RB - Università degli Studi della Tuscia

Cdc28 Regulate Many Cell Cycle
Events
Synchronized cdc28 ts cells can’t
initiate S-phase
Cdc28 drives entry into S-phase as
well as Mitosis
How can one enzyme regulate
multiple cell cycle events?
1

Cells Use Multiple Cyclins to Regulate
Different Cell Cycle Events
Cicline M
Cicline G1
2

Il complesso MPF regola l’ingresso mitotico
(studi su S. S. pombe) pombe
Il complesso MPF è formato da
Cdc28/cicline mitotiche in S. cerevisiae e
Cdc2/Cdc13 in S. pombe
Il complesso SPF regola l’ingresso in S
(studi su S. S. cerevisiae)
cerevisiae
Il complesso SPF è formato da
Cdc28/cicline G1 in S. cerevisiae
3

Modello di azione del complesso Cdc28/cicline
in S. S. cerevisiae
4

Cyclin
G1 Cyclins
Cln1
Cln2
Cln3
B-type Cyclins
Clb1
Clb2
Clb3
Clb4
Clb5
Clb6
Budding Yeast Cyclin Genes
Identification
mutant
mutant
mutant
Homology
Homology
Homology
Homology
Accidental
Accidental
Expression
G1
G1
G1
M
M
S
S
G1
G1
Function
START
START
START
Mitosis
Mitosis
Spindle
Spindle
S-phase entry
S-phase entry
5

Cyclins Nomenclature
All contain “cyclin box” domain cdc2 binding domain
Cyclin A
Cyclin B’s
S. pombe cdc13...
S. cerevisiae CLB1,2,3,4,5,6
G1 Cyclins
S. cerevisiae CLN1,2,3
A and B’s closely related
- destruction box
- required for S and M
- “A” accumulates earlier,
- degraded earlier
- G1’s more similar to each other
-PEST domains (Pro,Glu,SerThr)
6

Cyclins can be grouped by expression patterns
[Cyclin]
- CLN1 and CLN2 and CLB5 and CLB6 -
- CLB3 and CLB4
- CLB 1 and CLB2
CLN1,2,CLB5,6
CLB3,4
CLN3
G1 Start Metaphase
CLB1,2
7

Yeast Cyclin Genes: CLNs and CLBs
Clb3,4
Activate replication
origins
Clb5,6
G1 entry
Triple cln1,2,3 mutant
Quadruple clb1,2 mutant
CLN1,2,3
Sense cell size, commit to division
Dominant CLN Mutations
Clb1,2
Spindle assembly
anaphase

Vertebrate Cells, like budding yeast,
express multiple cyclins, but they
also express several CDK’s
9

Regulation of G1/S transition (start/restriction point)
Identification of mammalian G1 cyclins
Human cDNA libraries screened for ability to complement CLN3 mutant
lead to cloning of three human G1 cyclins:
Cyclin C
Cyclin D1
Cyclin E
Cyclin D2 and Cyclin D3 subsequently
identified
D-type cyclins: respond to growth factors
G 0 to G1 transition
E-type cyclin: expression is periodic
peaks at G1/S transition
controls ability of mammalian cells
to enter S-phase
10

Regulation of G1/S transition (start/restriction point)
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 coimmunoprecipitation
experiment
Unable to complement CDC28
11

Punti di controllo del ciclo cellulare
START PUNTO DI RESTRIZIONE
Budding yeast Mammiferi
13

Restriction point (START)
Point at which cell is irreversibly
committed to traversing the cell cycle
Mammals: restriction point
Yeast: START
Cell cycle proceeds without influence
from environment (only stopped by
damage)
Late in G 1
14

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
15

In assenza di fattori di crescita le
cellule di mammifero si arrestano in
G0 (cellule cellule quiescenti);
quiescenti
L’aggiunta di mitogeni causa il
passaggio attraverso il punto di
restrizione (dopo dopo 14-16 ore); ore
Le cellule in proliferazione entrano in
S (dopo dopo altre 6-8 ore)
ore
16

Cyclin D is required to pass restriction point
R point
Colorazione 16 ore dopo la microiniezione
e l’aggiunta di
BrdU al mezzo.
Colorante per DNA
Anticorpi anti-BrdU
Anticorpi anti-cicl.D
17

Continous expression of Cyclin E shortens G1
18

Attività dei complessi
Cdk/ciclina dei mammiferi
All’inizio della fase S le
cicline D ed E vengono
degradate. I livelli di Cdk4
e 6 crollano repentinamente
All’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.
Queste proteine non sono
necessarie per la progressione
nella fase S
Il complesso Cdk2/ciclina A è
indispensabile per la
progressione nella fase S
19

Cell-cycle phase-specific
Three classes:
G 1 Cdk complexes
Cdk complexes
S-phase Cdk complexes
Mitotic Cdk complexes (also known as MPF)
Cell-cycle phase specificity determined by
cyclin type and, in some cells, Cdk type
20

