1

Cdc28 Regulate Many Cell Cycle Events

How can one enzyme regulate multiple cell cycle events?

Synchronized cdc28ts cells can’t initiate S-phase

Cdc28 drives entry into S-phase as well as Mitosis

2

Cells Use Multiple Cyclins to Regulate Cells Use Multiple Cyclins to Regulate Different Cell Cycle EventsDifferent Cell Cycle Events

Cicline G1

Cicline M

3

Il complesso MPFMPF è formato da Cdc28/cicline mitotiche in S. cerevisiae e

Cdc2/Cdc13 in S. pombe

Il complesso SPFSPF è formato da Cdc28/cicline G1 in S. cerevisiae

Il complesso Il complesso MPFMPF regola l’ingresso mitotico regola l’ingresso mitotico (studi su (studi su S. pombeS. pombe))

Il complesso Il complesso SPFSPF regola l’ingresso in S regola l’ingresso in S (studi su (studi su S. cerevisiaeS. cerevisiae))

4

Modello di azione del complesso Cdc28/cicline Modello di azione del complesso Cdc28/cicline in in S. cerevisiaeS. cerevisiae

5

FunctionExpressionIdentificationCyclin

S-phase entryG1AccidentalClb6

S-phase entryG1AccidentalClb5

SpindleSHomologyClb4

SpindleSHomologyClb3

MitosisMHomologyClb2

MitosisMHomologyClb1

B-type Cyclins

STARTG1mutantCln3

STARTG1mutantCln2

STARTG1mutantCln1

G1 Cyclins

Budding Yeast Cyclin Genes

6

Cyclins NomenclatureCyclins Nomenclature

Cyclin ACyclin B’sS. pombe cdc13...S. cerevisiae CLB1,2,3,4,5,6

- G1’s more similar to each other-PEST domains (Pro,Glu,SerThr)

All contain “cyclin box” domain cdc2 binding domain

A and B’s closely related- destruction box- required for S and M- “A” accumulates earlier,- degraded earlier

G1 CyclinsS. cerevisiae CLN1,2,3

7

Cyclins can be grouped by expression patternsCyclins can be grouped by expression patterns

- CLN1 and CLN2 and CLB5 and CLB6 -

CLN1,2,CLB5,6

CLN3

CLB1,2

- CLB 1 and CLB2

- CLB3 and CLB4

CLB3,4

G1 Start Metaphase

[Cyc

lin]

[Cyc

lin]

Yeast Cyclin Genes: CLNs and CLBsYeast Cyclin Genes: CLNs and CLBs

Triple cln1,2,3 mutantTriple cln1,2,3 mutant

Quadruple clb1,2 mutantQuadruple clb1,2 mutant

Dominant CLN MutationsDominant CLN Mutations

CLN1,2,3CLN1,2,3Sense cell size, commit to divisionSense cell size, commit to division

Clb1,2Clb1,2Spindle assemblySpindle assembly

anaphaseanaphase

Clb5,6Clb5,6G1 entryG1 entry

Clb3,4Clb3,4Activate replication Activate replication originsorigins

9

Vertebrate Cells, like budding yeast, express multiple cyclins, but they also express several CDK’s

10

Regulation of G1/S transition (start/restriction point)

Identification of mammalian G1 cyclins

Human cDNA libraries screened for ability to complement CLN3 mutantlead to cloning of three human G1 cyclins:Cyclin CCyclin D1 Cyclin E

Cyclin D2 and Cyclin D3 subsequentlyidentified

D-type cyclins: respond to growth factors G0 to G1 transition

E-type cyclin: expression is periodic peaks at G1/S transition controls ability of mammalian cells to enter S-phase

11

Regulation of G1/S transition (start/restriction point)Regulation of G1/S transition (start/restriction point)

Identification of mammalian cyclin-dependent kinases (CDKs)Identification of mammalian cyclin-dependent kinases (CDKs)

Human cDNA libraries screened for ability to complement budding yeast CDC28 mutant

Three cDNA clones identified which could complement CDC28 mutant

Mammalian cdk1 acts at G2/M transition cdk2 acts at G1/S transition cdk3 unknown function

Cdk4 identified in an anti-cyclin D co-immunoprecipitation experiment

Unable to complement CDC28

12

13

Budding yeastBudding yeast MammiferiMammiferi

Punti di controllo del ciclo cellulare

START PUNTO DI RESTRIZIONE

14

Restriction point (START)

