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M = Mitosis
S = DNA-Synthesis
Go = Quiescent state
of differentiated cells
G1 = Cell growth
G2 = Control of
DNA-Synthesis
The cell cycle
How is the correct
sequence of the cell cycle
controlled ?
S = DNA-Synthesis
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
Go = Quiescent state
The cell cycle
How is the correct
sequence of the cell cycle
controlled ?
Checkpoints
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
S = DNA-Synthesis
Go = Quiescent state
The cell cycle
Restriction point
intrinsic: final cell size reached?
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
Go = Quiescent state
How is the correct
sequence of the cell cycle
controlled ?
S = DNA-Synthesis
The cell cycle
Restriction point
intrinsic: final cell size reached?
external: growth factors ?
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
Go = Quiescent state
How is the correct
sequence of the cell cycle
controlled ?
S = DNA-Synthesis
The cell cycle
Restriction point
intrinsic: final cell size reached?
external: growth factors ?
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
Go = Quiescent state
S = DNA-Synthesis
The cell cycle
G2-Control
Replication complete?
DNA intact?
Restriction point
intrinsic: final cell size reached?
external: growth factors ?
G2 = Control of
DNA-Synthesis
M = Mitosis
G1 = Cell growth
Go = Quiescent state
S = DNA-Synthesis
The cell cycle
G2-Control
Replication complete?
DNA intact?
Metaphase-Control
correct
annealing of chromosoms?
M = Mitosis
S
Go
G1 G2
Checkpoints of the cell cycle
Which biochemical factors
regulate the transition of
cell cycle steps ?
Cell fusion experiments show three results:
1. Only cells in the G1-phase are competent to enter the S-phase (DNA-replication)
2. Only cells in the S-Phase express a factor whichstimulates DNA-replication in competent cells in the G1-phase.
3. Cells in the G2-phase can`t execute DNA-replication until mitosis is not completed.
Initiation of DNA-synthesis by a soluble factor in the early S-phase
G1/S-Phase: S-Phase-promoting- factor (SPF)G2/M-Phase: Mitosis-promoting-factor (MPF)
Cell fusion experiments show three results:
1. Only cells in the G1-phase are competent to enter the S-phase (DNA-replication)
2. Only cells in the S-Phase express a factor whichstimulates DNA-replication in competent cells in the G1-phase.
3. Cells in the G2-phase can`t execute DNA-replication until mitosis is not completed.
Initiation of DNA-synthesis by a soluble factor in the early S-phase
G1/S-Phase: S-Phase-promoting- factor (SPF)G2/M-Phase: Mitosis-promoting-factor (MPF)
Cell fusion experiments show three results:
1. Only cells in the G1-phase are competent to enter the S-phase (DNA-replication)
2. Only cells in the S-Phase express a factor whichstimulates DNA-replication in competent cells in the G1-phase.
3. Cells in the G2-phase can`t execute DNA-replication until mitosis is not completed.
Initiation of DNA-synthesis by a soluble factor in the early S-phase
G1/S-Phase: S-Phase-promoting- factor (SPF)G2/M-Phase: Mitosis-promoting-factor (MPF)
Cell fusion experiments show three results:
1. Only cells in the G1-phase are competent to enter the S-phase (DNA-replication)
2. Only cells in the S-Phase express a factor whichstimulates DNA-replication in competent cells in the G1-phase.
3. Cells in the G2-phase can`t execute DNA-replication until mitosis is not completed.
Initiation of DNA-synthesis by a soluble factor in the early S-phase
G1/S-Phase: S-Phase-promoting- factor (SPF)G2/M-Phase: Mitosis-promoting-factor (MPF)
Cell fusion experiments show three results:
