Phosphorylation of CDK Targets Changes Their Activity

Now performsa cell cycle function

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 hormonesso that they lay eggs

2. Pack eggs into acentrifuge tube and spin

3. RemoveCytoplasmic Extract

4. Add sperm chromatinand away you go!

Cyclin Synthesis and DestructionDrives the Early Embryonic Cell Cycle

sea urchin!sea urchin!

Cyclin Destruction is Controlled by Ubiquitination

But is cyclin abundance the only

way to control CDK activity?

The Cell Cycle According to Cyclin Abundance

How are CDKs Regulated?

Isolate mutants that phenocopy cdc2- alleles

CDKs are Regulated by Phosphorylation

is a kinase is a phosphatase

CAK(CDKActivatingKinase)

Conformational Changes Associated with CDK Phosphorylation

The T-loop blockssubstrate access

Free CDK CDK + Cyclin T161 phosphorylation

Binding of cyclinmoves the T-loop

Phosporylation movesthe T-loop more

How does the G1-S Transition Work?

but…S. pombe cdc2+ can subsitute

for S. cerevisiae cdc28 and vice versa

cdc2 mutants arrest in G2

cdc28 mutants arrest in G1

Why?

and, cln1 and cln2 !!

The Identification of G1 Cyclins in S. cerevisiae

How Does High Copy Suppression Work?

The S. cerevisiae Cell Cycle

The G1-S Transition in S. cerevisiae

Growth Signal

Cln3/Cdc28

Cln1/Cdc28Cln2/Cdc28

make a bud

SIC1

G1

S

Clb5/Cdc28

Clb6/Cdc28

(cyclin/cdk)

Sic1 is Destroyed by Ubiquitination

(Cdc4,Cdc53)

Once and Only Once Replication is Controlled by CDKs

Human cdc2 (cdk1) Rescues cdc2 Mutant

cdk1cdk1

cdc2 mutantcells at 25C

Transformation with humancDNA library expressedwith SV40 viral promoter cdc2 transformed

mutant cells at 35C

Human cyclin E Rescues cln1,2,3 Mutant

cyc Ecyc E

cln1,2,3 triple mutantcells at 25C

Transformation with humancDNA library expressed

with yeast promoter cln1,2,3 transformed mutant cells at 35C

Figure 8.8 The Biology of Cancer (© Garland Science 2007)

Figure 8.9 The Biology of Cancer (© Garland Science 2007)

Figure 8.10 The Biology of Cancer (© Garland Science 2007)

Cyclin Dependent Kinase Inhibitors (CKIs)

Cyclin

CDKp21

Cyclin

CDK4

Cyclin

CDK

CDK4p16

p16

p21

The Discovery of p21 and p16

Cultured cells

Adding 35S[Met]

Metabolic labeling

Lysis cells midly

Immunoprecipitate

Add anti-CDK4 antibody

Add protein A-agarose beads

SDS-PAGE

Autoradiography

CDK4p16

Xiong et al. (1993) Genes & Dev. 7:1572

Cyclin

CDK4p21

CDK4

Cyclin D

p21

p16

Cell Line

No

rma

l

Tra

ns

form

ed

Tra

ns

form

ed

No

rma

l

Competing peptide - -+ +

-CDK4

The p21 Family of CDK inhibitors(p21CIP1/WAF1, p27KIP1, p57KIP2)

CDK

Cyclin

active

p21+

inactive

CDK

Cyclinp21

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

Jeffrey et al. (1995) Nature 376:313

The INK4 Family of CDK inhibitors(p16INK4a, p15INK4b, p18INK4c, p19INK4d)

INK4+CDK4/6

Cyclin D

active

CDK4/6Cyclin D

INK4+

inactive

Russo et al. (1998) Nature 395:237Brotherton et al. (1998) Nature 395:244

CKIs Regulate the G1-S Transition

(p16)

(p21, p27)

p16 is Frequently Mutated in Human Tumors

Tumor t ype L ines (n) Del etion s (n) D eleti ons (%) Astrocytoma 17 14 82 Bladder 15 5 33 Breast 10 6 60 Colon 20 0 0 G lioma 35 25 71 L eukemia 4 1 25 L ung 59 15 25 Melan oma 99 57 58 N euro blast oma 10 0 0 O steosarcoma 5 3 60 O vary 7 2 29 Renal 9 5 56 Total 29 0 13 3 46

Table 1. D eleti ons i n tumor cells and p rimary tumors.

See Kamb et al. (1994) Science 264: 436; Nobori et al. (1994) Nature 368:753 for detail

9p21

Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

Chemical structures of small molecular cdk inhibitors

Table 1. Pharmacologic effects of flavopiridol*

•IC50 = concentration that inhibits growth or activity by 50%;

•NCI = National Cancer Institute; DTP = Developmental Therapeutics Program; VEGF = vascular endothelial growth factor.

Effect IC50, nM

Growth inhibition, NCI DTP screen 66

cdk inhibition 40-200

Apoptosis 100-1000

Cell cycle arrest 100-300

Cyclin D1 depletion 100-300

Differentiation 100-300

VEGF depletion 50-100

Sensitization to standard chemotherapies 100-300

Epidermal growth factor receptor tyrosine kinase inhibition 21 000

Protein kinase A inhibition 122 000

Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

Table 2. Phase I trials with cdk modulators

* Free = concentration of UCN-01 in saliva.

Flavopiridol (96) UCN-01 (131)

Schedule 72-h continuous infusion every 2 wk 72-h continuous infusion every 4 wk (cycle 1) followed by 36-h continuous infusion every 4 wk (cycles 2 or higher)

Dose-limiting toxicity (maximal tolerated dose)

Diarrhea (50 mg/m2 per day for 3 days)

Hypotension (78 mg/m2 per day for 3 days)

Nausea/vomiting, hyperglycemia, and hypoxemia (42.5 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

Senderowicz, A. M. et al. J Natl Cancer Inst 2000;92:376-387

http://www.clinicaltrials.gov/