1. Phosphorylation of CDK Targets Changes Their Activity
Now performs
a cell cycle function

2. Yeasts have one CDK and several cyclins
Humans have 4 CDKs and 4 cyclins

3. CDK/Cyclin complex regulation controls the cell cycle
- Cyclin-Cdk complexes function in different phases
- G1/S-Cdk complexes commit the cell to a new cell cycle
- S-Cdk complexes promote S phase (and block it in G2!)
- M-Cdk complexes trigger entry into mitosis
- M-Cdk complexes are inactivated before anaphase

4. Example: cycB-Cdk1 appears in mitosis, phosphorylates lamin and leads to nuclear envelope breakdown during early mitosis
cycB-Cdk1 will be destroyed during mitosis to allow formation of a new nuclear envelop breakdown during telophase

5. How are CDK’s Regulated?
By cyclin synthesis and destruction
By phosphorylation
By binding to CDK inhibitory proteins (CKIs)

6. Generation of a “Cycling” Frog Egg Extract
Inject females with hormones
so that they lay eggs
2. Pack eggs into a
centrifuge tube and spin
4. Add sperm chromatin
and away you go!
3. Remove
Cytoplasmic Extract

7. Cyclin Synthesis and Destruction
Is Essential for Cell Cycle Progression
sea urchin!
sea urchin!

8. But what is ubiquitylation??!!
Cyclin Destruction is Controlled by Ubiquitylation
But what is ubiquitylation??!!

9. Ubiquitylation: a post-translational
protein modification
E3 ubiquitin ligase is a protein complex that confers specificity:
i.e. which protein to target
Proteasome: the cellular garbage can
E1, E2, and E3 enzyme cascade

10. An E3 ubiquitin ligase called the Anaphase Promoting Complex (APC) destroys mitotic cyclins (and other things)
Not to be confused with:
APC (adenomatous polyposis coli)
APC (antigen-presenting cell)

11. How does the APC function?M
APC M
Cyclin and securin must be destroyed in order for anaphase to take place

12. APC Activity changes during the cell cycle
Negative feedback generates a repeating oscillator

13. The Cell Cycle According to Oscillating Cyclin/CDK and APC Activity
A cyclin promotes synthesis of the next cyclin that in turn, promotes destruction of the previous one

14. Once and Only Once S phase is Controlled by CDKs

15. NO! The Cell Cycle According to Oscillating
Cyclin/CDK and APC Activity
But is cyclin
abundance the only
way to control CDK activity?
NO!

16. How else are CDKs Regulated? Genetic studies in fission yeast.

17. CDKs are Regulated by Phosphorylation is a kinase is a phosphatase CAK
Activating
Kinase)
is a kinase is a phosphatase

18. Conformational Changes Associated with CDK Phosphorylation
Free CDK
CDK + Cyclin
T161 phosphorylation
The T-loop blocks
substrate access
Binding of cyclin
moves the T-loop
Phosporylation moves
the T-loop more

19. How else are CDKs Regulated?
Biochemical studies
in mammalian cells.
Figure 8.8 The Biology of Cancer (© Garland Science 2007)

20. Cyclin Dependent Kinase Inhibitors (CKIs)
CDKs are regulated by
Cyclin Dependent Kinase Inhibitors (CKIs)
p21
Cyclin
CDK
CDK
p21
Cyclin
p16
Cyclin
CDK4
CDK4
p16

21. The Discovery of p21 and p16: what binds to CDKs?
Transformed
Transformed
Cultured cells
Cell Line
Normal
Normal
Competing - + - +
Adding 35S[Met]
peptide
Cyclin
CDK4
p21
Metabolic labeling
Lysis cells midly
Add anti-CDK4 antibody
Add protein A-agarose beads
Immunoprecipitate
Cyclin D
SDS-PAGE
CDK4
Autoradiography
p21
p16
Xiong et al. (1993) Genes & Dev. 7:1572

22. The p21 Family of CDK inhibitors
(p21CIP1/WAF1, p27KIP1, p57KIP2)
CDK
Cyclin
active
p21 +
inactive
CDK
Cyclin

23. Jeffrey et al. (1995) Nature 376:313
Russo et al. (1996) Nature 382:325
Figure 8.13b The Biology of Cancer (© Garland Science 2007)

24. + + The INK4 Family of CDK inhibitors
(p16INK4a, p15INK4b, p18INK4c, p19INK4d)
CDK4/6
Cyclin D
active
CDK4/6
Cyclin D
INK4 +
inactive
INK4 +
Russo et al. (1998) Nature 395:237
Brotherton et al. (1998) Nature 395:244