1. Only cells in the G1-phase are competent to enter the S-phase (DNA-replication)
2. Only cells in the S-Phase express a factor whichstimulates DNA-replication in competent cells in the G1-phase.
3. Cells in the G2-phase can`t execute DNA-replication until mitosis is not completed.
Initiation of DNA-synthesis by a soluble factor in the early S-phase
G1/S-Phase: S-Phase-promoting- factor (SPF)G2/M-Phase: Mitosis-promoting-factor (MPF)
25°C
35°C
Identification of temperature-sensitive CDC (Cell-Division-Cycle) Mutants
Model: Saccharomyces sereviciae
Mutagenized cells
Identification of cell cycle regulators: CDKs
25°C
35°C
35°C
non CDC-Mutants
budding
Entry into the
Cell cycle
Identification of temperature-sensitive CDC (Cell-Division-Cycle) Mutants
Model: Saccharomyces sereviciae
Mutagenized cells
Identification of cell cycle regulators: CDKs
25°C
35°C
35°C
CDC-START-Mutants
non CDC-Mutants
no entry into the
cell cycle
Identification of temperature-sensitive CDC (Cell-Division-Cycle) Mutants
Model: Saccharomyces sereviciae
Mutagenized cells
Identification of cell cycle regulators: CDKs
35°C 35°C
Identification of the CDC-START gene
Wt CDC-START-Gen
CDC-START-Mutant
Wt Gen X
Identification of cell cycle regulators: CDKs
35°C 35°C
Wt CDC START Gen
CDC-START-Mutant
Wt Gen X
Cyclin-dependent Kinase (CDK)
Analysis
Identification of the CDC-START gene
Identification of cell cycle regulators: CDKs
Discovery of Cyclin-dependent Kinases (CDKs):
Nobel price in medicine 2001
Leland H Hartwell Tim Hunt Paul Nurse
CDKs in yeast Cyclins in sea urch
Cyclin-dependent kinases (CDKs): Heterodimeric proteins
C.L. Card et al.,
EMBO Journal 2000
katalytische Untereinheit: CDK regulatorische Untereinheit: Cyclin
C.L. Card et.al.,
EMBO Journal 2000
catalytic subunit: CDK
- Serine/Threonine-Kinases
- Yeast: one CDK
- Mammals: CDK1 - CDK7
- high identity
- conserved cyclin-binding site
regulatorische Untereinheit: Cyclin
Cyclin-dependent kinases (CDKs): Heterodimeric proteins
C.L. Card et.al.,
EMBO Journal 2000
regulatory subunit: Cyclin
- Cyclin A-H
- heterogeneous protein family
- cyclic concentration changes
in the cell cycle
- Nuclear localisation
Cyclin-dependent kinases (CDKs): Heterodimeric proteins
catalytic subunit: CDK
- Serine/Threonine-Kinases
- Yeast: one CDK
- Mammals: CDK1 - CDK7
- high identity
- conserved cyclin-binding site
M
S
G1
G2
CDK/Cyclin-complexes in the cell cycle
Restriction point
Go
CDK2/CyclinD
CDK4/CyclinD
CDK6/CyclinD
CDK2/CyclinE
G1/S-Phase
transition
M
S
G2
Go
CDK2/CyclinE
CDK2/CyclinA
CDK2/CyclinD
CDK4/CyclinD
CDK6/CyclinD
CDK/Cyclin-complexes in the cell cycle
Restriction point G1/S-Phase
transition
M
S
G1
G2
Go
CDK2/CyclinE
CDK1/CyclinB
G2/M-Phase
transition CDK2/CyclinD
CDK4/CyclinD
CDK6/CyclinD
CDK2/CyclinA
Restriction point G1/S-Phase
transition
CDK/Cyclin-complexes in the cell cycle
CDKs: Motors of the cellcycle
How act CDKs as motors of te cell cycle?
Which substrates are phophorylated by CDKs during the cell cycle?
