Post on 29-Jul-2020
Cell Cycle – Control of Cell Proliferation Ludger Hengst
Sektion für Medizinische Biochemie - Biozentrum - Medizinische Universität Innsbruck
1 fertilized egg !! !! ! !100 000 000 000 000 cells �
Cell cycle – control of cell proliferation Ludger Hengst
Biozentrum - Medizinische Biochemie - Medizinische Universität Innsbruck
In Adults: ! ≈4.000.000 cell divisions / sec.�
1. Principles of the cell cycle �
Cell Cycle Checkpoints�
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
1. Duplication of the genome!
2. Separation of the duplicated genetic material (and other cellular compounds) into two daughter cells"
Central Aims of the Cell Cycle
Four Cell Cycle Phases �
M
Mitosis and Cytokinesis"
S
DNA Synthesis
G1
G2
Gap-phase 1"
Gap-phase 2"
M
S
G1
G2
Principles of the eucaryotic cell cycle:
1. One and only one replication of the entire genome per cell cycle
2. Initiation of a subsequent cell cycle phase requires proper termination of the preceding phase
M
S
G1
G2 G0
Restriction point"
G0-phase
Why seperate cell cycle phases?
The temporal separation of DNA replikation and mitosis permits the incorporation of control mechanisms in the eucaryotic cell cycle. These control mechanisms are called!Checkpoints.
Checkpoint: a process within the cell cycle, which controls the transition ! from one cell cycle state into the next state
!! - Checkpoints secure e.g. that DNA replication is completed ! before mitosis can be initiated.
!! - Checkpoints secure genomic stability by arresting cell ! cycle progression upon DNA damage.!
Checkpoints
G1/S Checkpoint"
√ size ? "
√ DNA damage ?"
G2 / M Checkpoint"√ DNA replicated?"
√ DNA damage?"
Spindel Checkpoint"√ all chromosomes attached to the spindle ?"
G1
G0
S
M
G2
Important Checkpoints
√ mitogens anti-mitogenic signals ?""√ differentiation - signals ?"
1. Restrict cell divisions to precisely the required numbers, e.g. induced divisions after wounding or cell death�"- Avoids hypo- and hyperproliferation�
�2. Warrant one, and only one DNA replication of all regions of
the genome per cell cycle. �"- Avoids genetic instability�
�3. Precise segregation of the genome into both daughter cells.�
"- Avoids genetic instability��4. "Prevention of replication or segregation of damaged DNA �
"- Avoids the multiplication of damaged DNA
Important aims of cell cycle control mechanisms: �
Consequences of misregulated cell cycle control: �Genetic instability of tumor cells
Aneuploidy : abnormal number of chromosomes (extra or missing)
Chromosomal translocations: Rearrangements of chromosome parts between nonhomologues chromosomes
1
3
14
Length of cell cycle phases"
Duplication of bacteria:"
Eucaryotes:"Early frog embryo cells " 30 minYeast " " " 1.5 - 3 hCells in the epithelium of the small intestine " 12 h"Proliferating mammalian cells18-24 h"Fibroblasts in culture " 20 h"Human liver cells " " 1 year"
E. coli: 20 - 25 min"
M
G1"
G2"
S
1 h"
6-12 h"
6-8 h"
2-4 h"
1. Principles of the cell cycle �
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
G1
G0
S
M
G2
Restriction Point"
The Cell Cycle �
Mitosis and cytokinesis"
DNA-Synthesis
Mitogens ?"Antimitogens ?"
Growth factors ?"Differentiation signals ?"
Exit from the cell cycle:"- senescence - terminal differentiation- quiescence""G0-phase / quiescence:"- no cell divisions"- no DNA-replikation"- can persist hours, days or years"
Cell Fusions
Rao, P.N. & Johnson, R.T. (1970). Mammalian Cell Fusion: Studies on the Regulation of DNA Synthesis and Mitosis. Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian Cell Fusion: Induction of Premature Chromosome Condensation in Interphase Nuclei. Nature 226, 717-722.
Fusion of G1, S or G2 cells with mitotic cells leads to nuclear envelope breakdown and DNA condensation in the heterocaryon.!!Mitotic cells contain a factor (“MPF” - Mitosis promoting factor) which can induce mitosis in cells of other cell cycle phases.!
Rao, P.N. & Johnson, R.T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis.Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei.Nature 226,
717-722.
Heterocaryon experiments"
G1, S or G2
+ M M
Fusion of G1 cells with S-phase cells results in heterocaryons, in which the G1 nuclei initiate DNA replikation. S-phase cells only initiate mitosis after completion of DNA!replication in G1 cells.!!S-phase cells contain a factor which can initiate DNA replication in G1 cells. Checkpoints prevent “premature” entry into mitosis before S-phase is completed in both nuclei.
