chmi 4237 e special topics in biochemistry
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CHMI 4237 E Special topics in Biochemistry. Cell proliferation 1 – basic machinery. Eric R. Gauthier, Ph.D . Dept . Chemistry - Biochemistry Laurentian University. Mitosis. Blue: DNA / Green: microtubules. Mitosis. Blue: DNA / Green: microtubules. - PowerPoint PPT PresentationTRANSCRIPT
CHMI 4237 ECHMI 4237 E
Special topics in Special topics in BiochemistryBiochemistry
Eric R. Gauthier, Ph.D.Dept. Chemistry-Biochemistry
Laurentian University
Cell proliferation1 – basic machinery
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MitosisMitosis
2CHMI 4237 E - Winter 2010Blue: DNA / Green: microtubules
MitosisMitosis
3CHMI 4237 E - Winter 2010Blue: DNA / Green: microtubules
Cell cycleCell cycle
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Mitosis: ~1 h
http://219.221.200.61/ywwy/zbsw%28E%29/edetail11.htm
Cell cycleCell cycle
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Signals
So, what are the BIG questions:So, what are the BIG questions:
1) How does the basic cell cycle machinery work?
2) How does the cell ensure that a given step in the cell cycle is properly completed before moving forward?
3) What are the signals that modulate the cell cycle?
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So, what are the BIG questions:So, what are the BIG questions:
1) How does the basic cell cycle machinery work?
2) How does the cell ensure that a given step in the cell cycle is properly completed before moving forward?
3) What are the signals that modulate the cell cycle?
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Maturation Promoting Factor Maturation Promoting Factor (MPF): the engine of the cell cycle(MPF): the engine of the cell cycle
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MPF can trigger mitosis when injected Into frog eggs
Works even in the presence of protein synthesis inhibitors (e.g. cycloheximide)
Cyclins: drivers of the cell cycleCyclins: drivers of the cell cycle
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Cyclin levels and MPF activity Cyclin levels and MPF activity fluctuate during the cell cyclefluctuate during the cell cycle
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MPF: dimer of a cyclin and a MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk)cyclin-dependent kinase (cdk)
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MPF: dimer of a cyclin and a MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk)cyclin-dependent kinase (cdk)
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Specific cyclins and cdks pair up Specific cyclins and cdks pair up to control specific cell cycle to control specific cell cycle eventsevents
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Fission yeast Mammals
Specific cyclins and cdks pair up Specific cyclins and cdks pair up to control specific cell cycle to control specific cell cycle eventsevents
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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | NOVEMBER 2001 | 815
Importance of CDKsImportance of CDKs
CHMI 4237 E - Winter 2010 15Nature Reviews | Cancer Volume 9 | March 2009
CDK regulationCDK regulation
While CDK activity varies according to the cell cycle, the level of CDKs is pretty constant;
This raises the question: what regulates the activity of the CDKs?
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Progress through cell cycle
CDK activity
Cyclin levels
CDK levels
CDK activationCDK activation1 – cyclin binding1 – cyclin binding
CHMI 4237 E - Winter 2010 17http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg
Protein kinase structureProtein kinase structure
Lower jaw Substrate binding and
phosphotransfer reaction
Activation loop (aka T loop): forms a barrier between ATP and substrate inactive kinase
Phosphorylation of T loop change in conformation kinase activation;
Upper jawATP binding
P loop: Gly-rich sequence which binds the phosphates of ATP
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Protein kinase structureProtein kinase structure
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http://www.new-science-press.com/info/illustration_files/nsp-protein-3-13-3_25.jpg
CDK activationCDK activation1 – cyclin binding1 – cyclin binding
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http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg
But: cyclin levels do not But: cyclin levels do not necessarily correlate with CDK necessarily correlate with CDK activation….activation….