NH2- -COOH A B
Retinoblastom-Protein (Rb)
- „Key“- substrate of the S-Phase -
nuclear protein, 110 kDa
CDK Substrates: Initiation of the S-Phase
NH2- -COOH A B
Binding of
transcription factor E2F
Retinoblastom-Protein (Rb)
- „Key“- substrate of the S-Phase -
nuclear protein, 110 kDa
CDK Substrates: Initiation of the S-Phase
NH2- -COOH
E2F: central transcription factor for the induction
of S-Phase genes
-COOH A B
Binding of
transcription factor E2F
Retinoblastom-Protein (Rb)
- „Key“- substrate of the S-Phase -
nuclear protein, 110 kDa
CDK Substrates: Initiation of the S-Phase
NH2- -COOH
Rb act as a tumor supressor gene
A B
Binding of
transcription factor E2F
Retinoblastom-Protein (Rb)
- „Key“- substrate of the S-Phase -
nuclear protein, 110 kDa
CDK Substrates: Initiation of the S-Phase
- Rb act as a tumor supressor gene
- Rb can be hyperphosphorylated
Retinoblastom-Protein (Rb)
- „Key“- substrate of the S-Phase -
nuclear protein, 110 kDa
CDK Substrates: Initiation of the S-Phase
NH2- -COOH A B
P P P P P P P P P P
Binding of
transcription factor E2F
Rb
E2F
Induktion
of E2F-controlled genes
P P P
CDK 2
Cyclin E
Rb
E2F
Rb
CDK Substrates: Initiation of the S-Phase
Repression
of E2F-controlled genes
E2F: Initiator of the S-Phase
E2F
E2F-controlled genes
DNA-Pol I
dNTP-Synth.
CDK 2
Cyclin E
Rb
P P P
E2F
Cyclin E
E2F
DNA-Pol I
dNTP-Synth.
CDK 2
Cyclin E
Rb
P P P
positive autoregulatory
feedback loop!
E2F: Initiator of the S-Phase
E2F-controlled genes
E2F
Cyclin E
E2F
DNA-Pol I
dNTP-Synth.
CDK 2
Cyclin E
Rb
P P P
„forced"
transition of
the restriction point
S-Phase
E2F: Initiator of the S-Phase
E2F-controlled genes
positive autoregulatory
feedback loop!
An important step in mitosis is the desintegration
of the nuclear membrane
Interphase Mitose
CDK substrate: Mitosis
desintegration
of the nuclear lamina
desintegration of the der
nuclear membran
mitosis
chromatin nuclear lamina
CDK substrate: Mitosis
CDK 1
Cyclin B
Mitose
ATP
chromatin nuclear lamina
CDK substrate: Mitosis
desintegration
of the nuclear lamina
desintegration of the der
nuclear membran
CDK 1
Cyclin B
lamin tetramer
Mitose
lamin network
ATP
desintegration
of the nuclear lamina
desintegration of the der
nuclear membran
chromatin nuclear lamina
CDK substrate: Mitosis
CDK 1
Cyclin B
lamin tetramer
-P P- -P P-
phosphorylated
lamin dimers
lamin netork
Mitose
ATP ATP
chromatin nuclear lamina
CDK substrate: Mitosis
desintegration
of the nuclear lamina
desintegration of the der
nuclear membran
M
S
G1
G2
CDK/Cyclin complexes in the cell cycle
restriction point
Go
CDK2/CyclinE
CDK1/CyclinB
G1/S-phase
transition
G2/M-phase
transition
CDK2/CyclinD
CDK4/CyclinD
CDK6/CyclinD
CDK2/CyclinA
Lamin-P
Rb-P E2F
CDKs: Switches of the cell cycle
How is the activity of CDKs
switched on- and off in the cell cycle?
P
only CDK-Cyclin complexes
are substrates
Threonin-Kinase (CAK)
active
T160 P CDK
Cyclin
P ATP
Regulation of CDK activity: Cyclin concentration
inactive CDK
Cyclin Cyclin
CDK inactive
inactive CDK
Cyclin Cyclin
CDK inactive
P
Cyclin concentration
active
T160 P CDK
Cyclin
ATP
Threonin-Kinase (CAK)
Regulation of CDK activity: Cyclin concentration
How is the concentration of cyclins regulated in the cell cycle?
Cyclin
Regulation of CDK activity: Cyclin concentration
De novo synthesis
by transcriptional
induction
Cyclin
How is the concentration of cyclins regulated in the cell cycle?
