RB and E2Fs

- linking trx with cell cycle

RB
- a tumour supressor

MBV4230

tumour suppressor genes

Fusion of normal cells with tumour cells

suppression of neoplastic properties tumour
suppressor genes must exist

Sincehealthycellsaredominantovertumourcellswhenitcomestogrowth
propertiestumourcellshavelostfunctionsassociatedwithtumoursuppressors

Rb, the retinoblastoma susceptibility gene, was cloned

and identified as the first tumour suppressor gene in
1986

Eyecancerinchildren(1:20000below3years)

TSG +/+

TSG /

TSG +/

MBV4230

RB = tumour suppressor

RB was the first tumour

suppressor to be
identified.
RB is absent or mutated
in at least one-third of
all human tumours.

MBV4230

Retinoblastoma and the Two-hit

model of carcinogenesis

Knudsons two-hit
hypothesis:

Ifamilialcases(highfrequency,early
onset):retinoblastomacausedbya
germlinemutationofoneRballele+an
acquiredsomaticmutationofthe
remainingalleleoftheRbgeneboth
inactivated
Isporadiccases(lowfrequency,late
onset):retinoblastomacausedbytwo
acquiredsomaticmutationsinboth
allelesbothinactivated

m
ut

*
m
ut

**

*
early
onset

late
onset

MBV4230

RB - structure of gene and protein

Gene
Theretinoblastomasusceptibilitygene,rb1gene,cloned198687
Highlycomplex:200kbwith27exonsandintronsfrom80bpto60kb

Mutated or lost in all cases of retinoblastomas

Protein
multiplebandsMw=110116kDa
nuclearphosphoprotein
bindsDNAnonspecifically

Rb contains several functional domains

DomainsAandBarehighlyconservedfromhumanstoplants,andtheyinteract
witheachotheralonganextendedinterdomaininterfacetoformthecentral
pocket,whichiscriticaltothetumoursuppressorfunctionofRb

MBV4230

Mechanisms of RB inactivation

RB functions as a molecular
scaffold for trx complexes. RB
inactivation may occur by four
known mechanisms.

TheRBgeneismutated(dashedline),
causingreleaseofitsassociatedfactors.
RBmutationshavebeendetectedin
retinoblastomaandasmallfractionof
sporadictumours.
RBissequesteredbyviraloncoproteins,
preventingbindingtootherfactors

SV40largeTantigen
adenovirusE1A
humanpapillomavirusE7

Phosphorylation(P)ofRBbyCDK
cyclincomplexesduringcellcycle
progressiondisruptsitsabilitytoassemble
trxcomplexes.
RBisdegradedbyacaspasedependent
proteolyticpathwayduringapoptosis.

RB
- controlling the cell cycle

MBV4230

RBs function: a signal transducer connecting the cell

cycle clock with the transcriptional machinery

Cell cycle clock

M
G2

Rb
G1
Transcriptional apparatus

RB constitutively expressed and relatively stable

halflife12hours
Stillsomeinductionunderspecificconditions:

restingG0cells+mitogenicstimuliRBlevelincreased46x

RB modified by phosphorylation during cell cycle

Cell cycle

MBV4230

Cell cycle - phases

The cell-division cycle is usually divided into four

distinct phases.
G1(gap1)isagrowthphasethatoccursbefore
S(synthesis)phasethestageofDNAreplication.Thisisfollowedby
asecondgapphase,G2,
whichprecedesM(mitosis)phase,duringwhichchromosomesegregationand
celldivisionoccurs.

