Regulation of the cell cycle - uni-potsdam.de · Regulation of the cell cycle . Cyclin-dependent Kinases (CDKs) Dr. F. Neuschäfer-Rube . Cyclin-dependent kinases: Motors and switches

Regulation of the cell cycle

Cyclin-dependent Kinases (CDKs)

Dr. F. Neuschäfer-Rube

Cyclin-dependent kinases: Motors and switches of the cell cycle

Dr. F. Neuschäfer-Rube

M

S

The cell cyle

S = DNA-Synthesis

(Replication)

The cell cyle

M

M = Mitosis

Dissection of the chromosoms

S = DNA-Synthesis

The cell cyle

The cell cycle: Steps of the Mitosis

Prophase Metaphase Anaphase Telophase

M = Mitosis

S = DNA-Synthesis

G1

The cell cycle

G2

S = DNA-Synthesis

G2 = Control of

DNA-Synthesis

The cell cycle

M = Mitosis

G1

G1 = Cell growth

The cell cycle

S = DNA-Synthesis

G2 = Control of

DNA-Synthesis

M = Mitosis

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


The cell cycle

How is the correct


controlled ?

Checkpoints

G2 = Control of

DNA-Synthesis

M = Mitosis

G1 = Cell growth

S = DNA-Synthesis


The cell cycle

Restriction point

intrinsic: final cell size reached?

G2 = Control of

DNA-Synthesis

M = Mitosis

G1 = Cell growth


How is the correct


controlled ?

S = DNA-Synthesis

The cell cycle

Restriction point


external: growth factors ?

G2 = Control of

DNA-Synthesis

M = Mitosis

G1 = Cell growth


How is the correct


controlled ?

S = DNA-Synthesis

The cell cycle

Restriction point



G2 = Control of

DNA-Synthesis

M = Mitosis

G1 = Cell growth


S = DNA-Synthesis

The cell cycle

G2-Control

Replication complete?

DNA intact?

Restriction point



G2 = Control of

DNA-Synthesis

M = Mitosis

G1 = Cell growth


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 ?

S-Phase

S-Phase

G1

G2

G1G2

Cellfusion experiments with synchronised HeLa cells

+

+

+

S-Phase

S-Phase

G1

G2

G1G2


+

+

+

S-Phase

S-Phase

G1

G2

G1G2


+

+

+

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)

























Leland H Hartwell

Identification of cell cycle regulators: CDKs

25°C

35°C

Identification of temperature-sensitive CDC (Cell-Division-Cycle) Mutants

Model: Saccharomyces sereviciae

Mutagenized cells


25°C

35°C

35°C

non CDC-Mutants

budding

Entry into the

Cell cycle



Mutagenized cells


25°C

35°C

35°C

CDC-START-Mutants

non CDC-Mutants

no entry into the

cell cycle



Mutagenized cells


35°C 35°C

Identification of the CDC-START gene

Wt CDC-START-Gen

CDC-START-Mutant

Wt Gen X


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


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


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


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


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


CDKs: Motors of the cellcycle

How act CDKs as motors of te cell cycle?

Which substrates are phophorylated by CDKs during the cell cycle?

CDK Substrates: Initiation of the S-Phase

Fuction of CDKs in

initiation of the S-Phase

NH2- -COOH A B

Retinoblastom-Protein (Rb)

- „Key“- substrate of the S-Phase -

nuclear protein, 110 kDa


NH2- -COOH A B

Binding of

transcription factor E2F





NH2- -COOH

E2F: central transcription factor for the induction

of S-Phase genes

-COOH A B

Binding of






NH2- -COOH

Rb act as a tumor supressor gene

A B

Binding of






- Rb act as a tumor supressor gene

- Rb can be hyperphosphorylated





NH2- -COOH A B

P P P P P P P P P P

Binding of


Rb

E2F

Rb

Repression

of E2F-controlled genes


Rb

E2F

Induktion


P P P

CDK 2

Cyclin E

Rb

E2F

Rb


Repression


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

Cyclin E

E2F

DNA-Pol I

dNTP-Synth.

CDK 2

Cyclin E

Rb

P P P

„forced"

transition of

the restriction point

S-Phase




feedback loop!

CDK substrate: Mitosis

Function of CDKs

in the mitosis

An important step in mitosis is the desintegration

of the nuclear membrane

Interphase Mitose


chromatin nuclear lamina

inner nuclear membrane


desintegration

of the nuclear lamina

desintegration of the der

nuclear membran

mitosis



CDK 1

Cyclin B

Mitose

ATP



desintegration



nuclear membran

CDK 1

Cyclin B

lamin tetramer

Mitose

lamin network

ATP

desintegration



nuclear membran



CDK 1

Cyclin B

lamin tetramer

-P P- -P P-

phosphorylated

lamin dimers

lamin netork

Mitose

ATP ATP



desintegration



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?

Regulation of CDK activity

CDK inactive

Regulation of CDK activity: Cyclin concentration

inactive CDK

Cyclin Cyclin

CDK inactive

P

only CDK-Cyclin complexes

are substrates

Threonin-Kinase (CAK)

active

T160 P CDK

Cyclin

P ATP


inactive CDK

Cyclin Cyclin

CDK inactive

Conformation change in CDK structureby cyclin binding

+ CAK

Conformation change in CDK structureby cyclin binding

inactive CDK

Cyclin Cyclin

CDK inactive

P

Cyclin concentration

active

T160 P CDK

Cyclin

ATP



How is the concentration of cyclins regulated in the cell cycle?

