16INK4b induced by negative growth factor TGF beta

42 INKs and KIPs inhibit CDKs in different ways CDK INK cyclin Conformation change in CDK blocks cyclin binding CDK cyclin Kip Binds CDK-cyclin, blocks ATP binding and substrate access Early G1--INKs keep CDK4/6 cyclin D inactive, p27 keeps CycE-CDK2 inactive As CycD accumulates it overcomes INK binding to CDK4/6 CycD-CDK4/6 complexes compete p27 from CycE-CDK2 promoting S phase

43 Phosphorylation of RB is a key step in S phase activation E2F-DP RB CycE CycA S phase genes Early G1 RB hypophosphorylated E2F-DP RB P E2F-DP RB P P P Mid G1 RB partially phosphorylated by CDK4/6-D cyclins (priming) E2F-DP RB P PP PP P Restriction point/late G1/S RB hyperphosphorylated by CDK2-CyclinE complexes dissociation from E2F-DP Loss of one copy of RB leads to tumors CycD and CycE overexpressed in many cancers Animal DNA tumor viruses produce proteins (e.g. SV40 T antigen) that inactivate RB Plant DNA viruses have evolved the same trick

44 Does RB phosphorylation=Restriction Point? Previous work on bulk synchronized cells indicates correlation between RB phosphorylation, Cyclin E transcription and Restriction Point Zetterberg and colleagues Time lapse videomicroscopy on single cells + immunofluorescence to look at timing of RB phosphorylation vs. R vs. S phase What is the molecular correlate of the Restriction point?

45 G1 control in mammalian cells CycA-CDK2 also blocks E2F DNA binding by phosphorylation

46 Parallel Mechanisms of G1 regulation in budding yeast and metazoans

47 Coupling cell size to cell cycle progression yeasts show evidence of size control nutritional shift experiment: move cells from rapid growth to slow growth conditions- observe a G1 (budding yeast) or G2 (fission yeast ) delay until a minmal size is reached Data on animal cells is controversial but evidence for G1 size control exists. Growth may also be cell cycle regulated: RB controls rRNA synthesis Tumors often have aberrant growth characteristics faster cell cycle, faster growth

48 Regulation of DNA replication I Properties of DNA replication in eukaryotes: Occurs at a specific phase of the cell cycle--S Initiates from specific locations termed origins--well defined in budding yeast poorly defined in other organisms Occurs once and only once per S phase Completion of S phase is ensured by checkpoints S phase is regulated by oscillating CDK activity Low CDK activity required to prime origins High CDK activity required to fire origins and block re-priming

49 Key Components of S phase Regulation Cdc6 and Cdt1-- origin priming proteins-activity is tightly regulated Orc--origin recognition complex--binds origins throughout cell cycle, required for origin firing Mcms--(mini-chromosome maintenance)-part of a hexameric origin unwinding complex required for initiation, AAA ATPase family S phase CDKs--Clb 5/6-Cdc28 in budding yeast, CycE-Cdk2, CycA-Cdk2 in mammals Cdc7-Dbf4--kinase complex analogous to CDK-cyclin required for origin firing Geminin (metazoans only) inhibits Cdt1 mediated MCM loading at origins

50 How to ensure one round of replication? Origin “Licensing” Blow, Laskey and coworkers Naked DNA + interphase Xenopus extract Chromatin assembly, NE assembly, 1 round of DNA replication Mitosis Next round of DNA replication Add replicated G2 nuclei to fresh interphase extract control No replication Next round of DNA replication +NE permeabilizing detergent lysolecithin Something present in early interphase extracts that allows replication: licensing factor Licensing factor cannot cross NE. Lf gets made in early interphase, destroyed during S remade during M

51 Model for Licensing Factor (A) licensing factor (+) generated during M-G1 transition (B) + binds to chromatin prior to NE assembly (C) further access to + restricted by NE (D) nuclear + destroyed (-) upon S phase initiation (E) cytoplasmic + decays during late S and G2 Principles of model proved correct: Details vary between organisms Highly regulated and labile factors are generated in G1 that bind to origins (Cdc6, Cdt1) Cdc6 and Cdt1 allow loading of MCM complex prior to S phase S phase CDK activity simultaneously fires loaded origins and inactivates or destroys Cdc6, Cdt1, MCMs Multiple redundant mechanisms are involved

52 Pre-RC formation (licensing) involves ordered loading of proteins Crystal structure of archeal MCM complex Blue indicates + charge region that could accommodate ss or ds DNA

53 Molecular View of Licensing in Metazoans Cdc6, Cdt1 and MCM complex can only load onto origins in G1 After S phase CDK activation: Geminin (A APC-Cdh1 substrate) is stabilized and inhibits MCM loading origin unwinding by MCM triggers SCF-Skp2 mediated degradation of Cdt1 phosphorylation of priming proteins blocks activity, nuclear entry or origin binding

54 Summary of Replication Control Mechanisms

55 Cell Cycle Checkpoints Checkpoint: mechanism to ensure that the next cell cycle stage is not entered until the events of the current stage are completed Examples of important checkpoints: Spindle assembly--ensures all chromosomes attached to spindle prior to anaphase S-phase completion--ensures that replication is complete prior to mitotic entry DNA damage-blocks S phase initiation in G1 cells, blocks G2-M in S phase cells until DNA damage repaired

56 S phase completion checkpoint in fission yeast

57 Cell Cycle Checkpoints II What defines a checkpoint? Problem that sends a signal: --DNA damage --kinetochore unattached to mitotic spindle Signal detector and transducer: --DNA damage sensing kinase cascade --spindle attachment monitors-BUB/MAD proteins Target or Effector: --Cdc25 inactivated by DNA damage--G2-M block --APC Cdc20 inactivated by spindle checkpoint (Metaphase block) Checkpoints are often not essential under normal circumstances i.e. unperturbed cell cycle How to find them?

58 Genetic screens for DNA damage checkpoint mutants Isolate mutants that are hypersensitive to DNA damaging agents Rescreen for those that don’t arrest cell cycle properly

59 DNA damage checkpoints Sensors-- Mre11 complex? signal transducers Targets Most of these proteins are conserved p53 is a metazoan protein that helps decide whether a damaged cell arrests or commits suicide

60 Enough Already! I will make lecture notes and references available on the class web site soon

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