Regulation of CDK activity: Cyclin concentration
Regulation of cyclin concentration: transcriptional Induktion
P
Example: Induction by growth factor signal chains
P
GF
-R
GF
Regulation of cyclin concentration: transcriptional Induktion
Example: Induction by growth factor signal chains
P
TF-OH TF-O-P
Protein kinases
GF
-R
GF
Regulation of cyclin concentration: transcriptional induction
Example: Induction by growth factor signal chains
P
TF-OH TF-O-P
immediate early
genes
Protein kinases
GF
-R
GF Example: Induction by growth factor signal chains
Regulation of cyclin concentration: transcriptional induction
P
TF-OH TF-O-P
immediate early
genes
c-jun/c-fos (TF) Protein kinases
GF
-R
GF Example: Induction by growth factor signal chains
Regulation of cyclin concentration: transcriptional induction
P
TF-OH TF-O-P
immediate early
genes
c-jun/c-fos (TF)
delayed genes
c-jun
c-fos
Protein kinases
GF
-R
GF Example: Induction by growth factor signal chains
Regulation of cyclin concentration: transcriptional induction
P
TF-OH TF-O-P
immediate early
genes
c-jun/c-fos (TF)
delayed genes
Cyclin D
Cyclin E
CDK2
CDK4
c-jun
c-fos
Protein kinases
GF
-R
GF Example: Induction by growth factor signal chains
Regulation of cyclin concentration: transcriptional induction
P
TF-OH TF-O-P
immediate early
genes
c-jun/c-fos (TF)
delayed genes
Restriction point
(G1 S-Phase)
c-jun
c-fos
Cyclin D
Cyclin E
CDK2
CDK4
Protein kinases
GF
-R
GF Example: Induction by growth factor signal chains
Regulation of cyclin concentration: transcriptional induction
Cyclin
Restriktion point
(G1 S-Phase)
Degradation
by proteolysis
De novo synthesis
by transcriptional
induction
How is the concentration of cyclins regulated in the cell cycle?
Regulation of CDK activity: Cyclin concentration
Regulation of cyclin concentration: proteolytic degradation
CD
K1
-ac
tiv
ity
Cell cycle-Phase
G1 S G2 M G1 S G2 M
Example: Regulation of CDK1 activity by degradation of cyclin B
Metaphase
CD
K1
-ac
tiv
ity
Cell cycle-Phase
G1 S G2 M G1 S G2 M
CDK1 concentration
Metaphase
Regulation of cyclin concentration: proteolytic degradation
Example: Regulation of CDK1 activity by degradation of cyclin B
CD
K1
-ac
tiv
ity
Cy
clin
B c
on
cen
tratio
n
Cell cycle-Phase
G1 S G2 M G1 S G2 M
CDK1 concentration
Degradation
of cyclinB
Metaphase
Regulation of cyclin concentration: proteolytic degradation
Example: Regulation of CDK1 activity by degradation of cyclin B
NH2 COOH
Cyclin A,B
Destruction-Box
NH2 COOH
Ubiquitin
Regulation of cyclin concentration: proteolytic degradation
NH2 COOH
Cyclin A,B
Destruction-Box
NH2 COOH
Ubiquitin Anaphase-promoting-complex
(APC, Ubiquitin-Ligase-complex)
Regulation of cyclin concentration: proteolytic degradation
NH2 COOH
Cyclin A,B
Destruction-Box
NH2 COOH
Ubiquitin
Labeling for proteolytic
degradation
Anaphase-promoting-complex
(APC, Ubiquitin-Ligase-Komplex)
Regulation of cyclin concentration: proteolytic degradation
NH2 COOH
Proteasom
degradation
NH2 COOH
Cyclin A,B
Destruction-Box
NH2 COOH
Ubiquitin
Labeling for
proteolytic degradation
Anaphase-promoting-complex
(APC, Ubiquitin-Ligase-Komplex)
Regulation of cyclin concentration: proteolytic degradation
How is the proteolytic degradation of cyclin B regulated?