Point at which cell is irreversibly committed to traversing the cell cycleMammals: restriction pointYeast: START

Cell cycle proceeds without influence from environment (only stopped by damage)

Late in G1

15

To divide or not to divide:that is the question

Yeast cells make decision based on cell size, which is dependent on nutrient availability

Mammalian cells make decision based on the presence of protein growth factors called mitogens that stimulate cell growth

16

In assenza di fattori di crescita le cellule di mammifero si arrestano in G0 (cellule quiescenticellule quiescenti);

L’aggiunta di mitogeni causa il passaggio attraverso il punto di restrizione (dopo 14-16 oredopo 14-16 ore);

Le cellule in proliferazione entrano in S (dopo altre 6-8 oredopo altre 6-8 ore)

17

Cyclin D is required to pass restriction point

Colorazione 16 ore dopo la microiniezione e l’aggiunta di BrdU al mezzo.

Colorante per DNA

Anticorpi anti-BrdU

Anticorpi anti-cicl.D

R point

18

Continous expression of Cyclin E shortens G1Continous expression of Cyclin E shortens G1

19

Attività dei complessi Attività dei complessi Cdk/ciclina dei mammiferiCdk/ciclina dei mammiferi

All’inizio della fase S le cicline D ed E vengono degradate. I livelli di Cdk4 e 6 crollano repentinamente

Queste proteine non sono Queste proteine non sono necessarie per la progressione necessarie per la progressione

nella fase Snella fase SAll’inizio della fase S viene sintetizzata la ciclina A che si associa a Cdk2. La distruzione della ciclina A o una sua modificazione inibiscono la sintesi di DNA. Il complesso Cdk2/ciclina A è Il complesso Cdk2/ciclina A è

indispensabile per la indispensabile per la progressione nella fase Sprogressione nella fase S

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Cell-cycle phase-specific Cell-cycle phase-specific Cdk complexesCdk complexes

Three classes:G1 Cdk complexes

S-phase Cdk complexesMitotic Cdk complexes (also known as MPF)

Cell-cycle phase specificity determined by cyclin type and, in some cells, Cdk type

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Controllo del ciclo cellulare dei mammiferiControllo del ciclo cellulare dei mammiferi

Ingresso in fase S (complesso SPF)Ingresso in fase S (complesso SPF)

Progressione in fase SProgressione in fase S

Ingresso in fase M (complesso MPF)Ingresso in fase M (complesso MPF)Cdk1/ciclina ACdk1/ciclina B

Cdk4,6/ciclina D punto di restrizioneingresso in S

Cdk2/ciclina A

Cdk2/ciclina E

22

In addition to forming complexes between In addition to forming complexes between specific Cdks and their cyclin binding specific Cdks and their cyclin binding partners, all complexes require partners, all complexes require phosphorylation and dephosphorylation to phosphorylation and dephosphorylation to become fully activebecome fully active

phosphorylation of thr160/161 is phosphorylation of thr160/161 is important for the activation of all major important for the activation of all major Cdk/cyclin complexesCdk/cyclin complexes

catalysed by Cdk activating kinase CAKcatalysed by Cdk activating kinase CAK

23

Regolazione della Cdk1 mediante Regolazione della Cdk1 mediante fosforilazione-defosforilazionefosforilazione-defosforilazione

Equilibrio tra fosfatasi (Cdc25) e kinasi (Wee1)

Siti di inattivazione

Sito di attivazione

24

Another mechanism by which Another mechanism by which CDKs canCDKs can

regulate Multiple Transitions regulate Multiple Transitions is by usingis by using

different CDK inhibitors different CDK inhibitors (CKI’s)(CKI’s)

25

CDK activity is controlled by chemical modification, CDK activity is controlled by chemical modification, cyclin synthesis/proteolysis, and:cyclin synthesis/proteolysis, and:

CKIs (Cyclin-dependent kinase Inhibitors)CKIs (Cyclin-dependent kinase Inhibitors)