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CDK activationCDK activation2- Phosphorylation by CDK-2- Phosphorylation by CDK-activating kinases (CAK)activating kinases (CAK)
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http://www.new-science-press.com/info/illustration_files/
CDK activationCDK activation2- Phosphorylation by CDK-2- Phosphorylation by CDK-activating kinases (CAK)activating kinases (CAK)
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http://www.new-science-press.com/info/illustration_files
CDK regulationCDK regulation3- Phosphorylation status of Tyr 3- Phosphorylation status of Tyr 1515
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Wee 1 (kinase): phosphorylates Tyr 15, inactivating cdk2
Cdc25 (phosphatase): de-phosphorylates Tyr 15, activating cdk2
CDK regulationCDK regulation4- Inhibition by CDK inhibitory 4- Inhibition by CDK inhibitory proteins (CKIs)proteins (CKIs)
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p27-/-
p27-/-
Wild type
Wild type
CDK regulationCDK regulation4- Inhibition by CDK inhibitory 4- Inhibition by CDK inhibitory proteins (CKIs)proteins (CKIs)
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p27, p57, p21: obstruct ATP- binding site
INK4 family: decrease affinity of CDK 4/6 for D-type cyclins
CDK regulationCDK regulation4- Inhibition by CDK inhibitory 4- Inhibition by CDK inhibitory proteins (CKIs)proteins (CKIs)
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CDK regulationCDK regulation4- Modulation by CDK inhibitory 4- Modulation by CDK inhibitory proteins (CKIs)proteins (CKIs)
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CDK regulationCDK regulation4- Modulation by CDK inhibitory 4- Modulation by CDK inhibitory proteins (CKIs)proteins (CKIs)
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CDK regulationCDK regulation5- Subcellular localization5- Subcellular localization
Cyclin B is kept in the cytosol, away of its targets;
Just prior to the onset of mitosis, Cyclin B is phosphorylated;
Cyc B phosphorylation masks nuclear export sequences, resulting in its accumulation in the nucleus
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CDK regulationCDK regulation6- Controlled proteolysis6- Controlled proteolysis
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Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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Ubiquitin:◦ 76 aa / 8.5 kDa peptide
Reversible modification
In yeast: 20% of all proteins can e ubiquitylated
Outcome:◦ PolyUb ( Lys48): Protein degradation◦ MonoUb or PolyUb (Lys 63):
protein/protein interactions
Often works in tandem with phosphorylation;
Enzymatic machinery rivals the one used for phosphorylation:◦ 500 E3 ligases vs 518 kinases◦ 80 DUBs vs 120 phosphatases
Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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E1: Activating enzyme◦ Very few in the cell
E2: Conjugating enzyme◦ Catalyses the addition of Ub to substrate proteins
E3: Ub ligases◦ Responsible for the substrate specificity of the E2 enzyme◦ Lots of them in the cell
E1
E2a E2b E2c
E3bE3a E3c
Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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http://www.scills.ac.uk/images/whatis-1.jpg
Ubiquitin-mediated Ubiquitin-mediated proteolysisproteolysis
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CDK regulationCDK regulation
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http://www.cella.cn/book/13/images/image027.jpg
Coordinated regulation of CDKs Coordinated regulation of CDKs during the cell cycleduring the cell cycle
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But: what do CDKs actually do?But: what do CDKs actually do?1- G1 phase1- G1 phase
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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | AUGUST 2007
pRb:◦ Retinoblastoma protein
◦ Family of 3 proteins: pRb 105 kDa p107 P130
◦ pRb proteins bind proteins of the E2F family
E2F:◦ Family of transcription factors
◦ When bound to pRb: suppresses expression of genes required for cell cycle progression
◦ After dissociation from pRb: E2F activates the expression of cell cycle-related genes
pRbpRb
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In G0: pRb is unphosphorylated pRb binds E2F
In early G1:◦ pRb is hypophosphorylated by cyclin D-cdk4/6◦ pRb binds E2F
In late G1:◦ pRb is hyperphosphorylated by Cyclin E/Cdk2◦ Allows progression through the cell cycle past the restriction (« R »)
point: point of no return when the cell is committed to complete the cell cycle:
Before the R point: cells require growth signals to progress in G1 After the R point: growth signals are no longer necessary
pRb and E2FpRb and E2F
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Gene modulation by pRb and E2FGene modulation by pRb and E2F
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p107/p130: pRb
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Current Opinion in Cell Biology 2007, 19:658–662
E2F gene targetsE2F gene targets
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Cdk2Cyclin ACyclin E
Passing the R pointPassing the R point
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Passing the R point: Passing the R point: Positive feedback loops ensure the Positive feedback loops ensure the irreversibility of the cell cycle irreversibility of the cell cycle
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But: what do CDKs actually do?But: what do CDKs actually do?2- S phase2- S phase
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http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-4-0-4_1.jpg
DNA replication occurs once (and only once) during the cell cycle, during S phase;
All the enzymes required for DNA synthesis (nucleotide synthesis, DNA synthesis proper, etc) have been produced prior to entering S phase;
At the end of S phase: the two copies of a duplicated chromosomes are physically kept together as sister chromatids via a protein called cohesin.
But: what do CDKs actually do?But: what do CDKs actually do?2- S phase2- S phase
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In eukaryotes, replication forks progress at a rate of ~10-100 bp /sec;
The massive size of eukaryotic genome requires the presence of multiple replication initiation sites;
But: what prevents a given replication origin to be more than once during the same S phase?