Regulation of cyclin concentration: proteolytic degradation
Cyclin B
synthesis
Cyclin B
S, G2-Phase
G1-Phase CDK1
APC
inactive
Regulation of cyclin concentration: proteolytic degradation
CDK1
Cyclin B
Cyclin B
synthesis
P
Metaphase
Cyclin B
APC APC
P
inactiv active
S, G2-Phase
G1-Phase CDK1
Regulation of cyclin concentration: proteolytic degradation
CDK1
Cyclin B
Cyclin B
synthesis
P
Metaphase
Cyclin B
APC APC
P
inactive active
S, G2-Phase
G1-Phase CDK1
ATP
Regulation of cyclin concentration: proteolytic degradation
CDK1
Cyclin B
CDK1
Cyclin B
Cyclin B
synthesis
P
polyubiquitinylation
Metaphase
Cyclin B
APC APC
P
inactive active
proteasomal
degradation
S, G2-Phase
G1-Phase CDK1
ATP
Regulation of cyclin concentration: proteolytic degradation
CDK1
Cyclin B
CDK1
Cyclin B
Cyclin B
synthesis
P
Metaphase
Anaphase
Cyclin B
APC APC
P
inactive active
S, G2-Phase
G1-Phase CDK1
ATP
Cyclin B
polyubiquitinylation
proteasomal
degradation
Regulation of cyclin concentration: proteolytic degradation
CDK1
Cyclin B
CDK1
Cyclin B
Cyclin B
synthesis
P
Metaphase
Anaphase
Cyclin B
APC APC
P
inactive active
S, G2-Phase
G1-Phase CDK1
ATP
Cyclin B
polyubiquitinylation
proteasomal
degradation
Regulation of cyclin concentration: proteolytic degradation
negative autoregulatory
feedbackloop
Regulation of CDK activity: Cyclin concentration
Cyclin
Degradation
by proteolysis
Metaphase Anaphase
completion of mitosis
Growth factors Autoregulation
Restriktion point
(G1 S-Phase)
De novo synthesis
by transcriptional
induktion
Regulation of CDK activity: Phosphorylation
inactive CDK
Cyclin Cyclin
aktiv
T160 P CDK
Cyclin
CDK
Cyclin
CDK
T14
Y15
P
P
inactive
inactive P
T160 P
Threonin/Tyrosin-
Kinase
Cyclin concentration ATP
ATP
Threonin-Kinase (CAK)
Regulation of CDK activity: Dephosphorylation
inactive CDK
Cyclin Cyclin
aktiv
T160 P CDK
Cyclin
CDK
Cyclin
CDK
T14
Y15
inactive
inactive P
T160 P
Threonin/Tyrosin-
Kinase
Cyclin concentration ATP
ATP
Threonin-Kinase (CAK)
P
P
Threonin/Tyrosin
Phosphatase
Pi
How is the activation of CDKs by Dephosphorylation
regulated?
Regulation of CDK activity: Dephosphorylation
CDK1
CyclinB
T14
Y15
P
P
inactive
P
T160 P G2-Phase
Threonin/Tyrosin
Phosphatase inactive
G2-Phase
Regulation of CDK activity: Dephosphorylation of CDK1
P P
Threonin/Tyrosin
Phosphatase active
Threonin/Tyrosin
Phosphatase inactive
CDK1
CyclinB
T14
Y15
P
P
inactive
P
T160 P
CDK1
CyclinB
active
T160 P
G2-Phase
Mitosis
G2-Phase
Mitosis
Pi
Regulation of CDK activity: Dephosphorylation of CDK1
activation
P P
Threonin/Tyrosin
Phosphatase active
Threonin/Tyrosin
Phosphatase inactive
CDK1
CyclinB
T14
Y15
P
P
inactive
T160 P
CDK1
CyclinB
active
T160 P
P
G2-Phase
Mitosis
G2-Phase
Mitosis
Pi
ATP
Regulation of CDK activity: Dephosphorylation of CDK1
activation
P P
Threonin/Tyrosin
Phosphatase active
Threonin/Tyrosin
Phosphatase inactive
CDK1
CyclinB
T14
Y15
P
P
inactive
T160 P
CDK1
CyclinB
active
T160 P
P
G2-Phase
Mitosis
G2-Phase
Mitosis
Pi
ATP
Regulation of CDK activity: Dephosphorylation of CDK1