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CDK

Cyclin

CKI

T-Loop

CKI’s can be specific for particular CDK CKI’s can be specific for particular CDK complexes because their binding can depend upon complexes because their binding can depend upon

a specific cyclina specific cyclin

27

Cdc28/Cln1,2Sic1

Down-regulation of a CKI can, in turn, be Down-regulation of a CKI can, in turn, be regulated by a CDK-mediated phosphorylationregulated by a CDK-mediated phosphorylationbecause its ubiquitin-dependent proteolysis because its ubiquitin-dependent proteolysis can be regulated by its phosphorylation statecan be regulated by its phosphorylation state

28

Wee1 kinases

Cdc25 phosphatases

Cdk-Activating Kinases (CAK)

Kap1 phosphatases

T160

Y15

PhosphorylationControl

Ubiquitin-dependent Proteolytic Control

Transcriptional Control

Cyclin-dependent KinaseInhibitors (CKI’s)

We can add another limb to our regulation treeWe can add another limb to our regulation tree

Transcriptional Control

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Inibitori delle CdkInibitori delle CdkMembri diversi di questa famiglia inibiscono Cdk diverse

Cdc2/Clb P40

Cdc2/ClnFar1

Inibitore lievito

Cdk4/ciclinaDCdk6/ciclinaDCdk2/ciclinaECdk2/ciclinaA

p21, p27

Cdk4/ciclinaDCdk6/ciclinaD

p15, p16

Cyclin-Cdk complexInibitore cellule animali

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Meccanismi di controllo dell’attività dei complessi ciclina/CDK

Concentrazione delle cicline -livello di sintesi tramite controllo trascrizionale-proteolisi-localizzazione cellulare

Fosforilazione delle CDK

Inibitori delle CDK

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Proliferazione neoplastica: innesco della proliferazione in assenza di

programma, per mutazione di un gene cellulare o infezione di virus

Embriogenesi: proliferazione attivacicli cellulari brevirapida successione di fasi di replicazione (S) e mitosi (M) crescita "logaritmica".

Differenziamento: graduale attivazione di funzioni specializzate espansione clonale di cellule che si specializzano. contemporaneamente il ciclo di divisione viene modulato.

Differenziamento terminale: arresto programmato del ciclo (GO).

Reversibilità: cellule differenziate possono ripristinare il programma di divisione in risposta a stimoli (danni meccanici, agenti fisici etc.).

Situazioni biologiche in cui la proliferazione è controllata

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2 principali livelli di controllo della proliferazione di cellule eucariotiche:

controllo della scelta tra destino proliferativo / entrata in quiescenza (G0) ed eventuale differenziamento della cellula.

REGOLATO DALL’ ESTERN0

controllo delle fasi del ciclo e coordinamento tra i diversi eventi necessari

alla divisione cellulare.MECCANISMI DI CONTROLLO INTRINSECI

Controllo del ciclo cellulare e controllo della proliferazione

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A cell continues through the cell cycle after passing the restriction point (STARTSTART) unless it encounters genetic damage

If the cell receives a go-ahead signal, it completes the cell cycle and divides otherwise it switches to a nondividing state, the G0 phase. Most human cells are in this phase. Liver cells can be “called back” to the cell cycle by external cues (growth factors), but highly specialized nerve and muscle cells never divide.

Progress though the cell cycle is monitored at four checkpoints

Regulation of cell cycleRegulation of cell cycle

34

Cell cycle checkpointsCell cycle checkpoints ensure integrity of the genome

cell does not enter mitosis until DNA replication is complete and DNA damage is repaired

ensure chromosome segregation does not occur if chromosomes are incorrectly aligned on the mitotic spindle and spindle formation is inhibited

during the cell cycle a number of processes take place and they need to be co-ordinated

each process involves synthesis, assembly and correct function; all the changes that take place during the cell cycle have to integrated and correctly regulated

35

Look out for defect Look out for defect and emit a signal and emit a signal SENSORSSENSORS

Transmission of signals throughout Transmission of signals throughout the nucleus or cell and amplification the nucleus or cell and amplification TRANSDUCERSTRANSDUCERS

Delay cell-cycle progression Delay cell-cycle progression EFFECTORSEFFECTORS

Checkpoint machineryCheckpoint machinery

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Four checkpointsFour checkpoints

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THE THE G1/S CHECKPOINTG1/S CHECKPOINT