Rao, P.N. & Johnson, R.T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis.Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei.Nature 226,
717-722.
Heterocaryon experiments"
G1, S or G2
+ M
G1
+
S S
M
Rao, P.N. & Johnson, R.T. (1970). Mammalian cell fusion: studies on the regulation of DNA synthesis and mitosis.Nature 225, 159-164. Johnson, R.T. & Rao, P.N. (1970). Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei.Nature 226,
717-722.
Heterocaryon experiments"
G1, S or G2
+ M
G1
+
S S
S
+
G2 no DNA synthesis in the G2 nucleus, delayed entry into mitosis
M
G2 nuclei do not re-initiate DNA replication!Existence of a “licensing system”, which labels replicated DNA
and prevents re-replication.
1. Principles of the cell cycle �
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
The Nobel Prize in Physiology or Medicine 2001 "for their discoveries of key regulators of the cell cycle"
R. Tim Hunt
Fission yeast
Sir Paul M. Nurse
Sea Urchin
Leland H. Hartwell
Bakers yeast
The Cell Cycle Engine�
Cyclin A (N-term.)
Cdk2
Cdk
Cyclin
The Cell Cycle Engine: Cyclin-Dependent Kinases�
time!
rel. Cdk-kinase activity
Cell Cycle Transitions are driven by the oscillating Activity� of Cyclin-Dependent Kinases (CDKs) �
G1-phase kinase! S-phase kinase! M-phase kinase!
G 1 S G 2 M
G1
G0
S
M
G2
Cdk2 Cyc E
Cdk2 Cyc A
Cdk1 Cyc A
Cdk1 Cyc B
Cdk4 Cyc D
Restriction Point"
The Cell Cycle Engine: Cyclin-Dependent Kinases�
inactive! inactive! inactive!active!
Assembly factors ?spontanious"
CAK"KAP!
Wee1 / Myt1"Cdc25!
Cdk
Cyclin
Cdk
Cyclin
Cdk
Cyclin
Cdk
Cyclin
P"P"P"P"
Kinases"Phosphatases!
CDKs are regulated by activating and inhibitory phosphorylations �
1. Principles of the cell cycle �
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
M
G2
S
G1
G1 arrest"(Lovastatin; TGF-β)"
Molecular Brakes: CDK Inhibitor Proteins �
G1
G2 S
M
100!
75!
50!
25!
0!
% E-
kina
se a
ctiv
ity!
Cdk
Cyclin p27
Hengst et al., PNAS 1994!
Mechanism of CDK inhibition by p27 �
Cyclin A
Cdk2
p27
Cyclin A Cdk2
p27Kip1 Cdk
Cyclin p27
Mechanism of CDK inhibition by p27 �
ATP"p27"
Lacy et al., Nature Structural & Molecular Biology. 2004
Cip/Kip Inhibitors are Intrinsically Unstructured Proteins (IUPs)!
p27 198 AA
CDK inhibitory domain
p21 p27 p57
22 30 40 50 60 70 80 90 PCGSK ACR R LFGPVD S E Q L S R D SDALM A GCIQ E ARE R W N F D F VTET PL E-- G DFA W ER V RGLG L P KL Y LPTGP EHPKP S ACR N LFGPVD H E E L T R DL EKHC RDMEE AS Q RK W N F D F Q NHK PL E-- G KYE W Q E V EKG SL P EF Y YRPPR VLVRT S ACR S LFGPVD H E E L G R E L RMRA A ELNA E D Q N R W D F N F Q QDV PL RGP G RLQ W M E V DSE S V P AF Y RET-V
p57
Cyclin binding domain CDK binding domain
316 AA
p21 164 AA
The Cip/Kip-Family of CDK Inhibitors
Cdk
Cyclin p27
Linker helix
Two Families of CDK Inhibitors �
Cdk p16
Ink4 family!(Inhibitor of Cdk4)!
p15, p16, p18, p19 (Ink4 a-d)!!- specific for cyclin D / CDK4,6!- bind the CDK subunit!- ankyrin repeat structure!!
Cdk
Cyclin p27
Cip/Kip family (Cdk interacting protein;!Kinase inhibitory protein)!
p21, p27, p57 (Cip1, Kip1, Kip2)!!- bind to a broad spectrum of CDK/ cyclins!- bind the CDK / cyclin complex!- conserved N-terminal CDK- inhibitory domain!- may act as actvators for cyclin D/Cdk4,6 !
