1. CHMI 4237 E Special topics in Biochemistry Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry Laurentian University Cell proliferation 2- cell cycle checkpoints 1CHMI 4237 E - Winter 2010

2. 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? CHMI 4237 E - Winter 20102

3. Checkpoints: Go or No-go CHMI 4237 E - Winter 20103 MOLECULAR AND CELLULAR BIOLOGY, Jan. 2010, p. 22–32

4. Checkpoints: Go or No-go CHMI 4237 E - Winter 20104

5. Checkpoints: Go or No-go CHMI 4237 E - Winter 20105

6. DNA damage checkpoint CHMI 4237 E - Winter 20106 http://atlasgeneticsoncology.org/Deep/Images/DoubleStrandBreak4.jpg 1) Minor damage: ◦Single strand breaks ◦Base mismatch ◦DNA crosslinks 2) Major damage: ◦Double strand breaks

7. DNA damage checkpoint 1- Response to minor damage CHMI 4237 E - Winter 20107 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 | molecular cell biology volum e 9 | AUGUST 2008 | 617

8. 1-G2 checkpoint Response to minor damage CHMI 4237 E - Winter 20108 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;

9. 14-3-3 proteins CHMI 4237 E - Winter 20109 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

10. 2-G1 checkpoint Response to double strand breaks CHMI 4237 E - Winter 201010 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

11. 2-G1 checkpoint Response to double strand breaks CHMI 4237 E - Winter 201011 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

12. 2-G1 checkpoint CHMI 4237 E - Winter 201012 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;

13. p53 CHMI 4237 E - Winter 201013 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;

14. p53 CHMI 4237 E - Winter 201014

15. p53 CHMI 4237 E - Winter 201015 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;

16. p53 CHMI 4237 E - Winter 201016 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

17. p53 CHMI 4237 E - Winter 201017 ↓ Cyclin B ↓ Cdc2

18. p53 CHMI 4237 E - Winter 201018 http://www.brc.riken.go.jp/lab/dna/en/GENESETBANK/p53- G2_arrest.png

19. p53 regulation CHMI 4237 E - Winter 201019 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

20. p53 regulation CHMI 4237 E - Winter 201020 Because of its importance in modulating the cell cycle, the activation of p53 is regulated at multiple level: ◦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;

21. p53 regulation CHMI 4237 E - Winter 201021 Because of its importance in modulating the cell cycle, the activation of p53 is regulated at multiple level: ◦p14Arf protein:  Encoded by the same gene as the CKI p16 Ink4A  p14 Arf acts as an inhibitor of MDM-2: triggers the re- localization of MDM-2 to the nucleolus

22. p53 regulation CHMI 4237 E - Winter 201022 http://www.nature.com/nrc/journal/v3/n2/images/nrc991-f2.gif

23. Spindle-assembly checkpoint CHMI 4237 E - Winter 201023 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;

24. Microtubules and the mitotic spindle CHMI 4237 E - Winter 201024 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

25. The Microtubule Organization Center (MTOC) CHMI 4237 E - Winter 201025

26. The Microtubule Organization Center (MTOC) CHMI 4237 E - Winter 201026

27. Microtubules show « dynamic instability » CHMI 4237 E - Winter 201027 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/Dyna mic-instability-of-microtubules--under- microscope-

28. Microtubules can « treadmill » CHMI 4237 E - Winter 201028

29. Microtubule motors DyneinsKinesins (-) 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; CHMI 4237 E - Winter 201029

30. Microtubule motors DyneinsKinesins CHMI 4237 E - Winter 201030

31. Kinesins CHMI 4237 E - Winter 201031 http://www.youtube.com/watch?v=686qX5yzksU

32. Kinesins and dyneins CHMI 4237 E - Winter 201032

33. Kinetochore CHMI 4237 E - Winter 201033 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.

34. Kinetochore CHMI 4237 E - Winter 201034 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

35. Kinetochore CHMI 4237 E - Winter 201035 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

36. Metaphase CHMI 4237 E - Winter 201036 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;

37. Metaphase CHMI 4237 E - Winter 201037 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.

38. Control of MT attachment CHMI 4237 E - Winter 201038 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; The EMBO Journal VOL 28 | NO 17 | 2009

39. Control of MT attachment CHMI 4237 E - Winter 201039 Developmental Cell, Vol. 7, 637–651, November, 2004

40. Control of MT attachment CHMI 4237 E - Winter 201040 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; The EMBO Journal VOL 28 | NO 17 | 2009

41. Control of MT attachment CHMI 4237 E - Winter 201041 The EMBO Journal VOL 28 | NO 17 | 2009

42. Spindle assembly checkpoint CHMI 4237 E - Winter 201042 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;

43. Spindle assembly checkpoint CHMI 4237 E - Winter 201043 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 p31 comet ; Active p31 comet binds the MAD2/Cdc20 complex, and releases active Cdc20; Cdc20 then turns on the APC/C to cleave cohesin and lead to chromatid separation;

44. Anaphase CHMI 4237 E - Winter 201044 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.