38

GROWTH FACTORS ARE INVOLVED GROWTH FACTORS ARE INVOLVED IN PASSING THE IN PASSING THE GG11 CHECKPOINT CHECKPOINT

Cyclin Cyclin

CyclinCyclin

Cyclin

Cyclin

CyclinCdK

CdK

CdK

ATP

ADP

Targetprotein

Pi

1. Arrival ofgrowth factorsfrom other cells.

2. Growth factors cause increase in cyclin concentration.

3. Cyclin activatescyclin-dependent kinase.

4. Kinases activate S phase proteins, leading to cell division.

39

Growth factor signalling and Growth factor signalling and transcriptiontranscription

Growth factors increase the expression of specific genes:

1. early response genes: rapid increase in mRNA and protein levels include transcription factors such as E2F, c-myc, c-

fos, c-jun

2. delayed response genes: include cell cycle proteins such as Cdks, cyclins expression regulated by early response genes

40

Regulated expression of two classes of Regulated expression of two classes of genes returns Ggenes returns G00 mammalian cells to mammalian cells to

the cell cyclethe cell cycle

Early-response genes:

Transcription factors (E2F)

Delayed-response genes:

CyclinD, ECdk2, 4, 6

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Il passagggio attraverso il punto di Il passagggio attraverso il punto di restrizione dipende dalla attivazione di restrizione dipende dalla attivazione di

fattori di trascrizione E2Ffattori di trascrizione E2F

I fattori E2F attivi stimolano la propria I fattori E2F attivi stimolano la propria sintesi e quella della Cdk2 e della Ciclina E;sintesi e quella della Cdk2 e della Ciclina E;

Il complesso Cdk2/CyclE porta all’aumento di Il complesso Cdk2/CyclE porta all’aumento di fattori E2F attivi fattori E2F attivi

42

E2F is a transcription factor that by itself E2F is a transcription factor that by itself activates transcriptionactivates transcription

E2F

However, Rb binds to E2F and represses its activation function

Rb

gene expressiongene expression

43

When Rb repression is inhibited by phosphorylation of Rb by early G1 Cyclin-CDK protein kinases (Cyclin D-CDK4/6), E2F stimulates the expression of genes required for S-phase, including genes encoding DNA polymerases and other proteins required for DNA synthesis, enzymes that synthesize dNTPs, and genes encoding late G1 Cyclin-CDKs (Cyclin E and CDK2) and the major S-phase Cyclin (Cyclin A).

Rb repression is regulated by Rb repression is regulated by early Gearly G11 Cyclins-CDKs Cyclins-CDKs

PP P P

E2FE2F

RbRbS-phase gene transcription

44

The G1/S transition in human cells: Rb (retinoblastoma protein) regulates E2F proteins, which are transcription

factors for S phase genes

45

Tumor-suppressor genes Tumor-suppressor genes Genes capable of suppressing the tumor-

forming potential of transformed cellsTumor suppressor genes normally function

to suppress cell growth and division.Tumor-suppressor genes encode :

Regulatory proteins eg rbrb, p53p53Intra-cellular signaling proteins (nf1-

Neurofibrosarcomas)Cell adhesion proteins (dcc–colon carcinomas)

46

Cells become malignant by losing tumor suppressor gene activity.

These act as Mendelian recessive traits. A cell has to be homozygous for a nonfunctional (or missing) tumor suppressor gene for it to have an effect.

AnalogyTumor suppressor brake pedalLack of suppressor no brake pedal

Tumor-suppressor genes Tumor-suppressor genes

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Gene oncosoppressore RbGene oncosoppressore Rb• Raro tumore dell’occhio che

colpisce circa un bambino su 14000 nati

• Provocato da due mutazioni consecutive che interessano entrambe le copie del gene Rb

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What is the defect in hereditary retinoblastoma?

Rb Rb Rb

Normal person:2 good copies ofRb gene

Hereditary retinoblastomapatient: 1 good copy ofRb gene, 1 defective copy

Compromise this good one, then problems!

or

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Il retinoblastoma erediatrio viene ereditato Il retinoblastoma erediatrio viene ereditato come carattere autosomico dominantecome carattere autosomico dominante

Poiché il gene Rb è localizzato sul cromosoma 13 sia i maschi che le femmine hanno la stessa

predisposizione alla malattia

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Perdita dell’eterozigosi degli antioncogeniPerdita dell’eterozigosi degli antioncogeni

51

Meccanismi genetici alla base del retinoblastoma

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Retinoblastoma and the “Two-hit” Retinoblastoma and the “Two-hit” model of carcinogenesismodel of carcinogenesis

Knudsons “two-hit” hypothesis:familial cases (high frequency, early onset): retinoblastoma caused by a germline mutation of one Rb allele + an acquired somatic mutation of the remaining allele of the Rb gene = both inactivatedsporadic cases (low frequency, late onset): retinoblastom caused by two acquired somatic mutations in both alleles = both inactivated

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Knudson’s 2-hit mutation model for retinoblastoma

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RB = tumor suppressor geneRB was the first tumor

suppressor to be identified.