Cdk6
p16Ink4a
INK4 CDK4,6 Inhibitors�
Cdk4 p16
Cyclin D
What are critical CDK substrates at the G1/S transition ? �
1. Principles of the cell cycle �
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
Rb
The CDK substrat retinoblastom protein (Rb) is a central cell cycle regulator
E1A G1
G0
S
M
G2
- Retinoblastoma is a rapidly developing childhood cancer (1/20.000) arising from immature retinal cells - The Rb protein is a tumor suppressor protein; 45% of the patients carry a heterozygous mutation in Rb1 - Children with (heteroygous) mutations in Rb frequently develop tumours in other tissues (Knudsons two-hit hypothesis) - Viral oncoproteins like E1A and HPV E7 bind Rb and induce cell division in cultured cells
Rb binds E2F and inhibits cell cycle progression
Rb E2F
E2F binding site
inactive
Rb
E2F binding site
E2F
active
E1A
Phosphorylation of Rb releases E2F
inactive
P
CDK
Rb E2F
E2F binding site
P Rb
E2F binding site
E2F
active
1. Principles of the cell cycle �
2. The restriction point�
3. CDKs – central cell cycle regulators�
4. CDK inhibitors�
5. The retinoblastoma protein�
6. The RB-E2F pathway�
Rb E2F
mitogens
P
Mitogen induced expression and activation of cyclin D/ CDK initiates cell cycle progression
G1
G0
S
M
G2
Cdk2 Cyc E
Cdk2 Cyc A
Cdk1 Cyc A
Cdk4 Cyc D
P
Cdk4 Cyc D
Cdk2 Cyc E
mitogen independent
CDK inhibitors prevent CDK activation
Rb E2F
G1
G0
S
M
G2
Cdk2 Cyc E
Cdk4 Cyc D
p16 p15
p18 p19
p27 p57
p21
Cdk4
STOP
The Rb pathway during the G0/G1/S transition"
S-Phase Cell
S G0/G1
p27
Antimitogens
Cdk4,6 p16
Antimitogens
E2Fs Rb
HDAC
Cdk4,6
Cdc37
p27 degradation
P
S-Phase Cell
S
Cdk2 Cyc E
Cdk4,6 p27
Cyc D
Cdk2 Cyc E
p27
HDAC
E2Fs Rb P
P P
Cyclin E DHFR DNA Pol.α
G1
Cyclin A Cdk1
E2Fs
P
P Rb P
P P
P P
Cyc D
Mitogens
Cdk4,6 p16
Antimitogens
Antimitogens
Der Rb Signalweg des G0/G1/S Übergangs"
Head and neck!
Prostate!
Lung! Melanoma!
Gastrointestinal!
Breast!
Liver!Pancreas!
Bladder! Bone (osteosarcoma)!
Bone marrow (leukemia)!
Lymphoma!
p130"
p130" p130"
p16"
p16"
p16"
p15"
p15"
p15"
D1"
Pituitary!
Testis/Ovary!
p16"
D2"
Cdk6"
Cdk6"
D1"
D1"
D1"
p16"
p16"
p130"
D1"
Cdk4"p16"
p16"
D1"D3"
p16"
Cdk4"
p16"D1"
Cdk4" D1"
D1"
Cdk4" p16"
D1"
Cdk4" D1" p16"
pRb"E1"
p27"
p27"
p27"
p27" E1"
E1"
E1"p27"
E1"p27"
p27"
E1" p27"
E1" p27"
Cdk4"Cdk2"D2"
Other Sarcomas!p16"
Endometrium!p130"Cdk4"
p27"D1"
E1"
Glioma/blastoma!Cdk4"Cdk6"E1" p16"p15"
70%
> 80%
> 90%
> 80% > 90%
> 90%
> 80%
> 90%
> 90%
D2" D3" E1"> 90%
20%
> 80%
> 80%
p16" > 80%
E1"70%
Cdk4" E1"90%
> 90%
pRb"
pRb"
pRb"
pRb"
pRb"
pRb"
pRb"
pRb"
pRb"
pRb"
p27"
p27"
D1"
p27"
E1"
p16"
p27"
p27"
*
*
*
* *
*
*
*
*
*
*
* *
*
*
Cdk2"
*
Cdk4"p15"
* * * * *
* * D1"
* *
E1"
* *
* *
D3"
*
* *
*
pRb"D1"p16"
* * *
Cancer is a disease of the cell cycle �
From: Malumbres and Barbarcid: Nature Reviews Cancer (01). Vol1, 222
Questions ��
???"