M
G2

G1
S

MBV4230

Cell cycle - driven by cdks

Orderly progression through these cell-cycle phases is controlled
by the sequential activation of the Cdks.
Cyclines and cyclin-dependent kinases (cdk)

cyclines+cdkcellcycledependentvariationsintheactivityofthekinases
phosphorylationofnuclearfactorssuchasRBchangesduringthecycle
Thesubsequentphasesarecontrolledbycyclincdkpairsasshownbelow

Cellular stress activation of checkpoint pathways cell-cycle

progression is disrupted
The R-point: restriction point 2/3 into G1
M

G2

G1
S

MBV4230

Cyclins

Cyclines and cyclin-dependent kinases (cdks)

Thecyclineshaveoscillatinglevelsduringcellcycle
ThecyclinesareregulatorysubunitsoftheCDKkinases

cyclines+ cdk cell cycle-dependent variations in the

activity of the kinases
Cyclin E Cyclin A Cyclin B

determined
by
mitogenic
growth
factors

Cyclin D

G0

G1

G2 M

MBV4230

Restriction point of the cell cycle

Growth factors (both positive and negative) exert their

effect during the G1 phase.
Beyond the restriction (R) point = committed

Therestriction(R)pointdefinesacriticaltimeinlateG1afterwhichacellis
committedtoundergoDNAreplicationandisnolongersensitivetogrowthfactor
signalling.AftertheRpoint,cellcycleprogressioncanonlybehaltedby
conditionsofcellularstress,suchasDNAdamageormitoticspindledefects.
Beforetherestrictionpoint,thecellhasachoicebetweencelldivision(growth)by
continuingthecellcycle,andrestbygoingintoG0
Beyondtherestrictionpointthecelliscommitedtoproceeduntilcelldivision(M)

Growthfactorsensitive

Committedinsensitive

MBV4230

Regulating
cell cycle

Cdk regulation
cyclins,
inhibitoryandactivatingphosphorylationevents,
association/dissociationofinhibitorymolecules
calledCdkinhibitors(CDIs).

Mitogenic growth factors

exerttheireffectbypromotingthesynthesisofthe
Dtypecyclins.
cyclinEistriggeredbyinternalsignalling
theappearanceofCdk2cyclinEkinaseactivity
seemstobesynonymouswiththerestrictionpoint.

The ordered activation of the

remaining Cdkcyclin complexes
seems to be self-regulating:

eachCdkcyclincomplextriggerstheactivationof
thenextCdkcyclinspecies.

RB - gatekeeper of the
cell cycle

MBV4230

RB is active only within a limited

time window during the cell cycle
Before the R-point in G1: Rb = hypophosphorylated
active repressor of growth (inhibits cell cycle progression)

SDSPAGE:110kDa

After the R-point in G1: Rb = hyperphosphorylated

inactive repressor of growth (facilitates cell cycle
progression)

SDSPAGE:112116kDa

G2

Rb
G1

Rb is dephosphorylated at the end of mitosis

active
repressor

Coupling phosphorylation status/function

OncoproteinsfromDNAtumourvirusbind/inactivateprefhypoRB
OnlyhypoRbbind/inactivatesandrecellulreproteins/TFs
StimulithatenhanceRbphosphorylationfacilitateproliferation

Rb
Inactive
repressor

P
P
PPPP

MBV4230

Gate-keeper model for RB

The R-point functions as a door that is kept closed by Rb

G1arrestuponoverexpressionofRb

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

Suchcellswillalsohavelosttheabilitytorespondtogrowthpromoting/inhibitory
signals

Mitogenes(+),TGF(),contactinhibition()

Two key elements in this model:

upstreamsignalsRbsphosphorylationstatus
Rbsphosphorylationsstatusdownstreameffects

Rb as signal transducer
CellcycleclockRBsphosphorylationstatus
RBsphosphorylationstatustranscriptionapparatusinvolvedinproliferation

MBV4230

Gate keeper model

M
G2

G1
S

Cdk4/6
Cyclin D

Rb

E2F released
S-phase genes expressed

Signaling to RB
- Upstream events

MBV4230

Cell cycle clock RBs

phosphorylation status

Multiple Ser/Thr sites in RB are phosphorylated

multiplekinasesconvergeonRB

Multiple sites typical CDK sites

M
G2

G1
S

Cyclin D most involved in RB phosphorylation

G1CyclinsD1,D2andD3areregulatorsofCDK4andCDK6
TheDcyclinsformphysicalcomplexeswithRB
RegulatorswhichinhibitCDK4/6willblockRBphosphorylation

