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CHMI 4237 E Special topics in Biochemistry

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.

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CHMI 4237 E Special topics in Biochemistry

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  1. CHMI 4237 ESpecialtopics in Biochemistry Cellproliferation 1 – basic machinery Eric R. Gauthier, Ph.D. Dept. Chemistry-Biochemistry LaurentianUniversity CHMI 4237 E - Winter 2010

  2. Mitosis Blue: DNA / Green: microtubules CHMI 4237 E - Winter 2010

  3. Mitosis Blue: DNA / Green: microtubules CHMI 4237 E - Winter 2010

  4. http://219.221.200.61/ywwy/zbsw%28E%29/edetail11.htm Mitosis:  ~1 h Cell cycle CHMI 4237 E - Winter 2010

  5. Signals Cell cycle CHMI 4237 E - Winter 2010

  6. 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

  7. 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

  8.  MPF can trigger mitosis when injected • Into frog eggs • Works even in the presence of protein • synthesis inhibitors (e.g. cycloheximide) Maturation Promoting Factor (MPF): the engine of the cell cycle CHMI 4237 E - Winter 2010

  9. Cyclins: drivers of the cell cycle CHMI 4237 E - Winter 2010

  10. Cyclinlevels and MPF activityfluctuateduring the cell cycle CHMI 4237 E - Winter 2010

  11. MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk) CHMI 4237 E - Winter 2010

  12. MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk) CHMI 4237 E - Winter 2010

  13. Fission yeast Mammals Specificcyclins and cdks pair up to control specificcell cycle events CHMI 4237 E - Winter 2010

  14. Specificcyclins and cdks pair up to control specificcell cycle events NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | NOVEMBER 2001 | 815 CHMI 4237 E - Winter 2010

  15. Importance of CDKs CHMI 4237 E - Winter 2010 Nature Reviews | Cancer Volume 9 | March 2009

  16. 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? CDK activity Cyclinlevels CDK levels Progress through cell cycle CDK regulation CHMI 4237 E - Winter 2010

  17. CDK activation1 – cyclinbinding http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg CHMI 4237 E - Winter 2010

  18. Upper jaw Lower jaw Substratebinding and phosphotransferreaction Activation loop (aka T loop): forms a barrierbetween ATP and substrate inactive kinase Phosphorylation of T loop change in conformation  kinase activation; • ATP binding • P loop: Gly-richsequencewhichbinds the phosphates of ATP Protein kinase structure

  19. http://www.waimr.uwa.edu.au/images/misc/Src-Family-lge.gif Protein kinase structure http://www.nature.com/nrm/journal/v5/n1/images/nrm1280-i1.jpg http://www.new-science-press.com/info/illustration_files/nsp-protein-3-13-3_25.jpg

  20. CDK activation1 – cyclinbinding http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-3-4-3_12.jpg CHMI 4237 E - Winter 2010

  21. But: cyclinlevels do not necessarilycorrelatewith CDK activation…. CHMI 4237 E - Winter 2010

  22. CDK activation2- Phosphorylation by CDK-activating kinases (CAK) http://www.new-science-press.com/info/illustration_files/ CHMI 4237 E - Winter 2010

  23. CDK activation2- Phosphorylation by CDK-activating kinases (CAK) http://www.new-science-press.com/info/illustration_files CHMI 4237 E - Winter 2010

  24. CDK regulation3- Phosphorylation status of Tyr 15  Wee 1 (kinase): phosphorylates Tyr 15, inactivating cdk2  Cdc25 (phosphatase): de-phosphorylates Tyr 15, activating cdk2 CHMI 4237 E - Winter 2010

  25. p27-/- Wild type p27-/- Wild type CDK regulation4- Inhibition by CDK inhibitoryproteins (CKIs) CHMI 4237 E - Winter 2010

  26. CDK regulation4- Inhibition by CDK inhibitoryproteins (CKIs) INK4 family: decrease affinity of CDK 4/6 for D-type cyclins p27, p57, p21: obstruct ATP- binding site CHMI 4237 E - Winter 2010

  27. CDK regulation4- Inhibition by CDK inhibitoryproteins (CKIs) CHMI 4237 E - Winter 2010

  28. CDK regulation4- Modulation by CDK inhibitoryproteins (CKIs) CHMI 4237 E - Winter 2010

  29. CDK regulation4- Modulation by CDK inhibitoryproteins (CKIs) CHMI 4237 E - Winter 2010

  30. 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 CDK regulation5- Subcellularlocalization CHMI 4237 E - Winter 2010

  31. CDK regulation6- Controlledproteolysis CHMI 4237 E - Winter 2010

  32. Ubiquitin-mediatedproteolysis • Ubiquitin: • 76 aa / 8.5 kDa peptide • Reversible modification • In yeast: 20% of all proteinscan e ubiquitylated • Outcome: • PolyUb ( Lys48): Proteindegradation • MonoUb or PolyUb (Lys 63): protein/protein interactions • Oftenworks in tandem with phosphorylation; • Enzymaticmachineryrivals the one used for phosphorylation: • 500 E3 ligases vs 518 kinases • 80 DUBs vs 120 phosphatases CHMI 4237 E - Winter 2010

