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 proliferation2- cell cycle checkpoints

1CHMI 4237 E - Winter 2010

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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Checkpoints: Go or No-goCheckpoints: Go or No-go


MOLECULAR AND CELLULAR BIOLOGY, Jan. 2010, p. 22–32





DNA damage checkpointDNA damage checkpoint

CHMI 4237 E - Winter 2010 6http://atlasgeneticsoncology.org/Deep/Images/DoubleStrandBreak4.jpg

1) Minor damage:◦Single strand breaks◦Base mismatch◦DNA crosslinks

2) Major damage:◦Double strand

breaks

DNA damage checkpointDNA damage checkpoint1- Response to minor damage1- Response to minor damage


Damage is first sensed by ATR (ATM and Rad3-related):

◦ Single-stranded damaged DNA is bound by RPA (Replication protein A – a single stranded DNA-binding protein);

◦ ATR binds to RPA-coated DNA (done via ATRIP)

◦ The damage-specific DNA sliding clamp protein 9-1-1 is loader at the damaged site by the Rad17 clamp loader.

◦ This allows the recruitment of the ATR activator protein TOPBP1;

◦ ATR then phosphorylates (and activates) the DNA damage-transducer protein Chk1

natur e reviews | m

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1-G2 checkpoint1-G2 checkpointResponse to minor damageResponse to minor damage


Activated Chk-1 targets and phosphorylates the protein phosphatase Cdc25;

This results in the inhibition of Cdc25;

In the absence of active Cdc25, Cdk phosphorylated on Tyr15 accumulates, resulting in cell cycle arrest

Phosphorylated Cdc25 is exported to the cytosol, where it is sequestered by the protein 14-3-3;

14-3-3 proteins14-3-3 proteins


Highly conserved 30 kDa protein;

14-3-3 dimers bind phosphorylated Ser residues;

www.stke.org/cgi/content/full/sigtrans;2005/296/re10

NATURE REVIEWS | CANCER VOLUME 3 | DECEMBER 2003

2-G1 checkpoint2-G1 checkpointResponse to double strand breaksResponse to double strand breaks

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ATM is kept inactive as a dimer, coupled with PP2A (a protein phosphatase) and TIP60 (an acetyltransferase);

DNA double strand breaks (DSBs) are recognized by the MRN (Mre11-Rad50-NBS1) complex;

ATM is recruited at this complex and accumulates at DSBs

NATURE REVIEWS | Molecular Cell Biology Volume 9 | OCTOBER 2008

2-G1 checkpoint2-G1 checkpointResponse to double strand breaksResponse to double strand breaks

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At DSBs, ATM is acetylated on Lys 3016 (by TIP60) and autophosphorylated (on Ser367,Ser1893 and Ser 1981);

Activation of ATM requires its conversion to a monomer;

Acetylation seems the be crucial for ATM activation, while phosphorylation is more consequence on its activation;

Activated ATM then activates downstream pathways, some of them involving the protein kinase CHK2; NATURE REVIEWS | Molecular Cell Biology Volume 9 | OCTOBER 2008

2-G1 checkpoint 2-G1 checkpoint

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Activated ATM phosphorylates and activates the protein kinase CHK2;

CHK2 then phosphorylates and stabilizes the transcription factor p53 (MUCH more on this one in a second…);

p53 then modulates the expression of a bunch of genes, among them the CKI p21, which inhibits G1 cyclin/CDKs and causes cell cycle arrest;

p53p53

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Mutated form found in over 50% of all human cancers;

Transcription factor with a short half life (~ 20 min);

DNA binding requires the formation of p53 tetramers;

p53p53

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p53p53

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Mutated p53 is dominant negative: the presence of only one mutated subunit in a p53 tetramer may be sufficient to disturb the normal function of the protein;

p53p53

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Multiple signals trigger p53 accumulation, mostly by stabilization of the protein;

Activated p53 can lead to several outcomes:◦ Cell cycle arrest◦ DNA repair◦ Cell death◦ Cell senescence

p53p53

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↓ Cyclin B↓ Cdc2

p53p53

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p53 regulationp53 regulation

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Because of its importance in modulating the cell cycle, the activation of p53 is regulated at multiple level:

◦ Stability: MDM-2 is a protein which

ubiquitylates p53 and targets it for degradation

MDM-2 is itself a transcriptional target of p53


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◦ Phosphorylation: p53 phosphorylation by

(CHK2, ATR, ATM) prevents its inhibition by MDM-2 ;

MDM-2 is also phosphorylated (and inhibited) by phosphorylation by CHK2, ATM and ATR;


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◦ p14Arf protein: Encoded by the same gene

as the CKI p16Ink4A

p14Arf acts as an inhibitor of MDM-2: triggers the re-localization of MDM-2 to the nucleolus


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http://www.nature.com/nrc/journal/v3/n2/images/nrc991-f2.gif

Spindle-assembly checkpointSpindle-assembly checkpoint

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MOLECULAR AND CELLULAR BIOLOGY, Jan. 2010, p. 22–32

At anaphase: MT attachment to sister chromatids must be coordinated with the cleavage of cohesin;

The spindle assembly checkpoint ensures that

◦ The mitotic spindle forms properly;

◦ all chromosomes are properly aligned at the equatorial plate

If this is not the case, the SAC will then delay mitosis;

Microtubules and the mitotic Microtubules and the mitotic spindlespindle

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The mitotic spindle is made of microtubules:◦ Dimers of and tubulin;◦ GTP-binding proteins:

tubulin: does not hydrolyse GTP tubulin: can hydrolyse GTP into

DP◦ Form tubular aggregates with a

polarity: Exposed tubulin :