Cyclin E-CDK2 also contributes to RB

phosphorylation
EctopicexpressionofcyclinERBphosphorylation
cyclinEincreasessignificantlytowardstheendofG1
viraloncoproteinswhichblockcyclinDbindingdonotabolishRB
phosphorylation

Cdk4/6
+cyclin D

Rb

R
Cdk2
+cyclin E

MBV4230

Cell cycle-watch RBs

phosphorylation status
Expression of RB in yeast normal RB phosphorylation requires
two types of cyclins

requirestwodifferentG1cyklines:CLN3+(CLN1orCLN2)
CLN3RBsphosphorylationnormalizedbyintroductionofmammaliancyclinD1
CLN1/2RBsphosphorylationnormalizedbyintroductionofmammaliancyclinE

Different models for cooperation of D and E cyclins

cyclinDCDK4/6formationofhyperphosphorylatedRB,whilecyclinECDK2
maintenanceofhyperphosphorylatedRB
cyclinDCDK4/6formationofpartiallyphosphorylatedRBbettersubstrateforcyclin
ECDK2formationofhyperphosphorylatedRB

Continuous turnover of phosphate

t1/2forphosphateonRB15min(duetophosphataseactivity)maintenanceof
phosphorylatedstatusnecessary

MBV4230

RB as an integrator of positive
growth signals

general: physiological signals that promote proliferation

enhanced RB phosphorylation

Growthfactors/mitogenicsignalsreceptorintracellularsignallingpathwaysRB
phosphorylationcellcycleprogression/proliferation

Abundance of extracellular mitogenes sensed as [cyclin D1]

sufficientD1RBphosphorylation
lowD1RBunphosphorylated

RB as repressor

MBV4230

E2F liberated by Rb inactivation

Rb excert its effects through E2F TFs

Rb=inactivated
Rb=activerepressor

Rpoint

E2F=activated!

MBV4230

RBs phosphorylation status

= a signal to the trx 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

MBV4230

Repressor-mechanism:
through chromatin

mechanism for repression

E2FbindsDNARB
RBactsasanactiverepressorassociated
withDNAboundE2F
RBrecruitsHDACcomplexesthatcause
repression

MBV4230

Repression in several stages

1. Blocking TAD

2. Recruitment of HDAC

3. Recruitment of HMT

MBV4230

Local repression by RB:

first deacetylation, then methylation
Step1:deacetylation

Step2:methylation

MBV4230

RBs Pocket-domain important

Pocket-properties

Model

HDAC1bindstoRbspocketdomain
(379792)
TherepressorfunctionofRbis
locatedtothepocketdomain
Pocketalsobindingsiteforviral
oncoproteinsviaLxCxEmotif
Alldiseaserelatedmutationslocated
tothepocketdomain
RbHDAC1associationinterrupted
andRbsrepressorfunctionlostwhen
1.Rbisphosphorylated
2.Pocketdomainmutated
3.Viraleoncoproteinsbindpocket

TheNineResiduesOfPapilloma
VirusE7PeptideContainThe
LxCxEMotif

MBV4230

Rb related pocket proteins

3 members in the pocket-family:

RB, p107, p130

Common:A+Bdomainsformingthepocketdomain

similaritiesincellcycledependentphosphorylation
Unequalwithregardtoassociatedcyclinsand
expression
Fewornomutationsinp107andp130foundinhuman
cancers

Parallel controls through several

pocket-proteins and multiple E2Fs

allnaturalRbmutationsinAorB

RBbindsE2F1,2and3
p107bindsE2Fs4
p130bindsE2Fs4and5
differentE2Fshavedifferentfunctions(sebelow)

Downstream RB
- the effectors: E2Fs

MBV4230

E2F liberated by Rb inactivation

Rb excert its effects through E2F TFs

Rb=inactivated
Rb=activerepressor

Rpoint

E2F=activated!

