Phosphorylation of CDK Targets Changes Their Activity
Now performs
a cell cycle function
Yeasts have one CDK and several cyclins
Humans have 4 CDKs and 4 cyclins
CDK/Cyclin complex regulation controls the cell cycle
X
- 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
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
How are CDK’s Regulated?
1. By cyclin synthesis and destruction
2. By phosphorylation
3. By binding to CDK inhibitory proteins (CKIs)
Generation of a “Cycling” Frog Egg Extract
1. 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
Cyclin Synthesis and Destruction
Is Essential for Cell Cycle Progression
sea urchin!
sea urchin!
Cyclin Destruction is Controlled by Ubiquitylation
But what is ubiquitylation??!!
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
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)
How does the APC function?
M
APC
M
Cyclin and securin must be destroyed
in order for anaphase to take place
APC Activity changes during
the cell cycle
Negative feedback generates a
repeating oscillator
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
Once and Only Once S phase is Controlled by CDKs
The Cell Cycle According to Oscillating
Cyclin/CDK and APC Activity
But is cyclin
abundance the only
way to control CDK activity?
NO!
How else are CDKs Regulated?
Genetic studies in fission yeast.
CDKs are Regulated
by Phosphorylation
is a kinase
is a phosphatase
CAK
(CDK
Activating
Kinase)
Conformational Changes Associated
with CDK Phosphorylation
Free CDK
The T-loop blocks
substrate access
CDK + Cyclin
Binding of cyclin
moves the T-loop
T161 phosphorylation
Phosporylation moves
the T-loop more
How else are CDKs Regulated?
Biochemical studies
in mammalian cells.
Figure 8.8 The Biology of Cancer (© Garland Science 2007)
CDKs are regulated by
Cyclin Dependent Kinase Inhibitors (CKIs)
p21
CDK
CDK
Cyclin
Cyclin
p16
CDK4
Cyclin
CDK4
p16
p21
The Discovery of p21 and p16: what binds to CDKs?
Adding
Transformed
Transformed
35S[Met]
Cell Line
Normal
Cultured cells
Normal
a-CDK4
Competing
peptide
-
+
-
+
CDK4
Metabolic labeling
Cyclin
p21
Lysis cells midly
Add anti-CDK4 antibody
Add protein A-agarose beads
Immunoprecipitate
SDS-PAGE
Cyclin D
CDK4
CDK4
p16
Autoradiography
p21
p16
Xiong et al. (1993) Genes & Dev. 7:1572
The p21 Family of CDK inhibitors
(p21CIP1/WAF1, p27KIP1, p57KIP2)
active
inactive
CDK
CDK
Cyclin
+
p21
Cyclin
p21
Jeffrey et al. (1995) Nature 376:313
Figure 8.13b The Biology of Cancer (© Garland Science 2007)
Russo et al. (1996) Nature 382:325
The INK4 Family of CDK inhibitors
(p16INK4a, p15INK4b, p18INK4c, p19INK4d)
active
inactive
CDK4/6
CDK4/6
Cyclin D
+
Russo et al. (1998) Nature 395:237
Brotherton et al. (1998) Nature 395:244
INK4
INK4
+
Cyclin D
CKIs Regulate the G1-S Transition
(p16)
(p21, p27)
p16 is Frequently Mutated in Human Tumors
Table 1. D eleti ons i n tumor cells and p rimary tumors.
9p21
Tumor t ype
L ines (n)
Del etion s (n) D eleti ons (%)
Astrocytoma
Bladder
Breast
Colon
G lioma
L eukemia
L ung
Melan oma
N euro blast oma
O steosarcoma
O vary
Renal
17
15
10
20
35
4
59
99
10
5
7
9
14
5
6
0
25
1
15
57
0
3
2
5
82
33
60
0
71
25
25
58
0
60
29
56
Total
29 0
13 3
46
See Kamb et al. (1994) Science 264: 436; Nobori et al. (1994) Nature 368:753 for detail
Therapeutic Targeting of the Hallmarks of Cancer
Hanahan and Weinberg, Cell 144:646 (2011)
Chemical structures of small molecular cdk inhibitors
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387
Table 1. Pharmacologic effects of flavopiridol*
Effect
Growth inhibition, NCI DTP screen
cdk inhibition
Apoptosis
IC50, nM
66
40-200
100-1000
Cell cycle arrest
100-300
Cyclin D1 depletion
100-300
Differentiation
100-300
VEGF depletion
Sensitization to standard chemotherapies
Epidermal growth factor receptor tyrosine kinase inhibition
Protein kinase A inhibition
50-100
100-300
21 000
122 000
•IC50 = concentration that inhibits growth or activity by 50%;
•NCI = National Cancer Institute; DTP = Developmental Therapeutics Program; VEGF = vascular endothelial growth factor.
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387
Table 2. Phase I trials with cdk modulators
Flavopiridol (96)
UCN-01 (131)
Schedule
72-h continuous infusion every 2 wk
72-h continuous infusion every 4 wk (cycle 1) followed by 36h continuous infusion every 4 wk (cycles 2 or higher)
Dose-limiting toxicity (maximal
tolerated dose)
Diarrhea (50 mg/m2 per day for 3 days)
Nausea/vomiting, hyperglycemia, and hypoxemia
(42.5 mg/m2 per day for 3 days)
Hypotension (78 mg/m2 per day for 3
days)
Other toxic effects
Anorexia, proinflammatory syndrome
Headache, myalgias
Suggestion of activity
Non-Hodgkin's lymphoma and renal,
colon, gastric, or prostate cancer
Melanoma, non-Hodgkin's lymphoma, or leiomyosarcoma
Median plasma concentration at
maximal-tolerated dose
271 nM (50 mg/m2 per day for 3 days)
344 nM (78 mg/m2 per day for 3 days)
Total = 36.4 µM (42.5 mg/m2 per day for 3 days)
Free* = 111 nM (42.5 mg/m2 per day for 3 days)
Plasma half-life, h
11.6
588
*
Free = concentration of UCN-01 in saliva.
Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387
http://www.clinicaltrials.gov/
51 studies for CDK inhibitors