◦ (-) end◦ Points toward the nucleus

Exposed tubulin : ◦ (+) end◦ Points toward the cell surface

The Microtubule Organization The Microtubule Organization Center (MTOC)Center (MTOC)

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The Microtubule Organization The Microtubule Organization Center (MTOC)Center (MTOC)

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Microtubules show « dynamic Microtubules show « dynamic instability »instability »

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Microtubules show dynamic instability: ◦ They can rapidly grow and shrink

in size;

Microtubule elongation and shortening occurs preferentially at the (+) end;

http://www.dnatube.com/video/118/Dynamic-instability-of-microtubules--under-microscope-

Microtubules can « treadmill »Microtubules can « treadmill »

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Microtubule motorsMicrotubule motorsDyneins Kinesins

(-) end-directed motor

Powered by ATP hydrolysis

Binds MT at one end, and cargo at the other;

(+) end-directed motor

Powered by ATP hydrolysis

Binds MT at one end, and cargo at the other;

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Microtubule motorsMicrotubule motorsDyneins Kinesins

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KinesinsKinesins

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http://www.youtube.com/watch?v=686qX5yzksU

Kinesins and dyneinsKinesins and dyneins

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KinetochoreKinetochore

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Comprises 4 modules: First (inner) module: interfaces with the centromere Second (outer) module: binds microtubules Third module (spindle assembly checkpoint [SAC]

complex): senses the kinetochore-microtubule attachment; Fourth module: detects correct vs incorrect kinetochore-

microtubule attachments; stabilizes the former and disrupts the latter.


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Kinetochores form at the centromere:

Nucleosome with histone H3 variant CENP-A

Inner kinetochore: A protein complex called « constitutive centromere associated network (CCAN) binds CENP-A;

Outer kinetochore: the KMN complex, a set of MT-binding proteins is recruited during mitosis;

The EMBO Journal VOL 28 | NO 17 | 2009


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The KMN complex serves also as a landing pad for the « spindle assembly checkpoint » (SAC )complex;

SAC: MAD1/BUB1/ MPS1/Aurora-B/MCC (contains MAD2/BUBR1 and BUB3)

The « chromosome passenger complex » (CPC) (senses MT attachment to kinetochore) includes Aurora B/Survivin/INCENP/Borealin

Finally, the APC/cyclosome sub-unit Cdc20 is also found at the kinetochore;NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | MAY

2007 | 379

MetaphaseMetaphase

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During mitosis, microtubules show increased dynamic instability;

Upon binding a kinetochore, MTs are greatly stabilized;

The kinetochore dynein dynactin moves the chromosome pair towards the closest centrosome. This exposes the other side of the kinetochore and increases the chances of MT binding

Once a pair of chromosomes is bound by MTs from both poles, it is said to be bi-orientated;

MetaphaseMetaphase

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Chromosomes congression at the equatorial plate is due to continuous MT lenghtening and shortening, powered by the combined action of MT motors and MT treadmilling:

Dynactin pulls the chromosomes toward the most distant pole;

This requires MT shortening, stimulated by kinesin 13;

Kinesin 7 holds the chromosome onto the (+) end of the MT;

Kinesin 4 helps in moving the chromosome towards the (+) end of MTs by walking on adjacent MTs.

Control of MT attachmentControl of MT attachment

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MT attachment to the kinetochore can occur in different ways:

◦ Amphitelic (Bi-orientated, normal);

◦ Syntelic: MTs from the same pole bind to both kinetochores of a chromosome

◦ Monotelic: MTs binds to a single kinetochore

◦ Merotelic: MTs from one pole bind to one kinetochore, while MTs from the opposite pole bind to both kinetochores;



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Developmental Cell, Vol. 7, 637–651, November, 2004


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In the absence of tension:

◦ several kinetochore proteins are phosphorylated by the CPC complex;

◦ These phosphorylated residues are landing pads for the SAC complex (reviewed later)

◦ Kinetochores have a low affinity for MTs

Bi-orientated kinetochores are under tension:

◦ a number of kinetochore proteins can no longer be phosphorylated because they are pulled away from the CPC complex

◦ This prevents the recruitment of SAC proteins;

◦ MTs are stably attached after dephosphorylation of kinetochore proteins;



CHMI 4237 E - Winter 2010 41The EMBO Journal VOL 28 | NO 17 | 2009

Spindle assembly checkpointSpindle assembly checkpoint

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The spindle assembly checkpoint is activated when the chromosome is NOT under tension by being pulled by both centromeres;

MAD2 binds MAD1 on unattached kinetochore, converting MAD2 from an « open » to a « closed » form;

Closed MAD2 binds the APC/C subunit Cdc20, resulting in the inactivation of the latter;

Closed MAD2 bound to Cdc20 can bind « open » Mad2 molecules and convert them into a « closed » form, which can in turn bind and inactivate another Cdc20 molecule;

Spindle assembly checkpointSpindle assembly checkpoint

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The spindle assembly checkpoint is inactivated when all the chromosomes have been properly aligned;

MT attachment releases the MAD2/MAD1 complex from the kinetochore;

The MAD2/MAD1 complex binds and activates another protein called p31comet;

Active p31comet binds the MAD2/Cdc20 complex, and releases active Cdc20;

Cdc20 then turns on the APC/C to cleave cohesin and lead to chromatid separation;

AnaphaseAnaphase

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At this point chromatids are no longer tied-up together by cohesin and are no longer under tension;

Kinesin-13 powers the depolymerization of the microtubules at both the kinetochore and spindle pole;

This leads to the migration of the chromatids to the most proximal pole.

CHMI 4237 E Special topics in Biochemistry

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