The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006. Outline of Class 1. History and fundamentals 7....

The Cell CycleJim UmenBGGN222

Feb. 21, 2006

Outline of Class1. 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

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 . . . .

What is a Cell Cycle?

1850’s Schultze cytosol (protoplasm) and nucleus defined as separate entities in

animal and plant cells

1830’s Schleiden & Schwannorganisms are made of cells

1850’s Remak and Virchowomnis cellula e cellula --all cells come from other cells

1880’s Fleming and Strasburgermitosis (chromosomes look like threads)

1950’s Stages of cell cycle are definedDNA replication as a discrete event (S phase)

1960’s Continuous and discontinuous eventsRNA synthesis, protein synthesis, cell growth--continuousDNA synthesis, Mitosis--discontinuous

Process by which cells replicate themselves

Cell Cycle Fundamentals 1

M

G1

S

G2

Mitosis

Interphase

1

2

G1 MG2S

Rela

tive A

mount

time

ProteinRNADNAMass/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

RestrictionPoint

Cell Cycle Fundamentals 2Mitosis is subdivided into different stages

Cell Cycle Fundamentals 3The 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 replication S phase

4. Cell Division (cytokinesis) Mitosis

3. Nuclear Division (karyokinesis) Mitosis

5. Cell Growth Throughout

2. Centrosome duplication (microtubule organizing center)

S phase

In a typical somatic cell 1-5 are all regulated and coupled

Cell Cycle Fundamentals 4Non-canonical cell cycles are found throughout nature and play a criticalrole 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)

Cell Cycle Fundamentals 5Cell Cycle States are Regulated

I. Johnson and Rao (1975) Cell fusion experiments

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Cell Cycle Fundamentals 5Cell Cycle States are Regulated:nucleocytoplasmic ratio controls replicationII. (Sudbery and Grant, 1976) Physarum (slime mold)



Starved plasmodiumno growth,no DNA replication

UV irradiate~50% of nuclei inactivated

Remaining nuclei replicate and divide untilnucleo-cytoplasmic ratio is restored

Cell Cycle Fundamentals 6Models for Cell Cycle Analysis

Xenopus oocytes/extractsSimple biochemical system

budding yeast (Saccharomyces cerevisiae)Powerful genetics, G1-S regulation

fission yeast (Schizosaccharomyces pombe)Powerful genetics, G2-M regulation

mammalian tissue culture cellse.g. HeLa cells, NIH3T3Closest model for human cells, regulation is more complex

Details vary between organisms, general principles are similar

Xenopus oocytes/extracts

Properties of Xenopus system:

Cell free extracts can cycle betweenS phase and Mitosis

Extracts can be manipulated to effect amitotic arrest: chelate calcium > stabilize cytostatic factor (CSF)

Feedback controls/checkpoints are missing*-extracts can cycle without nuclei

G1 regulation is absent

Discovery of MPF (maturation promoting factor)

Yoshio Masui and co-workers

Serial injections can be repeatedindefinitely until original sourceof MPF is diluted away

MPF activity fluctuates and is presentin both oocytes and fertilized embryos

Tim Hunt andco-workers

Cyclin synthesis and abrubtdegradation mirrorsrise and fall of MPF activity

Biochemical nature of MPF I

Lohka andMaller Stabilize MPF in vitro from

Xenopus extracts and fractionate:MPF has two components 32 kd and 45 kd; MPF posseses kinaseactivity

45 kd protein = cyclin B

Kinase activity only present when both subunits are present

Several groups:

Murray andKirschner

Take cell free extract and RNase treatto destroy all endogenous mRNAs

Inactivate RNase with inhibitor and add no RNA or cyclin mRNA

activate extract to induce interphase and add sperm nuclei

Biochemical nature of MPF II

Murray andKirschner

∆13 equiv. to wt cyclin B

∆90=nondegradable cyclin missing destruction box

Cyclin synthesis drivesactivation of MPF

Cyclin destruction is requiredto inactivate MPF and drivecells into interphase

MPF is required for cyclin destruction

Cyclin destruction is NOTrequired to initiate anaphase

Oscillator behavior of cell cycleis explained:

cyclin MPF

Yeast Genetics and Unification of the Cell Cycle



Main control point is G1-S boundaryMain control point is G2/Mboundary

cdc mutants are critical for identifying cell cycle components

Hartwell and colleagues, Nurse and colleaguesbudding yeast fission yeast

mitotic cdc mutant

cdc28 (budding yeast) has two arrest pointspre-S phase, and pre-M

cdc2 (fission yeast) shows arrest at G2/M, butalso has dominant alleles that give a weephenotype

phenotypes suggest that these two cdcs havea critical role in cell cycle regulation