MBV4230

The E2F/DP-family of
transcription factors

E2F/DPs = a group of bHLH-ZIP factors

E2F/DP - heterodimers of E2F + DP
E2F:6distinctrelatedTFs(E2F16)
DPpartners:2TFs(DP1,DP2)
Allpossiblecombinations

3 subgroups
Activating E2Fs

Repressive E2Fs

Potentactivators
Activerepressors

E2F6 - repressor?
Pocketindependent
Asspolycombcomplex

MBV4230

Target genes controlled by

activating E2Fs

E2F sites

commonkonsensusbindingsite:TTTCCCGC

NodifferenceinsequencepreferencebetweendifferentE2Fs

target genes: E2F controls the transcription of

cellular genes that are essential for cell division:

cellcycleregulators

suchasdihydrofolatereductase,thymidylatesynthetaseandthymidinekinase

themaincomponentsoftheDNAreplicationmachinery

suchascyclinE,cyclinA,Cdc2,Cdc25A,RBandE2F1,

enzymesthatareinvolvedinnucleotidebiosynthesis

optimalbindingtoTTTCGCCGCCAAAA(tomotsattorienterteoverlappendesites)

Cdc6,ORC1andtheminichromosomemaintenance(MCM)proteins.

E2F knock-out - a paradox

MBV4230

The activating E2F1, E2F2 & E2F3

Key role: the activation of genes that are essential for

cellular proliferation and the induction of apoptosis.
Overexpression proliferation
quiescentcellsreenterthecellcycle
Overridevariousgrowtharrestsignals
Transformationofprimarycells

Knock-outs reduced proliferation

E2f3/MEFs:defectiveinthemitogeninducedactivationofalmostallknown
E2Fresponsivegenes
thecombinedmutationofE2f1,E2f2andE2f3issufficienttocompletelyblock
cellularproliferation.

MBV4230

The activating E2F1, E2F2 & E2F3

apoptosis ??

Key role: the activation of genes that are essential for cellular
proliferation and the induction of apoptosis.

The threshold model of the activating E2Fs.

TheactivatingE2FscontributetoapoolofE2Factivity.Oncethisreachesacriticallevel,
ittriggersproliferation(threshold1)orapoptosis(threshold2).

MBV4230

The activating E2Fs

are key targets of RB

E2F1-3 interact specifically with RB

TheactivatingE2FsarespecificallyregulatedbytheirassociationwithRB,butnotwith
therelatedpocketproteinsp107orp130.

RBbindstransactivationdomain(TAD)inE2F

ReleasefromRbistriggeredbythephosphorylationofRBinlateG1andcorrelates
closelywiththeactivationofE2Fresponsivegenes.
ThefunctionalinactivationofRBinducesthesamephenotypeastheoverexpressionof
E2F:

inappropriateproliferation,p53dependentandp53independentapoptosis

Mutation of either E2f1 or E2f3 in RB-deficient embryos is

sufficient to suppress all these defects.

Rbbinding

MBV4230

The repressive E2F4 & E2F5

regulated in a different fashion

Significant levels of E2F4 and E2F5 are detected in quiescent

(G0) cells,

The E2F subgroups bind to different pocket proteins.

itaccountsforatleasthalfoftheRB,p107andp130associatedE2Factivity.

The subcellular localization of the endogenous E2F4 and E2F5

complexes is also regulated,

WhereastheactivatingE2FsarespecificallyregulatedbyRB,E2F5ismainlyregulated
byp130,andE2F4associateswitheachofthepocketproteinsatdifferentpointsinthe
cellcycle.