RB is absent or mutated in at least one-third of all human tumors.

Cloning of the retinoblastoma gene mapped to 13q14 (loss of heterozygosity) rb-1 gene cloned 1986-87 Mutated or lost in all cases of retinoblastomas Also found mutated in osteosarcoma and small-cell lung cancer

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Il gene Rb-1Il gene Rb-1GeneticaFrequenza del tumore: 1/14.000 nasciteLe cellule tumorali sono prive di entrambi gli alleli Rb-1 funzionaliAssetto più frequente: una piccola delezione trasmessa dalla linea germinale, poi una mutazione di senso sull’allele omologo dà origine a un clone somatico Associazione di molti casi di tumore con la delezione della banda 13q1.4

Trasmissione mendeliana semplice: predisposizione allo sviluppo di tumori dominante: un cromosoma "difettivo" predispone tutti i portatori a sviluppare tumori;sviluppo di tumori recessivo: il tumore si sviluppa in completa assenza del prodotto, quando entrambi gli alleli sono deleti e/o mutati

Studi in sistemi modello trasfezione del gene Rb1 in cellule proliferanti, microiniezione di proteina pRb, inibiscono il superamento della transizioneG1/S

Topo Rb-/- : letale (difetti nell’eritropoiesi e nei tessuti neuronali)

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RB - structure of gene and proteinRB - structure of gene and protein Gene

– Highly complex: 200 kb with 27 exons and introns from 80bp to 60kb

Protein– multiple bands Mw= 110-116 kDa– nuclear phosphoprotein– binds DNA non-specifically

Rb contains several functional domains– Domains A and B are highly conserved from humans to

plants, and they interact with each other along an extended interdomain interface to form the central “pocket”, which is critical to the tumour suppressor function of Rb

57

La proteina Rb può essere suddivisa in quattro domini. Il dominio N La proteina Rb può essere suddivisa in quattro domini. Il dominio N è responsabile dell’oligomerizzazione della proteina in vitro. I è responsabile dell’oligomerizzazione della proteina in vitro. I domini A e B, detti “A/B pocket”, sono responsabili del legame di domini A e B, detti “A/B pocket”, sono responsabili del legame di Rb a vari fattori trascrizionali, come E2F e varie oncoproteine Rb a vari fattori trascrizionali, come E2F e varie oncoproteine virali, e risultano spesso mutati in vari tumori. Il quarto dominio, virali, e risultano spesso mutati in vari tumori. Il quarto dominio, detto “C pocket” è un sito di legame per c-Abl .detto “C pocket” è un sito di legame per c-Abl .

Caratteristiche strutturali di RbCaratteristiche strutturali di Rb

58

RB FAMILYRB FAMILY p105 RB; p107; p130 pRB binds to the transcriptional activation

domain of E2F and blocks activation E2Fs transactivate expression of genes

that are important for S phase:

dihydrofolate reductasedihydrofolate reductasethymidine kinasethymidine kinasepolymerase polymerase histoneshistones

59

RB´s function: “a signal transducer connecting the cell cycle clock with the

transcriptional machinery”

RB constitutively expressed and relatively stable half-life ≥ 12 hours Still induced increase in levels

resting G0 cells + mitogenic stimuli = RB level increased 4-6x

RB modified by phosphorylation during cell cycle

RbMM

G1G1G2G2

SS

Cell cycle clock

Transcriptional apparatus

60

Rb a substrate of CycD-CDKCycE-CDKCycA-CDK

CycC-Cdk3pRb

G0

61

RB is active only within a limited time RB is active only within a limited time window during the cell cyclewindow during the cell cycle

Before the R-point in G1: Rb hypophosphorylated = active repressor of growth (inhibits cell cycle progression) SDS-PAGE: 110 kDa