Controllo del ciclo cellulare dei mammiferi
Ingresso in fase M (complesso MPF)
Cdk1/ciclina A
Cdk1/ciclina B
Ingresso in fase S (complesso SPF)
Cdk4,6/ciclina D punto di restrizione
Cdk2/ciclina E
Progressione in fase S
Cdk2/ciclina A
ingresso in S
21

In addition to forming complexes between
specific Cdks and their cyclin binding
partners, all complexes require
phosphorylation and dephosphorylation to
become fully active
phosphorylation of thr160/161 is
important for the activation of all major
Cdk/cyclin complexes
catalysed by Cdk activating kinase CAK
22

Regolazione della Cdk1 mediante
fosforilazione-defosforilazione
Siti di inattivazione
Sito di attivazione
Equilibrio tra fosfatasi (Cdc25) e kinasi (Wee1)
23

Another mechanism by which
CDKs can
regulate Multiple Transitions
is by using
different CDK inhibitors
(CKI’s)
24

CDK activity is controlled by chemical modification,
cyclin synthesis/proteolysis, and:
CKIs (Cyclin-dependent kinase Inhibitors)
25

CKI’s can be specific for particular CDK
complexes because their binding can depend upon
a specific cyclin
CDK
CKI
T-Loop
Cyclin
26

Sic1
Down-regulation of a CKI can, in turn, be
regulated by a CDK-mediated phosphorylation
because its ubiquitin-dependent proteolysis
can be regulated by its phosphorylation state
Cdc28/Cln1,2
27

We can add another limb to our regulation tree
Wee1 kinases
Cdc25 phosphatases
Kap1 phosphatases
Phosphorylation
Control
Transcriptional
Control
Y15
T160
Ubiquitin-dependent
Proteolytic Control
Cdk-Activating
Kinases (CAK)
Cyclin-dependent Kinase
Inhibitors (CKI’s)
Transcriptional
Control
28

Inibitori delle Cdk
Membri diversi di questa famiglia inibiscono Cdk diverse
Inibitore cellule animali
p15, p16
p21, p27
Inibitore lievito
Far1
P40
Cyclin-Cdk complex
Cdk4/ciclinaD
Cdk6/ciclinaD
Cdk4/ciclinaD
Cdk6/ciclinaD
Cdk2/ciclinaE
Cdk2/ciclinaA
Cdc2/Cln
Cdc2/Clb
29

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
30

Situazioni biologiche in cui la proliferazione è controllata
Embriogenesi: proliferazione attiva
cicli cellulari brevi
rapida 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.).
Proliferazione neoplastica: innesco della proliferazione in assenza di
programma, per mutazione di un gene cellulare o infezione di virus
31

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.
Controllo del ciclo cellulare e controllo della
proliferazione
MECCANISMI DI CONTROLLO INTRINSECI
32

Regulation of cell cycle
A cell continues through the cell cycle after passing
the restriction point (START START) 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 G 0 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
33

Cell 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
34

Checkpoint machinery
Look out for defect
and emit a signal
Transmission of signals throughout
the nucleus or cell and amplification
SENSORS
TRANSDUCERS
Delay cell-cycle progression EFFECTORS
35

Four checkpoints
36

THE G1/S CHECKPOINT
37

GROWTH FACTORS ARE INVOLVED
IN PASSING THE G1 CHECKPOINT
1. Arrival of
growth factors
from other cells.
Cyclin
Cyclin
Cyclin
Cyclin
2. Growth factors
cause increase in
cyclin concentration.
CdK
CdK
Cyclin
Cyclin
3. Cyclin activates
cyclin-dependent
kinase.
ATP
ADP
CdK
P i
Cyclin
Target
protein
4. Kinases activate S
phase proteins, leading
to cell division.
38

Growth factor signalling and
transcription
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, cfos,
c-jun
2. delayed response genes:
include cell cycle proteins such as Cdks, cyclins
expression regulated by early response genes
39

Regulated expression of two classes of
genes returns G0 G mammalian cells to
the cell cycle
Early-response
genes:
Transcription factors
(E2F)
Delayed-response
genes:
CyclinD, E
Cdk2, 4, 6
40

Il passagggio attraverso il punto di
restrizione dipende dalla attivazione di
fattori di trascrizione E2F
I fattori E2F attivi stimolano la propria
sintesi e quella della Cdk2 e della Ciclina E;
Il complesso Cdk2/CyclE porta all’aumento di
fattori E2F attivi
41

E2F is a transcription factor that by itself
activates transcription
E2F
However, Rb binds to E2F and represses its
activation function
Rb
gene expression
42