25. CKIs Regulate the G1-S Transition
(p16)
(p21, p27)

26. p16 is Frequently Mutated in Human Tumors

27. Therapeutic Targeting of the Hallmarks of Cancer
Hanahan and Weinberg, Cell 144:646 (2011)

28. Chemical structures of small molecule cdk inhibitors
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:

29.
141 studies for CDK inhibitors

30. Growth Factors Induce Cell Proliferation
act Prior to
the Restriction Point
Modified from The Biology of Cancer (© Garland Science 2007)

31. Cell Division + Growth =
Let’s think about the Difference Between Growth and Cell Division
Growth with
No Cell Division
Cell Division
No Growth
Cell Division + Growth =
Proliferation!

32. A Differentiated Neuron
Growth with
No Cell Division:
A Differentiated Neuron

33. Cell Division with No Growth: Early Development
OOCYTE GROWS WITHOUT DIVIDING
(MONTHS)
FERTILIZED EGG DIVIDES WITHOUT GROWING
(HOURS)
FERTILIZATION
1 mm
sperm
tadpole feeds, grows
and bcecomes an adult frog

34. Unregulated Proliferation: Cancer!
A Typical Solid Tumor…Needs to Grow
The size of a tumor first detectable by X-ray: 108 cells
The size of a tumor first palpable: cells
The size of tumor at death of patient: cells
Unregulated Proliferation: Cancer!

35. Budding Yeast Saccharomyces cerevisiae

36. Cdc Mutants Affect the Cell Cycle, Not Growth
Permissive (low) temperature
Restrictive (high) temperature

37. Mammalian Cells Growing in Cell Culture
Amino Acids
Vitamins
Salts
Miscellaneous
Arginine
Cystine
Glutamine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine
Biotin
Choline
Folate
Nicotinamide
Pantothenate
Pyridoxal
Thiamine
Riboflavin
NaCl
KCl
NaH2PO4
NaHCO3
CaCl2
MgCl2
Glucose
Penicillin
Streptomycin
Phenol red
Whole serum
Specific growth factors
1961Hayflick and Moorhead
Showed that human fibroblasts die after a finite number of divisions in culture.
This is called “The Hayflick Limit”

38. Some Commonly Used Cell Lines
Cell Type and Origin
3T 3
fibroblast (mouse)
BHK 21
fibroblast (Syrian hamster)
MDCK
epithelial cell (dog)
HeLa
epithelial cell (human)
PtK 1
epithelial cell (rat kangaroo)
L 6
myoblast (rat)
PC 12
chromaffin cell (rat)
SP 2
plasma cell (mouse)
*Many of these cell lines were derived from tumors. All of them are capable of indefinite replication in culture and express at least some of the differentiated properties of their cell of origin. BHK 21 cells, HeLa cells, and SP 2 cells are capable of growth in suspension; the other cell lines require a solid culture substratum in order to multiply.

39. Growth Factors Induce Cell Proliferation
Growth + Cell Cycle Progression: But How?
Growth Factors
act Prior to
the Restriction Point
Modified from The Biology of Cancer (© Garland Science 2007)

40. Growth Factors Induce Gene Expression

41. Growth Factors Induce Oncogene Expression As Early Response Genes

42. Growth Factors Induce Cyclin D Expression They also act by
inhibiting CKIs
We actually have:
3 D-type cyclins
2 E-type cyclins
2 A-type cyclins
3 B-type cyclins
And the D-type cyclins respond to different cues

43. Growth Factors Get the Cell Cycle Going

44. ONCOGENES!!!! Growth Factors Induce Cyclin D Expression
So, is Cyclin D an oncogene?

45. YES! Cyclin D is also an Oncogene
Disease: INVOLVED IN B-LYMPHOCYTIC MALIGNANCY (PARTICULARLY MANTLE-CELL LYMPHOMA)
BY A CHROMOSOMAL TRANSLOCATION T(11;14)(Q13;Q32) THAT INVOLVES CCND1 AND IMMUNOGLOBULIN
GENE REGIONS (BCL1 ONCOGENE).
Disease: INVOLVED IN A SUBSET OF PARATHYROID ADENOMAS BY A CHROMOSOMAL TRANSLOCATION T(11;11)(Q13;P15) THAT INVOLVES CCND1 AND THE PARATHYROID HORMONE (PTH) ENHANCER (PRAD1 ONCOGENE).

46. Translocations Cause One Gene to be Controlled by Another