0. Molecular Cell Biology (Bio 5068)
Cell Cycle I
Ron Bose, MD PhD
November 10, 2016

1. CELL DIVISION CYCLE
MITOSIS G1 M G2 S

3. DISCOVERY AND NAMING OF CYCLINS
A protein (called “cyclin”) was observed to increase as cells approached mitosis, peak in mitosis and then precipitously disappear as cells exited mitosis.

4. Two proteins (cyclins A and B) increased as cells approached mitosis, peaked in mitosis and precipitously disappeared as cells exited mitosis.

5. The cell cycle is primarily regulated by cyclically activated protein kinases
Figure 17-15, Molecular Biology of the Cell, 4th Edition

6. Overview of major cyclins and Cdks of vertebrates and yeast
Table Molecular Biology of the Cell, 4th Edition

7. Evolution of cell cycle control: from yeast to humans
Malumbres M, Nature Reviews Cancer 2009

8. Cdk activity is regulated by inhibitory phosphorylation and inhibitory proteins
Why is cell cycle progression governed primarily by inhibitory regulation?
Figure 17-18, Molecular Biology of the Cell, 4th Edition

9. Cell cycle control depends on cyclical proteolysis
Figure Molecular Biology of the Cell, 4th Edition

10. UBIQUITIN-MEDIATED PROTEOLYSIS
Deshaies RJ and Joazeiro CA. Annu Rev Biochem. 2009
E1 (Ubiquitin activating enzyme)
Binds to Ubiquitin in an ATP-dependent manner
Passes Ubiquitin to E2
E2(Ubiquitin conjugating enzyme or UBC)
At least 12 in yeast
some are specific to a given target
E3 (ubiquitin protein ligase)
Large complex in both clam (cyclosome) and in frog (APC
= anaphase promoting complex). Final transfer of
ubiquitin to substrate can be mediated by E2 alone or E2
acting in concert with E3
Proteosome (26S complex) Structure from archaebacterium solved.
UBIQUITIN-MEDIATED PROTEOLYIS

11. Required for degradation of substrates at Metaphase to
APC = A
naphase P
romoting C
omplex
Required for degradation of
substrates
at Metaphase to
Anaphase transition ( ie
: B-type
Cyclins and securin)
substrate
Ring finger
UBC ( E2 )
for
ubiquitination
Apc10
Cdc20
Apcx
Apc4
Apc5
Apc7
Cdc27
Apc3
Apc1 /
BimE
Cdc23
Apc8
Cdc16
Apc6
Have D or
KEN Box
Cullin =
Apc2
Apc11 or
Cdh1
Hct1 WD
repeat-
containing
proteins
Ubiq
Ubiq
Ubiq
Ubiq
Cdc20
: targets
cyclin
A and B-type
Cyclins, securin
Cdh1 /
Hct1
: targets
Plk1 and B-type cyclins

12. SCF Ubiquitin Ligases Components: F Box: adapter
Brings substrate to E3 ligase.
F-box binds to Skp1
Additional protein interaction domains
(PID: WD repeat, leucine-rich repeat) binds to substrate
E2:
UBIQ. Conjugating enzyme (transfers UB to substrate)
Skp1:
Bridges F-box to cullin
Cullin:
Organizes and activates E3 complex
Recruits E2-UBIQ conjugating enzyme
Ring finger protein
Participates in E2 binding and catalysis

13. SCF E3 Ubiquitin Ligases
O'Connell BC, Harper JW. Curr Opin Cell Biol. 2007

14. CELL CYCLE REGULATION OF Cdc2
Reversible phosphorylation
Inhibitory kinase(s)
Phosphatase(s)
Protein-protein interactions
T14 Y15 T161
T161 P P P P
Cdc2
Cdc2
Cdc2
Cdc2
Cdc2
Cyclin B
Cyclin B
Cyclin B
INACTIVE
INACTIVE
INACTIVE
INACTIVE
ACTIVE
Cyclin B
Ubiquitin-mediated proteolyis
Activating Kinase(s)

15. Cyclin-dependent Kinase Inhibitor Proteins (CKI’s)
CIP/KIP family (p21Cip1, p27Kip1, p57Kip2):
Binds to Cdk2 and inhibits activity.
Binds Cdk4/6 and helps assemble complexes with cyclins.
INK4 family (p16, p15, p18, p19).
Specific for Cdk4 and Cdk6.
Binds Cdk subunit alone and prevents cyclin binding
Bind and inhibit Cdk4/6-Cyclin D heterodimers.