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But: what do CDKs actually do?But: what do CDKs actually do?2- S phase2- S phase
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Replication origines are licensed by becoming loaded with MCM proteins;
MCM loading requires a proteins called CDC6, itself recruited to replication origins by the protein ORC (Origin Replication Complex);
MCM/ORC/CDC6 proteins participate in recruiting the DNA replication machinery
However, S-phase cyclin (Cyclin A/Cdk2 in mammals) phosphorylates Cdc6, tagging it for degradation.
M-phase cyclins (Cyclin B/Cdk2 in mammals) also phosphorylate CDC6, preventing replication initiation during mitosis;
The MCM proteins are displaced by the moving replication fork.
But: what do CDKs actually do?But: what do CDKs actually do?3- M phase - nuclear membrane dissolution3- M phase - nuclear membrane dissolution
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One of the most easily recognized event in early mitosis is the dissolution of the nuclear membrane;
This requires the dissolution of the nuclear lamina, a mesh of proteins covering the intra-nuclear side of the nuclear membrane;
This is accomplished the phosphorylation of lamins by Cyclin B/cdk2;
http://www.bc.biol.ethz.ch/people/groups/ulkutay
But: what do CDKs actually do?But: what do CDKs actually do?3- M phase - nuclear membrane dissolution3- M phase - nuclear membrane dissolution
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But: what do CDKs actually do?But: what do CDKs actually do?4- M phase – separation of sister chromatids4- M phase – separation of sister chromatids
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During prophase: cyclin B/CDK1 phosphorylate and inhibit separase;
The protein securin also participates in inhibiting separase
This ensure that both sister chromatids stay together during the early part of mitosis;
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But: what do CDKs actually do?But: what do CDKs actually do? 4- M phase – separation of sister chromatids4- M phase – separation of sister chromatids
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Upon reaching anaphase, cyclin B and securin are degraded via the APC/cyclosome (a ubiquitin ligase);
This results in separase activation, which cleaves cohesin, allowing the separation of sister chromatids;
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But: what do CDKs actually do?But: what do CDKs actually do? 5- M phase – Exiting mitosis5- M phase – Exiting mitosis
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In order to complete mitosis, several events triggered by cyclin/cdks have to be reversed:◦ Disassembly of the mitotic spindle◦ Reformation of the nuclear membrane◦ Decondensation of the chromosomes
Also: MCM proteins need to be available for licensing the next DNA replication event;
This requires that the activation of cyclins/Cdks be terminated;
This is accomplished via the « Anaphase-promoting complex/cyclosome » (APC/C)
The Anaphase-promoting The Anaphase-promoting complex/cyclosomecomplex/cyclosome
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APC/C is a gigantic ubiquitin ligase (the size of a ribosome);
Exists in two separate forms:◦ Bound to Cdc20 (APC/Ccdc20)◦ Bound to Cdh1 (APC/Ccdh1)
Recognizes proteins with an amino acid sequence dubbed the Destruction box (D-box)
Two main targets of the APC/c:◦ Cyclins◦ Securin
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
PNAS December 22, 1998 vol. 95 no. 26 15374-15381
APC/CAPC/CCdc20Cdc20 - modulates - modulates anaphase and mitotic exitanaphase and mitotic exit
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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
The Anaphase-promoting The Anaphase-promoting complex/cyclosomecomplex/cyclosome
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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
Mitosis: APC/C is phosphorylated by cyclins/cdk1, promoting its association with CDC20;
Conversely, cyclin/cdk-mediated phosphorylation of Cdh1 prevent it from associating with APC/C;
So: APC/Ccdh1 only arises in late mitosis, after cyclins have been destroyed by APC/Ccdc20
The Anaphase-promoting The Anaphase-promoting complex/cyclosomecomplex/cyclosome
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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
APC/Ccdc20 levels decrease at the end of mitosis because:◦ Low cdk1 activity result in
its APC/C dephosphorylation
◦ Cdc20 is targeted for degradation by APC/Ccdh1.
APC/Ccdh1 ensures that cyclin levels stay low for most of G1, permitting the licensing of the DNA replication origins;
Inactivation of APC/CInactivation of APC/Ccdh1cdh1
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A) Phosphorylation by cyclin A/cdk2
B) EMI-1 expression◦ During G1, E2F triggers the expression of
EMI1 (early mitotic inhibitor-1);
◦ EMI1 inhibits APC/Ccdh1, permitting the increase of G1 cyclins;
C) UBCH10 degradation◦ UBCH10 is an E2 enzyme associated with
APC/C
◦ UBCH10 is essential for cyclin A degradation. It is also a target of APC/Ccdh1;
◦ So, the APC/Ccdh1-mediated degradation of UBCH10 allows the accumulation of cyclin A required in late G1;
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
The Anaphase-promoting The Anaphase-promoting complex/cyclosomecomplex/cyclosome
CHMI 4237 E - Winter 2010 61Genes Dev. 2006 20: 3069-3078