Cyclin


De novo synthesis

by transcriptional

induction

Cyclin



Regulation of cyclin concentration: transcriptional Induktion

P

Example: Induction by growth factor signal chains

P

GF

-R

GF

Regulation of cyclin concentration: transcriptional Induktion


P

TF-OH TF-O-P

Protein kinases

GF

-R

GF

Regulation of cyclin concentration: transcriptional induction


P

TF-OH TF-O-P

immediate early

genes

Protein kinases

GF

-R

GF Example: Induction by growth factor signal chains


P

TF-OH TF-O-P

immediate early

genes

c-jun/c-fos (TF) Protein kinases

GF

-R



P

TF-OH TF-O-P

immediate early

genes

c-jun/c-fos (TF)

delayed genes

c-jun

c-fos

Protein kinases

GF

-R



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



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



Cyclin

Restriktion point

(G1 S-Phase)

Degradation

by proteolysis

De novo synthesis

by transcriptional

induction



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



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



NH2 COOH

Cyclin A,B

Destruction-Box


NH2 COOH

Cyclin A,B

Destruction-Box

NH2 COOH

Ubiquitin


NH2 COOH

Cyclin A,B

Destruction-Box

NH2 COOH

Ubiquitin Anaphase-promoting-complex

(APC, Ubiquitin-Ligase-complex)


NH2 COOH

Cyclin A,B

Destruction-Box

NH2 COOH

Ubiquitin

Labeling for proteolytic

degradation

Anaphase-promoting-complex

(APC, Ubiquitin-Ligase-Komplex)


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)


How is the proteolytic degradation of cyclin B regulated?


CDK1

Cyclin B

G1-Phase

APC

inactive


Cyclin B

synthesis

Cyclin B

S, G2-Phase

G1-Phase CDK1

APC

inactive


CDK1

Cyclin B

Cyclin B

synthesis

P

Metaphase

Cyclin B

APC APC

P

inactiv active

S, G2-Phase

G1-Phase CDK1


CDK1

Cyclin B

Cyclin B

synthesis

P

Metaphase

Cyclin B

APC APC

P

inactive active

S, G2-Phase

G1-Phase CDK1

ATP


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


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


proteasomal

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


proteasomal

degradation


negative autoregulatory

feedbackloop


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


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


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


CDK1

CyclinB

T14

Y15

P

P

inactive

P

T160 P

CDK1

CyclinB

active

T160 P

G2-Phase

Mitosis

G2-Phase

Mitosis

Pi


activation

P P

Threonin/Tyrosin

Phosphatase active

Threonin/Tyrosin


CDK1

CyclinB

T14

Y15

P

P

inactive

T160 P

CDK1

CyclinB

active

T160 P

P

G2-Phase

Mitosis

G2-Phase

Mitosis

Pi

ATP


activation

P P

Threonin/Tyrosin

Phosphatase active

Threonin/Tyrosin


CDK1

CyclinB

T14

Y15

P

P

inactive

T160 P

CDK1

CyclinB

active

T160 P

P

G2-Phase

Mitosis

G2-Phase

Mitosis

Pi

ATP



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


How is the concentration of CKI p21 regulated?


Regulation of CDK activity: Induction of the inhibitor p21

p53 Transcription factor, tumor supressor gene

p53

CKI p21



p53

CKI p21

CDK 2

Cyclin E

CKI p21



p53

CKI p21

T1/2

= 3

0 m

in

p53

degradation

CDK 2

Cyclin E

CKI p21



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



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



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


p53

Mdm2p53

Mdm2

Degradation

Ubiquitin ligase

Regulation of CDK activity: regulation of p53

p53

Mdm2p53

Mdm2p53

P

Degradation


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


p53

Mdm2p53

Mdm2p53

P

DNA-damage

DNA-PK

ATM ATR

Mdm2

P

MAPK

Degradation


protein kinase


p53

Mdm2p53

Mdm2 p19/Arfp53

P

DNA-damage

DNA-PK

ATM ATR

Mdm2

P

MAPK

Degradation



p53

Mdm2p53

Mdm2 p19/Arf

Mdm2 p19/Arf

p53

P

DNA-damage

DNA-PK

ATM ATR

Mdm2

P

MAPK

Dgradation

Growth factorhyperstimulation


p53

Mdm2p53

Mdm2 p19/Arf

Mdm2 p19/Arf

p53

P

DNA-damage

DNA-PK

ATM ATR

Mdm2

P

MAPK

Dgradation

Growth factorhyperstimulation


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


Phosphorylation

Dephosphorylation

G2 M

G1 S M G1

G2 M

ATP

CDK

Cyclin

T14

Y15

P

P

inactive

T160 P

ATP

Pi


inactive CDK

Cyclin Cyclin

CDK inactive

active

T160 P CDK

Cyclin

P


Phosphorylation

Dephosphorylation

G2 M

G1 S M G1

G2 M

ATP

CDK

Cyclin

T14

Y15

P

P

inactive

T160 P

ATP

Pi


inactive CDK

Cyclin Cyclin

CDK inactive

inactive

T160 P CDK

CKI

CKI CDK-Inhibitors

G1 S

M

S

G1 G2

Cyclin-dependent kinases: Motors and switches of the cell cycle

R

Dephosphorylation Cyclin degradation

Cyclin synthesis

Inhibitors

pRb/E2F

Lamin-P

Regulation of the cell cycle - uni-potsdam.de · Regulation of the cell cycle . Cyclin-dependent Kinases (CDKs) Dr. F. Neuschäfer-Rube . Cyclin-dependent kinases: Motors and switches

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