  33. E1: Activating enzyme • Very few in the cell • E2: Conjugating enzyme • Catalyses the addition of Ub to substrateproteins • E3: Ub ligases • Responsible for the substratespecificity of the E2 enzyme • Lots of them in the cell E1 E2a E2b E2c Ubiquitin-mediatedproteolysis E3b E3a E3c CHMI 4237 E - Winter 2010

  34. http://www.nature.com/nrm/journal/v6/n8/images/nrm1701-i1.jpghttp://www.nature.com/nrm/journal/v6/n8/images/nrm1701-i1.jpg Ubiquitin-mediatedproteolysis CHMI 4237 E - Winter 2010

  35. http://www.mshri.on.ca/tyers/about_the_lab.html http://www.wormbook.org/chapters/www_ubiquitinpathways/ubiqfig2.jpg Ubiquitin-mediatedproteolysis CHMI 4237 E - Winter 2010

  36. Ubiquitin-mediatedproteolysis http://www.scills.ac.uk/images/whatis-1.jpg CHMI 4237 E - Winter 2010

  37. Ubiquitin-mediatedproteolysis CHMI 4237 E - Winter 2010

  38. http://www.cella.cn/book/13/images/image027.jpg CDK regulation CHMI 4237 E - Winter 2010

  39. http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/1940.jpghttp://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-19/1940.jpg Coordinatedregulation of CDKsduring the cell cycle CHMI 4237 E - Winter 2010

  40. CHMI 4237 E - Winter 2010

  41. pRb: • Retinoblastomaprotein • Family of 3 proteins: • pRb 105 kDa • p107 • P130 • pRbproteinsbindproteins of the E2F family • E2F: • Family of transcription factors • Whenbound to pRb: suppresses expression of genesrequired for cell cycle progression • After dissociation frompRb: E2F activates the expression of cell cycle-relatedgenes But: what do CDKsactually do?1- G1 phase NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | AUGUST 2007 CHMI 4237 E - Winter 2010

  42. In G0: • pRbisunphosphorylated • pRbbinds E2F • In early G1: • pRbishypophosphorylated by cyclin D-cdk4/6 • pRbbinds E2F • In late G1: • pRbishyperphosphorylated by Cyclin E/Cdk2 • Allows progression through the cell cycle past the restriction (« R ») point: point of no returnwhen the celliscommitted to complete the cell cycle: Before the R point: cellsrequiregrowthsignals to progress in G1 After the R point: growthsignals are no longer necessary pRb CHMI 4237 E - Winter 2010

  43. pRb and E2F CHMI 4237 E - Winter 2010

  44. Repression p107/p130: pRb Gene modulation by pRb and E2F Activation CHMI 4237 E - Winter 2010 Current Opinion in Cell Biology 2007, 19:658–662

  45. Cdk2 Cyclin A Cyclin E E2F genetargets CHMI 4237 E - Winter 2010

  46. Passing the R point CHMI 4237 E - Winter 2010

  47. Passing the R point:  Positive feedback loopsensure the irreversibility of the cell cycle CHMI 4237 E - Winter 2010

  48. DNA replicationoccurs once (and only once) during the cell cycle, during S phase; • All the enzymes required for DNA synthesis (nucleotidesynthesis, DNA synthesisproper, etc) have been producedprior to entering S phase; • At the end of S phase: the two copies of a duplicated chromosomes are physicallykepttogether as sisterchromatids via a proteincalledcohesin. But: what do CDKsactually do?2- S phase http://www.new-science-press.com/info/illustration_files/nsp-cellcycle-4-0-4_1.jpg CHMI 4237 E - Winter 2010

  49. In eukaryotes, replicationforksprogressat a rate of ~10-100 bp /sec; • The massive size of eukaryoticgenomerequires the presence of multiple replication initiation sites; • But: whatprevents a givenreplicationorigin to be more than once during the same S phase? http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cooper&part=A2433&rendertype=figure&id=A2448 But: what do CDKsactually do?2- S phase CHMI 4237 E - Winter 2010

  50. Replication origines are licensed by becomingloadedwith MCM proteins; • MCM loadingrequires a proteinscalled CDC6, itselfrecruited to replicationorigins by the protein ORC (OriginReplicationComplex); • MCM/ORC/CDC6 proteinsparticipate in recruiting the DNA replicationmachinery • However, S-phase cyclin (Cyclin A/Cdk2 in mammals) phosphorylates Cdc6, taggingit for degradation. • M-phase cyclins (Cyclin B/Cdk2 in mammals) alsophosphorylate CDC6, preventingreplication initiation duringmitosis; • The MCM proteins are displaced by the movingreplicationfork. But: what do CDKsactually do?2- S phase CHMI 4237 E - Winter 2010

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