positive autoregulatory
feedbackloop
Regulation of CDK activity: Inhibitors
inactive
inactive CDK
Cyclin Cyclin
active
T160 P CDK
Cyclin
T160 P CDK
CKI
CDK inactive
CKI
P
Cyclinconcentration
Phosphorylation
Dephosphorylation CDK-Inhibitors
CDK
Cyclin
T14
Y15
P
P
inactive
T160 P
ATP
Pi
ATP
Example: CKI p21
Isosteric inhibition of CDK activity by binding in the active centre
CDK 2
Cyclin E
CKI p21
G1 S-Phase
Regulation of CDK activity: Inhibitors
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
CKI p21
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
CKI p21
CDK 2
Cyclin E
CKI p21
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
CKI p21
T1/2
= 3
0 m
in
p53
degradation
CDK 2
Cyclin E
CKI p21
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
CKI p21
T1/2
= 3
0 m
in
p53
degradation
DNA
damage
T1/2
= 1
50 m
in
CDK 2
Cyclin E
CKI p21
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
CKI p21
T1/2
= 3
0 m
in
p53
degradation
DNA
damage
T1/2
= 1
50 m
in
CDK 2
Cyclin E
CKI p21
G1-Phase arrest
Regulation of CDK activity: Induction of the inhibitor p21
p53 Transcription factor, tumor supressor gene
p53
p53
CKI p21
T1/2
= 3
0 m
in
p53
Degradation
DNA
damage
T1/2
= 1
50 m
in
Transkriptiorfactor, tumor supressorgene
CDK 2
Cyclin E
CKI p21
Time for DNA repair
before replication G1-Phase Arrest
Regulation of CDK activity: Induction of the inhibitor p21
p53
Mdm2p53
Mdm2p53
P MAPK
Abbau
Wachstumsfaktor-hyperstimulation
Regulation der CDK-Aktivität: Regulation von p53
p53
Mdm2p53
Mdm2p53
P
DNA-damage
DNA-PK
ATMMAPK
Degradation
Growth factor-hyperstimulation
protein kinase
Regulation of CDK activity: regulation of p53
p53
Mdm2p53
Mdm2p53
P
DNA-damage
DNA-PK
ATM ATR
Mdm2
P
MAPK
Degradation
Growth factor-hyperstimulation
protein kinase
Regulation of CDK activity: regulation of p53
p53
Mdm2p53
Mdm2 p19/Arfp53
P
DNA-damage
DNA-PK
ATM ATR
Mdm2
P
MAPK
Degradation
Growth factor-hyperstimulation
Regulation of CDK activity: regulation of p53
p53
Mdm2p53
Mdm2 p19/Arf
Mdm2 p19/Arf
p53
P
DNA-damage
DNA-PK
ATM ATR
Mdm2
P
MAPK
Dgradation
Growth factorhyperstimulation
Regulation of CDK activity: regulation of p53
p53
Mdm2p53
Mdm2 p19/Arf
Mdm2 p19/Arf
p53
P
DNA-damage
DNA-PK
ATM ATR
Mdm2
P
MAPK
Dgradation
Growth factorhyperstimulation
Regulation of CDK activity: regulation of p53
induktion of p21
↓
cell cyle arrest
Regulation of CDK activity: summary
inactive CDK
Cyclin Cyclin
active
T160 P CDK
Cyclin
CDK inactive
P
Cyclin concentration P
G1 S M G1 ATP
active
T160 P CDK
Cyclin
P
Cyclin concentration
Phosphorylation
Dephosphorylation
G2 M
G1 S M G1
G2 M
ATP
CDK
Cyclin
T14
Y15
P
P
inactive
T160 P
ATP
Pi
Regulation of CDK activity: summary
inactive CDK
Cyclin Cyclin
CDK inactive
active
T160 P CDK
Cyclin
P
Cyclin concentration
Phosphorylation
Dephosphorylation
G2 M
G1 S M G1
G2 M
ATP
CDK
Cyclin
T14
Y15
P
P
inactive
T160 P
ATP
Pi
Regulation of CDK activity: summary
inactive CDK
Cyclin Cyclin
CDK inactive
inactive
T160 P CDK
CKI
CKI CDK-Inhibitors
G1 S