CHMI 4237 E Special topics in Biochemistry

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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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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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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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