P
P P
Rb
E2F
P
Rb repression is regulated by
early G 1 Cyclins-CDKs
S-phase gene transcription
When Rb repression is inhibited by phosphorylation of Rb by
early G 1 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 G 1 Cyclin-CDKs (Cyclin E and
CDK2) and the major S-phase Cyclin (Cyclin A).
43

The G1/S transition in human cells: Rb (retinoblastoma
protein) regulates E2F proteins, which are transcription
factors for S phase genes
44

Tumor-suppressor genes
Genes capable of suppressing the tumorforming
potential of transformed cells
Tumor suppressor genes normally function
to suppress cell growth and division.
Tumor-suppressor genes encode :
Regulatory proteins eg rb, rb p53
Intra-cellular signaling proteins (nf1-
Neurofibrosarcomas)
Cell adhesion proteins (dcc–colon carcinomas)
45

Tumor-suppressor genes
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.
Analogy
Tumor suppressor brake pedal
Lack of suppressor no brake pedal
46

Gene 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
47

What is the defect in hereditary retinoblastoma?
Rb Rb
Normal person:
2 good copies of
Rb gene
Compromise this good one, then problems!
or
Rb
Hereditary retinoblastoma
patient: 1 good copy of
Rb gene, 1 defective copy
48

Il retinoblastoma erediatrio viene ereditato
come carattere autosomico dominante
Poiché il gene Rb è localizzato sul cromosoma 13
sia i maschi che le femmine hanno la stessa
predisposizione alla malattia
49

Perdita dell’eterozigosi degli antioncogeni
50

Meccanismi genetici alla base del
retinoblastoma
51

Retinoblastoma and the “Two-hit”
model 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
inactivated
sporadic cases (low frequency, late onset):
retinoblastom caused by two acquired somatic
mutations in both alleles = both inactivated
52

Knudson’s 2-hit mutation model for retinoblastoma
53

RB = tumor suppressor gene
RB 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
54

Il gene Rb-1
Genetica
Frequenza del tumore: 1/14.000 nascite
Le cellule tumorali sono prive di entrambi gli alleli Rb-1 funzionali
Assetto 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)
55

RB - 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
56

Caratteristiche strutturali di Rb
La proteina Rb può essere suddivisa in quattro domini. Il dominio N
è responsabile dell’oligomerizzazione della proteina in vitro. I
domini A e B, detti “A/B pocket”, sono responsabili del legame di
Rb a vari fattori trascrizionali, come E2F e varie oncoproteine
virali, e risultano spesso mutati in vari tumori. Il quarto dominio,
detto “C pocket” è un sito di legame per c-Abl .
57

RB 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 dihydrofolate reductase
thymidine thymidine kinase
polymerase
polymerase
histones histones
58

RB´s function: “a signal transducer
connecting the cell cycle clock with the
transcriptional machinery”
Cell cycle clock
G2
S
M
G1
Rb
Transcriptional apparatus
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
59

G0
CycC-Cdk3
pRb
Rb a substrate of
CycD-CDK
CycE-CDK
CycA-CDK
60

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)
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
G2
S
M
G1
active
repressor
Rb
Rb
R
P P P PPP
Inactive
repressor
61
P

Gate-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 growthpromoting/-inhibitory
signals
Mitogenes (+), 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
62

G2
P
P
S
P
M
Gate keeper model
G1
P
P
R
P
P
Cdk4/6
Cyclin D
Rb
E2F released
S-phase genes expressed
63

E2F liberated by Rb inactivation
• Rb excert its effects through E2F TFs
Rb = active repressor
R-point
Rb = inactivated
E2F = activated!
64

RB´s phosphorylation status
⇒ 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
65

E2F/DP
dimers
The functional state of the retinoblastoma protein
(pRb) controls cell proliferation
pRb
GFs, mitogens...
cyclin D1
synthesis
5’..TTTCCGCG…3’
wt pRb represses transcription of cell cycle genes
Cell cycle genes off
G0
cyclin D1
cdk4/6
Active cyclin/kinase
complex
Phosho-pRb becomes inactive
P
Cell cycle progression
G1
P
RNA pol
II
5’..TTTCCGCG…3’
66

Target genes controlled by
activating 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
67

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
– 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
68

HOW ARE S PHASE PROTEINS ACTIVATED?
Rb
E2F
1. In normal cells,
Rb protein binds
to E2F and shuts
down the cell cycle.
ATP
ADP
CdK
P i
Cyclin
Rb
E2F
2. If growth factors arrive and
activate the cyclin-CdK
complex, Rb becomes
phosphorylated.
P i
Rb
E2F
3. E2F is
released
E2F
S Phase
proteins
mRNA
DNA
4. E2F stimulates the
production of S phase
proteins.
When Rb is mutated, no “tie-up” of E2F, so constant S phase turn on
69

Effetti delle mutazioni del Gene Rb
Osteosarcomi
Carcinomi:
1) Polmonari
2) Mammari
3) Prostatici
70