E2F4 is expressed at higher levels than the others,

E2F1,E2F2andE2F3aareprimarilyrestrictedtoactivelydividingcells.

E2F1,E2F2andE2F3areconstitutivelynuclear,whereasE2F4andE2F5are
predominantlycytoplasmic.Incomplexwithpocketproteinsnuclear.

KO: repressive E2Fs are important in the induction of cell-cycle

exit and terminal differentiation.

MBV4230

Cell-cycle regulation of individual

E2F complexes

The spectrum and subcellular localization of the E2Fcomplexes from

G0 to the restriction point (late G1). The approximate abundance of
each complex is indicated by their relative size.

Activerepression
oftargetgenes
Repressive
Complexes
Replaced
With
Acitvating
ones

Derepression+activation
oftargetgenes

Cellcycle

MBV4230

E2F/DP only active in a window

of the cell cycle (late G1 early S)

Early G1: active RB E2F/DP turned OFF

The R-point: inactivated RB E2F/DP turned ON

Late S: E2F/DP turned OFF again

E2F/DPliberatedactivationofE2Fdependentpromoters

cyclinA/cdk2phosphorylationofE2F/DPreducedDNAbinding
targetgenesturnedoff

MBV4230

EF26 - another mode of repression

Less well studied

MBV4230

Summary

RB control:
beyond E2F

MBV4230

Other effector-functions of RB

RB is abundant in the cell

RB/E2F100

RB can bind opp a range of proteins other than E2F

consensusbindingmotif:LxCxE
TFs:Elf1,MyoD,PU.1,ATF2
nucleartyrosinekinase:cAbl

By binding up several different effector-proteins

coordinated control of several downstream growthrelated pathways
Still - the E2F-pathway plays a key role

hypoRBbindscatalyticdomaininactivateskinase

EctopicexpressionofE2FoverridesRBblock

RB negative growth control

MBV4230

RB as integrator of negative
growth inhibitory signals

general: physiological signals that inhibit

proliferation reduced RB phosphorylation cell
cycle dont pass R
actsindirectlythroughCDKinhibitors(CDKIs)reducedCDKactivity
reducedRBphosphorylation
Threewellknownphysiologicalgrowthinhibitorysignals

TGF
cAMP
contactinhibition

TGF growth inhibtion: 3 mechanisms

TGFposttranslationalmodification/activationofCDKIp27Kip1
inactivationofCDK2,4and6reducedRBphosphorylation
TGFinductionofCDKIp15INK4BinactivationofCDK4and6through
cyclinDcompetitionreducedRBphosphorylation
TGFreducedlevelofCDK4reducedRBphosphorylation

MBV4230

RB as integrator of negative
growth inhibitory signals

cAMP/contact inhibition / growth inhibition

Irradiation/DNA-damage

cAMPmobilizeCDKIp27Kip1inactivationofCDK2,4and6
reducedRBphosphorylation
DNAdamageenhancedp53inductionofCDKIp21Waf1/Cip1
inactivationofCDK4and6reducedRBphosphorylationG1
arresttimetorepairDNA

RB and cancer

MBV4230

RB and cancer - several ways of

killing RB-mediated cell cycle control

Rb mutation

RB inactivated by RB-binding oncoproteins

retinoblastoma,smallcelllungcarcinomer,sarcoma,kidneycarcinomas
cervicalcarcinomas:humanpapillomasvirusE7oncoprotein

amplification of cyclin D genes

esophageal,brystandsquamouscellcarcinomas
inBcelllymphomasduetochromosometranslocation

Virus-encoded D-type cyclins

Herpesvirussaimiri

amplification of the CDK4 gene

glioblastomas
gliomas

deletion of genes for p15 or p16

severalcarcinomas
alsogermlinemutationsinfamilialmelanomes

Iallecases:lostRBfunction
openRdoorfreeE2Fcell
cyclewithoutbrakes