16. G1 Control M Cdk 4 & 6 INK4a proteins Cyclin D1, 2, 3 (p15,16, 18, 19)
Assembly &
Sequestration G1 S
Cdk2
Cyclin E
Cip/Kip proteins
(p21, p27, p57)

17. Mechanisms controlling G1/S-phase transition
MITOGENIC or
HORMONAL
SIGNALS
E2F
Transcription
factor
CYCLIN D Stability and
CYCLIN D-dependent Kinases
(Cdk4/Cdk6)
Rb1
Figure Molecular Biology of the Cell, 4th Edition

19. G1 Control CYCLIN E CDK2 RB E2F CYCLIN E S-Phase MITOGENIC SIGNALS
CYCLIN D STABILITY
CYCLIN D-DEPENDENT KINASES
(Cdk4/Cdk6)
E2F
Relief of Rb- mediated
transcriptional repression
CYCLIN A &
S-PHASE GENES
S-PHASE GENES
S-Phase

20. G1 Control p27KIP1-Phosphorylation Ubiq-Mediated proteolysis p27KIP1
Assembly &
Sequestration
CYCLIN E
CDK2 RB
E2F
CYCLIN E
S-Phase
MITOGENIC
SIGNALS
CYCLIN D STABILITY
CYCLIN D-DEPENDENT KINASES
(Cdk4/Cdk6)
E2F
Relief of Rb- mediated
transcriptional repression
CYCLIN A &
S-PHASE GENES
S-PHASE GENES
S-Phase

21. Checkpoints What are they? How were they defined?
How does their derailment contribute to cancer?

22. STOP! CHECKPOINTS IMPROPER SPINDLE ASSEMBLY M UNREPLICATED DNA G2 G1 S
DNA DAMAGE
UNREPLICATED DNA
IMPROPER SPINDLE
ASSEMBLY
STOP!

23. DNA damage checkpoint: integrity of DNA
Checkpoints: intracellular signaling pathways that determine if previous steps
are complete before proceeding onto the next stage (complete DNA synthesis before entering mitosis; spindles must be assembled before exiting metaphase and entering into anaphase) and whether there has been any damage to the DNA.
DNA damage checkpoint: integrity of DNA
DNA damage is repaired before entering S, completing S or entering M.
DNA replication checkpoint: replication state of DNA
Complete DNA synthesis before mitosis.
Spindle assembly checkpoint: integrity of spindle
spindles must be assembled before exiting metaphase
into anaphase.

24. DNA DAMAGE RESPONSE PATHWAY
G2-PHASE CHECKPOINT
STOP! G2 G1 S
G1-PHASE CHECKPOINT
S-PHASE CHECKPOINT

25. & activation of DNA repair pathways
CELLULAR RESPONSES TO CHECKPOINT ACTIVATION
(IR, etoposide, HU, gemcitibine, irinotecan, carboplatin…) G1 S G2 M
CHECKPOINTS
APOPTOSIS
SENESCENCE
TEMPORARY
CELL CYCLE ARREST
& activation of DNA repair pathways

26. Chemo- & Radio-therapy Signal Signaling Cascade Cellular Response
Fig. 2
Gemcitabine
Cytarabine
5-Fluorouracil
Chemo- &
Radio-therapy IR
Etoposide
Irinotecan
Topotecan
Cisplatin
Carboplatin
DSBs
Stalled replication forks
SSBs
Signal
RPA-ssDNA
ATM
ATR
Signaling Cascade
CHK2
p53
CHK1
BAX
PUMA
p21
14-3-3s
CDC25A
RAD51
FAND2
FANCE
Cellular
Response
Senescence
Apoptosis
Cell Cycle Arrest
DNA Repair 26

27. DNA DAMAGE CHECKPOINTS
IR/VP16 replication
stress
DNA DSBs ssDNA
ATM
ATR
Chk2
p53
Mdm2
Chk1
Cdc25A
p21, s
Cyclin B/Cdk1
Cyclin
E /
Cdk2 G1 S G2 M
DEATH
G1-checkpoint S-phase checkpoint G2 checkpoint
Overproduced in certain cancers.
Inactivated in certain cancers.

28. DNA damage leads to cell cycle arrest in G1
Figure Molecular Biology of the Cell, 4th Edition

29. Mitogens stimulate cell division
Figure Molecular Biology of the Cell, 4th Edition

30. Excessive stimulation of mitogenic pathways can lead to cell cycle arrest or cell death
Figure Molecular Biology of the Cell, 4th Edition

31. Extracellular Survival Factors Suppress Apoptosis
Figure Molecular Biology of the Cell, 4th Edition

32. Cell cycle regulators are frequently disrupted in cancer
Malumbres M, Nature Reviews Cancer 2001

33. Overview of CDK inhibitors in clinical development for cancer therapy
O’Leary et al., Nature Reviews Clinical Oncology 2016

34. Turner et al, NEJM 2015

35. NEJM Nov. 3, 2016

36. Conclusions
The cell cycle is a coordinated and tightly organized process to ensure the successful replication of the cell.
Activity of CDK-Cyclins is determined by:
Synthesis of Cyclins.
Reversible phosphorylation/dephosphorylation of stimulatory and inhibitory sites on CDK.
Ubiquitin mediated degradation of Cyclins.
CDK inhibitors – INK4 and CIP/KIP families.
Checkpoints can halt the cell cycle if all steps have not been properly completed.
Cancers have many alterations in cell cycle proteins and selective CDK4/6 inhibitors are now used in cancer treatment.