G1-S blocked cdc mutant

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 acyclin dependent kinase complex (CDK) composed of acatalytic kinase subunit and a cyclin that activates the kinase

2001 Nobel Prize in Physiology or Medicine given toTim Hunt, Lee Hartwell, and Paul Nurse for their pioneering work incell cycle regulation

Since then things got more complicated: Multiple CDKs, Multiple cyclins andInteracting proteins discovered

All eukaryotes use the same set of proteins for cell cycle regulation withsome species specific variation

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 controlprogression through G1, S phase and mitosis

NomenclatureHow to deal with all these gene names

Protein yeasts budding fission

animals plants

G0/G1 CDKsCdc28cdc2

CDK3 (G0), CDK4, CDK6

CDKA

S CDKs Cdc28cdc2

CDK2, CDK1 CKDA

G2/M CDKs Cdc28cdc2

CDK1 CDKA, CDKB

G0/G1 cyclins Cln1, Cln2, Cln3puc1

Cyclin D, Cyclin C (G0)

D cyclins

S cyclins Clb,5,6cig2

Cyclin E, Cyclin A A cyclins

G2/M cyclins Clb 1,2,3,4cdc13

Cyclin B B cyclins

CDK Regulation

How is CDK activity controlled during the cell cycle?

Activation by cyclin binding

Activation by phosphorylation (CAK)

Inactivation by cyclin destruction (Anaphase PromotingComplex/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

CDK regulation I

Cyclin concentrationor CDK activity

time

Why is CDK kinase activitynon-linear with respect to cyclin concentration?

CDK activation and inactivation by feedback loops

Cdc2-cyclin B

Cell cycle targetproteins

wee1

Inhibitorykinase

cdc25

Activatingphosphatase

APC/CUbiquitinligase for cyclin

CAK (CDK Activating Kinase)

CKI/KIPICK/KRP

Structural basis for CDK activation by cyclins and phosphorylation I

Inactive monomerATP misoriented, substratebinding occluded by T-loop,PSTAIRE helix mispositioned

Cyclin boundATP properly oriented via interaction with repositioned T loop and PSTAIRE helix.Substrate binding cleft suboptimal.Tyr14 site in roof of ATP binding cleftis available for Wee1phosphorylation(not shown)

Structural basis for CDK activation by cyclins and phosphorylation II

CDK Thr160 +cyclinT loop flattened. Phospho T160forms stabilizing interactions that optimize binding site

With substrate peptideSPXK-containing peptidefits into pocket and interactswith T loop, including Phospho T160.

Phosphorylation/Dephosphorylation of CDKs



cdc2

CAK (CDK activating kinase) --largely unregulated

Wee1 Major negative regulator

Cdc25 Major positive regulator

Balance between Cdc25 and Wee1 activitiesregulates mitotic entry

Postive Feedback Loop for CDK Activation

inactive active

active

active

inactive

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

Mitotic Exit is Regulated APC/C I

APC/C is a E3 specificity factor for ubiquitin ligase pathway

Cdc20

Hct1

core complexactivator andspecificity factor

or+

RXXL destruction (D) box-containing substratesCdc20, Hct1 (Cdh1)

B cyclin

Pds1

RXXL

+

or

KEN-box substratesHct1 only

Cdc20

Targeting by APC/C leads to rapid degradation by the 20S proteasome

examples:

KEN

Cin8

Mitotic Exit is Regulated APC/C IIB Cyclins and Pds1(Securin) are key substrates of APC/C:

Nondegradable cyclin blocks MPF destructionbut 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

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 rolein exiting mitosis

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 andestablishment of a stable G1 state

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?

G1 and G1-S regulation IG1 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)

G1M

What triggers entry to S phase, what mechanisms prevent it?

In budding yeast and animal cells G1 CDK activity must reach athreshold 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)

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

Start

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)SerumDependent

SerumIndependent

G1 and G1-S regulation IIanimal cells

Growth factors in serum e.g. FGF,PDGF

activation of RTK signaling

transcription ofD 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 abundanceproteins are highly unstablelength of G1 highly sensitive to dosage and expression levelscontrol rate limiting step in G1-S transitionneither are essential!

