CHMI 4237 E Special topics in Biochemistry

1. CHMI 4237 ESpecialtopics in Biochemistry Cellproliferation 2- cell cycle checkpoints Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry LaurentianUniversity CHMI 4237 E - Winter 2010

2. 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? So, what are the BIG questions: CHMI 4237 E - Winter 2010

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

4. Checkpoints: Go or No-go CHMI 4237 E - Winter 2010

5. Checkpoints: Go or No-go CHMI 4237 E - Winter 2010

6. 1) Minor damage: • Single strand breaks • Base mismatch • DNA crosslinks • 2) Major damage: • Double strand breaks DNA damage checkpoint CHMI 4237 E - Winter 2010 http://atlasgeneticsoncology.org/Deep/Images/DoubleStrandBreak4.jpg

7. 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 DNA damage checkpoint1- Response to minor damage CHMI 4237 E - Winter 2010

8. 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; 1-G2 checkpointResponse to minor damage CHMI 4237 E - Winter 2010

9. Highly conserved 30 kDa protein; • 14-3-3 dimers bind phosphorylated Ser residues; www.stke.org/cgi/content/full/sigtrans;2005/296/re10 14-3-3 proteins CHMI 4237 E - Winter 2010 NATURE REVIEWS | CANCER VOLUME 3 | DECEMBER 2003

10. 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 2-G1 checkpointResponse to double strand breaks NATURE REVIEWS | Molecular Cell Biology Volume 9 | OCTOBER 2008 CHMI 4237 E - Winter 2010

11. 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; 2-G1 checkpointResponse to double strand breaks NATURE REVIEWS | Molecular Cell Biology Volume 9 | OCTOBER 2008 CHMI 4237 E - Winter 2010

12. 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; 2-G1 checkpoint CHMI 4237 E - Winter 2010

13. 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; p53 CHMI 4237 E - Winter 2010

14. p53 CHMI 4237 E - Winter 2010

15. 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; p53 CHMI 4237 E - Winter 2010

16. 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 p53 CHMI 4237 E - Winter 2010

17. ↓ Cyclin B ↓ Cdc2 p53 CHMI 4237 E - Winter 2010

18. http://www.brc.riken.go.jp/lab/dna/en/GENESETBANK/p53-G2_arrest.pnghttp://www.brc.riken.go.jp/lab/dna/en/GENESETBANK/p53-G2_arrest.png p53 CHMI 4237 E - Winter 2010

19. 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 p53 regulation CHMI 4237 E - Winter 2010

20. 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; p53 regulation CHMI 4237 E - Winter 2010

21. 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 p16Ink4A • p14Arf acts as an inhibitor of MDM-2: triggers the re-localization of MDM-2 to the nucleolus p53 regulation CHMI 4237 E - Winter 2010

22. p53 regulation http://www.nature.com/nrc/journal/v3/n2/images/nrc991-f2.gif CHMI 4237 E - Winter 2010

23. 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; Spindle-assemblycheckpoint MOLECULAR AND CELLULAR BIOLOGY, Jan. 2010, p. 22–32 CHMI 4237 E - Winter 2010

24. The mitotic spindle is made of microtubules: • Dimers of a and b tubulin; • GTP-binding proteins: • a tubulin: does not hydrolyse GTP • b tubulin: can hydrolyse GTP into DP • Form tubular aggregates with a polarity: • Exposed a tubulin : • (-) end • Points toward the nucleus • Exposed b tubulin : • (+) end • Points toward the cell surface Microtubules and the mitoticspindle CHMI 4237 E - Winter 2010

25. The Microtubule Organization Center (MTOC) CHMI 4237 E - Winter 2010

26. The Microtubule Organization Center (MTOC) CHMI 4237 E - Winter 2010

27. 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 show « dynamicinstability » CHMI 4237 E - Winter 2010

28. Microtubules can « treadmill » CHMI 4237 E - Winter 2010

29. Dyneins 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; Microtubule motors CHMI 4237 E - Winter 2010

30. Dyneins Kinesins Microtubule motors CHMI 4237 E - Winter 2010

31. Kinesins http://www.youtube.com/watch?v=686qX5yzksU CHMI 4237 E - Winter 2010

32. Kinesins and dyneins CHMI 4237 E - Winter 2010

33. Comprises 4 modules: • First (inner) module: interfaces with the centromere • Second (outer) module: binds microtubules • Third module (spindleassemblycheckpoint [SAC] complex): senses the kinetochore-microtubule attachment; • Fourth module: detects correct vs incorrect kinetochore-microtubule attachments; stabilizes the former and disrupts the latter. Kinetochore CHMI 4237 E - Winter 2010

34. Kinetochoresformat the centromere: • Nucleosomewith histone H3 variant CENP-A • Innerkinetochore: A proteincomplexcalled « constitutive centromereassociated network (CCAN) binds CENP-A; • Outerkinetochore: the KMN complex, a set of MT-bindingproteinsisrecruitedduringmitosis; Kinetochore The EMBO Journal VOL 28 | NO 17 | 2009 CHMI 4237 E - Winter 2010

35. The KMN complex serves also as a landing pad for the « spindleassemblycheckpoint » (SAC )complex; • SAC: MAD1/BUB1/ MPS1/Aurora-B/MCC (contains MAD2/BUBR1 and BUB3) • The « chromosome passengercomplex » (CPC) (senses MT attachment to kinetochore) includes Aurora B/Survivin/INCENP/Borealin • Finally, the APC/cyclosomesub-unit Cdc20 isalsofoundat the kinetochore; Kinetochore CHMI 4237 E - Winter 2010 NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | MAY 2007 | 379

36. Duringmitosis, microtubules show increaseddynamicinstability; • Uponbinding a kinetochore, MTs are greatlystabilized; • The kinetochoredyneindynactin moves the chromosome pair towards the closest centrosome. This exposes the otherside of the kinetochore and increases the chances of MT binding • Once a pair of chromosomes isbound by MTsfrombothpoles, itissaid to bebi-orientated; Metaphase CHMI 4237 E - Winter 2010

37. Chromosomescongressionat 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 mostdistant 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. Metaphase CHMI 4237 E - Winter 2010

38. 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 Control of MT attachment CHMI 4237 E - Winter 2010

39. Developmental Cell, Vol. 7, 637–651, November, 2004 Control of MT attachment CHMI 4237 E - Winter 2010

40. 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; Control of MT attachment The EMBO Journal VOL 28 | NO 17 | 2009 CHMI 4237 E - Winter 2010

41. Control of MT attachment The EMBO Journal VOL 28 | NO 17 | 2009 CHMI 4237 E - Winter 2010

42. 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; Spindleassemblycheckpoint CHMI 4237 E - Winter 2010

43. 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; Spindleassemblycheckpoint CHMI 4237 E - Winter 2010

44. 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. Anaphase CHMI 4237 E - Winter 2010