1. CHMI 4237 E Special topics in Biochemistry
Cell proliferation
1 – basic machinery
Eric R. Gauthier, Ph.D.
Dept. Chemistry-Biochemistry
Laurentian University
CHMI 4237 E - Winter 2010

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

3. Mitosis
Blue: DNA / Green: microtubules
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4. Cell cycle Mitosis:  ~1 h
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5. Cell cycle
Signals
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6. 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 2010

7. 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 2010

8. Maturation Promoting Factor (MPF): the engine of the cell cycle
 MPF can trigger mitosis when injected
Into frog eggs
Works even in the presence of protein
synthesis inhibitors (e.g. cycloheximide)
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9. Cyclins: drivers of the cell cycle
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10. Cyclin levels and MPF activity fluctuate during the cell cycle
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11. MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk)
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12. MPF: dimer of a cyclin and a cyclin-dependent kinase (cdk)
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13. Specific cyclins and cdks pair up to control specific cell cycle events
Fission yeast
Mammals
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14. Specific cyclins and cdks pair up to control specific cell cycle events
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 2 | NOVEMBER 2001 | 815
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15. Importance of CDKs Nature Reviews | Cancer Volume 9 | March 2009
CHMI 4237 E - Winter 2010
Nature Reviews | Cancer Volume 9 | March 2009

16. CDK regulation
Progress through cell cycle
CDK activity
Cyclin levels
CDK levels
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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17. CDK activation 1 – cyclin binding
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18. Protein kinase structure
Upper jaw
ATP binding
P loop: Gly-rich sequence which binds the phosphates of ATP
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;

19. Protein kinase structure

20. CDK activation 1 – cyclin binding
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21. But: cyclin levels do not necessarily correlate with CDK activation….
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22. CDK activation 2- Phosphorylation by CDK-activating kinases (CAK)
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23. CDK activation 2- Phosphorylation by CDK-activating kinases (CAK)
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24. CDK regulation 3- Phosphorylation status of Tyr 15
 Wee 1 (kinase): phosphorylates Tyr 15, inactivating cdk2
 Cdc25 (phosphatase): de-phosphorylates Tyr 15, activating cdk2
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25. CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs)
Wild type
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26. CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs)
p27, p57, p21: obstruct ATP-
binding site
INK4 family: decrease affinity of
CDK 4/6 for D-type
cyclins
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27. CDK regulation 4- Inhibition by CDK inhibitory proteins (CKIs)
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28. CDK regulation 4- Modulation by CDK inhibitory proteins (CKIs)
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29. CDK regulation 4- Modulation by CDK inhibitory proteins (CKIs)
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30. CDK regulation 5- 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
CHMI 4237 E - Winter 2010

31. CDK regulation 6- Controlled proteolysis
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32. Ubiquitin-mediated proteolysis
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
CHMI 4237 E - Winter 2010

33. Ubiquitin-mediated proteolysis
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
E3b
E3a
E3c
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34. Ubiquitin-mediated proteolysis
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35. Ubiquitin-mediated proteolysis
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36. Ubiquitin-mediated proteolysis
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37. Ubiquitin-mediated proteolysis
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38. CDK regulation http://www.cella.cn/book/13/images/image027.jpg
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39. Coordinated regulation of CDKs during the cell cycle
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40. CHMI 4237 E - Winter 2010

41. But: what do CDKs actually do? 1- G1 phase
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
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 8 | AUGUST 2007
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42. pRb In G0: pRb is unphosphorylated pRb binds E2F In early G1:
pRb is hypophosphorylated by cyclin
D-cdk4/6
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
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43. pRb and E2F
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44. Gene modulation by pRb and E2F
Repression
Activation
Current Opinion in Cell Biology 2007, 19:658–662
p107/p130: pRb
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45. E2F gene targets
Cdk2
Cyclin A
Cyclin E
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46. Passing the R point
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47. Passing the R point:.  Positive feedback loops ensure the
Passing the R point:  Positive feedback loops ensure the irreversibility of the cell cycle
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48. But: what do CDKs actually do? 2- S phase
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.
CHMI 4237 E - Winter 2010

49. But: what do CDKs actually do? 2- S phase
In eukaryotes, replication forks progress at a rate of ~ 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?
CHMI 4237 E - Winter 2010

50. But: what do CDKs actually do? 2- S phase
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.
CHMI 4237 E - Winter 2010

51. But: what do CDKs actually do
But: what do CDKs actually do? 3- M phase - nuclear membrane dissolution
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;
CHMI 4237 E - Winter 2010

52. But: what do CDKs actually do
But: what do CDKs actually do? 3- M phase - nuclear membrane dissolution
CHMI 4237 E - Winter 2010

53. But: what do CDKs actually do
But: what do CDKs actually do? 4- M phase – separation of sister chromatids
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;
Nature Reviews | Cancer Volume 9 | March 2009
CHMI 4237 E - Winter 2010

54. But: what do CDKs actually do
But: what do CDKs actually do? 4- M phase – separation of sister chromatids
Nature Reviews | Cancer Volume 9 | March 2009
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;
CHMI 4237 E - Winter 2010

55. But: what do CDKs actually do? 5- M phase – Exiting mitosis
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)
CHMI 4237 E - Winter 2010

56. The Anaphase-promoting complex/cyclosome
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
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
PNAS December 22, 1998 vol. 95 no
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57. APC/CCdc20 - modulates anaphase and mitotic exit
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
CHMI 4237 E - Winter 2010

58. The Anaphase-promoting complex/cyclosome
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
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
CHMI 4237 E - Winter 2010

59. The Anaphase-promoting complex/cyclosome
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;
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 7 | SEPTEMBER 2006
CHMI 4237 E - Winter 2010

60. Inactivation of APC/Ccdh1
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
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61. The Anaphase-promoting complex/cyclosome
Genes Dev :
CHMI 4237 E - Winter 2010