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

Triggering Start in budding yeast II

SBF or MBF

Cln1Cln2Clb5Clb6

Whi5

Cln3-Cdc28

Cln1/2-Cdc28

Sic1 Clb5/6-Cdc28 Hct1/Cdh1SCF-Cdc4

As Cln3-Cdc28 activity builds:

SBF/MBF become active

Whi5 is phosphorylated anddissociates from SBF/MBF

Cln1/2 and Clb5/6 are made

SBF/MBF are further activatedin a positive feed back loop

Sic1 and Hct1 are inactivated ina negative feedback loop

?

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 byabrupt degradation



Tyers and colleagues



Cdc4 binding of Sic1depends on6+ phosphorylations



Replacement with a high affinity Cdc4 binding site causes premature S phase initiation and genome instability



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)

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

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 inhibitorsINK4 (CDKN2) family specific for CDK4/6-Cyclin D complexesp21, p27,p57 proteins inhibit all CDKs

Two classes of negative regulators:

RB-related proteinsRB, p107, p130--bind to E2F complexes and repress S phase transcription

Regulators show some functional overlap and tissue specificitye.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 negativegrowth factor TGF beta

INKs and KIPs inhibit CDKs in different ways

CDK

INK

cyclin

Conformation change inCDK blocks cyclin binding

CDKcyclin

Kip

Binds CDK-cyclin, blocksATP binding and substrateaccess

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

Phosphorylation of RB is a key step in S phase activation

E2F-DP

RB

CycECycA

S phase genes

Early G1 RB hypophosphorylated

E2F-DP

RBP

E2F-DP

RB

P PP

Mid G1 RB partially phosphorylatedby CDK4/6-D cyclins (priming)

E2F-DP

RBP

P P

PPP

Restriction point/late G1/S RB hyperphosphorylatedby CDK2-CyclinE complexesdissociation from E2F-DPLoss 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 RBPlant DNA viruses have evolved the same trick

Does RB phosphorylation=Restriction Point?

Previous work on bulk synchronized cells indicates correlation betweenRB phosphorylation, Cyclin E transcription and Restriction Point

Zetterberg and colleagues

Time lapse videomicroscopy on single cells + immunofluorescenceto look at timing of RB phosphorylation vs. R vs. S phase

What is the molecular correlate of the Restriction point?

G1 control in mammalian cells

CycA-CDK2 alsoblocks E2F DNA bindingby phosphorylation

Parallel Mechanisms of G1 regulationin budding yeast and metazoans

Coupling cell size to cell cycle progression

yeasts show evidence of size controlnutritional shift experiment:

move cells from rapid growth to slow growth conditions-observe a G1 (budding yeast) or G2 (fission yeast ) delay until a minmalsize 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 synthesisTumors often have aberrant growth characteristics

faster cell cycle, faster growth

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 yeastpoorly 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 activityLow CDK activity required to prime originsHigh CDK activity required to fire origins and block re-priming

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, requiredfor 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

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 replicationMitosis

Next round ofDNA 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 Sremade during M

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 generatedin 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 andinactivates or destroys Cdc6, Cdt1, MCMs

Multiple redundant mechanisms are involved

Pre-RC formation (licensing) involves ordered loading of proteins

Crystal structure of archealMCM complex

Blue indicates+ charge regionthat could accommodatess or ds DNA

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 loadingorigin unwinding by MCM triggers SCF-Skp2 mediated degradation of Cdt1phosphorylation of priming proteins blocks activity, nuclear entry or origin

binding

Summary of Replication Control Mechanisms

Cell Cycle Checkpoints

Checkpoint: mechanism to ensure that the next cell cycle stage is notentered until the events of the current stage are completed

Examples of important checkpoints:

Spindle assembly--ensures all chromosomes attachedto spindle prior to anaphase

S-phase completion--ensures that replication is completeprior to mitotic entry

DNA damage-blocks S phase initiation in G1 cells,blocks G2-M in S phase cells until DNA damage repaired

S phase completion checkpoint in fission yeast



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?

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

DNA damage checkpoints

Sensors--Mre11 complex?

signal transducers

Targets

Most of these proteins are conserved

p53 is a metazoanprotein thathelps decide whether adamaged cell arrests orcommits suicide

Enough Already!

I will make lecture notes and referencesavailable on the class web site soon

The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006. Outline of Class 1. History and fundamentals 7....

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Transcript of The Cell Cycle Jim Umen BGGN222 Feb. 21, 2006. Outline of Class 1. History and fundamentals 7....