After the R-point in G1: Rb hyperphosphorylated = inactive repressor of growth (facilitates cell cycle progression) SDS-PAGE: 112 - 116 kDa

Rb is dephosphorylated at the end of mitosis

R

Rb

RbP P P PP

P

activerepressor

Inactiverepressor

MM

G1G1G2G2

SS

P

62

Gate-keeper model for RBGate-keeper model for RB The R-point functions as a door that is kept closed by Rb

G1 arrest upon overexpression of Rb Under conditions favourable for proliferation ⇒ Rb phosphorylated

⇒ R-door is opened In cells with lost Rb-function the door is left open all the time

Such cells will also have lost the ability to respond to growth-promoting/-inhibitory signalsMitogenes (+), TGFβ (-), contact-inhibition (-)

Two key elements in this model: upstream signals ⇒ Rb´s phosphorylation status Rb´s phosphorylations status ⇒ downstream effects

Rb as “signal transducer” Cell cycle-clock ⇒ RB´s phosphorylation status RB´s phosphorylation status ⇒ transcription apparatus involved

in proliferation

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M

G1G2

S RRb

Cdk4/6Cyclin D

E2F releasedS-phase genes expressed

Gate keeper modelGate keeper model

PPPP

PPPP

PP

PP

PP

64

E2F liberated by Rb inactivationE2F liberated by Rb inactivation

• Rb excert its effects through E2F TFs

Rb = active repressor

Rb = inactivated

R-point

E2F = activated!

65

RB´s phosphorylation status RB´s phosphorylation status ⇒⇒ a signal to the transcription apparatus a signal to the transcription apparatus

• Hypophosphorylated RB binds and inactivates the transcription factor E2F/DP

• Hyperphosphorylation of RB ➨ E2F/DP liberated and free to activate genes necessary for proliferation

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The functional state of the retinoblastoma protein The functional state of the retinoblastoma protein (pRb) controls cell proliferation(pRb) controls cell proliferation

GFs, mitogens...

cyclin D1 synthesis

Cell cycle progression

G1

RNA pol II

5’..TTTCCGCG…3’

cyclin D1

cdk4/6

Active cyclin/kinasecomplex

Cell cycle genes off

G0

pRb

E2F/DPdimers

5’..TTTCCGCG…3’

wt pRb represses transcription of cell cycle genes

Phosho-pRb becomes inactive

P

P

67

Target genes controlled by Target genes controlled by activating E2Fsactivating E2Fs

E2F sites common consensus binding site: TTTCGCGC No difference in sequence preference between different

E2Fs target genes: E2F controls the transcription of cellular genes

that are essential for cell division: cell cycle regulators

such as cyclin E, cyclin A, Cdc2, Cdc25A, RB and E2F1, enzymes that are involved in nucleotide biosynthesis

such as dihydrofolate reductase, thymidylate synthetase and thymidine kinase

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E2F/DP only active in a window E2F/DP only active in a window of the cell cycle (late G1 of the cell cycle (late G1 ➜➜ early S) early S)

• Early G1: active RB ➜ E2F/DP turned OFF• The R-point: inactivated RB ➜ E2F/DP turned ON

– E2F/DP liberated ➜ activation of E2F-dependent promoters

• Late S: E2F/DP turned OFF again– cyclin A/cdk2 ➜ phosphorylation of E2F/DP ➜ reduced

DNA-binding ➜ target genes turned off

69

HOW ARE S PHASE PROTEINS ACTIVATED?HOW ARE S PHASE PROTEINS ACTIVATED?

Rb

CyclinCdK

Rb

E2F

Rb

ATP

ADPPi

Pi

E2FE2FE2F

S Phaseproteins

mRNA

DNA

1. In normal cells,Rb protein binds to E2F and shutsdown the cell cycle.

2. If growth factors arrive and activate the cyclin-CdK complex, Rb becomes phosphorylated.

3. E2F is released

4. E2F stimulates the

production of S phase

proteins.

When Rb is mutated, no “tie-up” of E2F, so constant S phase turn on

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Effetti delle mutazioni del Gene RbEffetti delle mutazioni del Gene Rb

OsteosarcomiOsteosarcomiCarcinomi: Carcinomi: 1) Polmonari 1) Polmonari 2) Mammari2) Mammari3) Prostatici3) Prostatici